Understanding HPV tests and their appropriate applications

REVIEW DOI:10.1111/cyt.12083 Understanding HPV tests and their appropriate applications H. A. Cubie* and K. Cuschieri *HPV Research Group, The Queen s Medical Research Institute, University of Edinburgh MRC Centre for Reproductive Health, Edinburgh, UK, Scottish HPV Reference Laboratory, Royal Infirmary of Edinburgh, Edinburgh, UK Accepted for publication 7 June 2013 Understanding HPV tests and their appropriate applications Greater understanding of the role played by human papillomavirus (HPV) in the causation of disease has led to the development of an increasing number of HPV tests with different characteristics. The bewildering choice facing healthcare professionals and providers is daunting. Clearly, HPV testing is no longer simply of research interest, but can provide information that can be used for individual patient management and at the population level for cervical screening and vaccine surveillance. This review aims to provide the background to the development of HPV tests, to explain the different technologies and to discuss the challenges of the application of these optimally in the varied contexts of disease management. Few HPV tests are approved for clinical use and it is important that clinicians understand which test can be utilized, in what circumstances, with what specimens and the meaning of the report issued. HPV testing is no longer applicable only to cervical disease, and we have suggested additional areas, such as the oropharynx, in which HPV testing services might be implemented in the near future. New tests will continue to emerge and we have identified some of the indirect measures of HPV activity, or biomarkers, that could help in the risk stratification of HPV infection and associated disease. The challenges relating to the optimal application of the various HPV technologies are compounded by the lack of evidence regarding their performance in vaccinated populations. Currently published work, including modelling studies, has been undertaken in non-immunized populations. We therefore end by addressing the issues regarding appropriate strategies and tests for immunized populations. Keywords: human papillomavirus tests, molecular technologies, clinically approved/validated, human papillomavirus vaccine, cervical screening, oropharyngeal cancer management Introduction Human papillomaviruses (HPVs) are ancient and ubiquitous viruses. HPV was the first known human tumour virus, associated with cutaneous warts of the external and anogenital skin, which can be readily observed clinically and require no laboratory tests Correspondence: H. A. Cubie, HPV Research Group, The Queen s Medical Research Institute, University of Edinburgh MRC Centre for Reproductive Health, 47 Little France Crescent, Edinburgh EH16 4TJ, UK Tel.: +44-0-131-242-6625; Fax: +44-0-131 242 6008; E-mails: heather.cubie@nhslothian.scot.nhs.uk; heather. cubie@ed.ac.uk for confirmation. In the last three decades, an understanding of the causal association of HPV with cervical cancer and, more recently, with other anogenital and oropharyngeal cancers 1 has led to step changes in the clinical relevance of HPV detection to aid diagnosis, with a consequent impact on clinical management. Molecular advances, using nucleotide sequence homology in the L1 gene, have led to the official recognition of 120 different HPV types with others pending classification. 2 A new type is defined as one in which the L1 open reading frame (ORF) DNA sequence differs by more than 10% from the most closely related known papillomavirus type. The HPV family of viruses is subdivided into five genera, with 289

290 anogenital HPV types, of which there are about 40, mostly clustered in the a-genus and cutaneous HPV clustered in the b- and c-genera. In addition to a systematic, phylogenetic approach to classification, HPV types are often stratified according to their risk of causing cancer, ranging from those almost never associated with cancer [low-risk (LR) types] to those which carry a relatively high risk of progression [high-risk (HR) types]. The risk status of a particular type should be informed by both epidemiological and mechanistic evidence. An expert working group was tasked with the examination of the evidence for carcinogenicity of HPV types using groupings set out by the International Agency for Research on Cancer as follows (and as shown in Table 1): Group 1 (Carcinogenic to humans), Group 2A (Probably carcinogenic to humans), Group 2B (Possibly Carcinogenic to humans), Group 3 (Not classifiable) and Group 4 (Probably not carcinogenic). The most recent output of this group reported 12 types as being Group 1 Carcinogens: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59. 3 Although determination of the risk status of HPV types is clearly important, it can be challenging, particularly for rare types and for types which are infrequently found as single infections. Furthermore, as evidence accumulates, classifications can refine (as evidenced by the recent shift of HPV 66 from Group 1 to Group 2B). 3 More than 70% of invasive cervical carcinoma worldwide is associated with HPV 16 and/or 18. 4 Most people become infected with HPV at some point in their lives, but the ecological niche which HPV has carved for itself is one in which individuals usually do not know that they have been infected or when they have cleared the virus. Transmission requires close skin contact, and therefore sexual activity is the most common method of spread to anogenital sites. HPV can rightly be described as a sexually transmitted infection (STI), but not as a sexually transmitted disease. 5 It is widely accepted that there are four stages in the progression of disease HPV acquisition, HPV persistence, progression of persisting infection to pre-cancer and invasion. 6 There is a need to distinguish these in the laboratory by the use of appropriate tests. The very ubiquity of HPV is both a useful and difficult facet, making the sensitivity of detection easier, but clinical relevance harder to achieve. By acknowledging that HPV persistence is a critical factor in the development of cancer, it can be seen that detection of the virus at a single time point might simply be an indicator of transient infection and not of disease. Early methods of detection of HPV HPVs are not readily amenable to in vitro culture as they require differentiated epithelial cells in order to complete the virus life cycle. In addition, natural infection does not consistently lead to the production of a detectable antibody response. 7 Thus, the traditional tools commonly used by diagnostic virology laboratories are not suitable for the detection of HPV. However, HPV has a stable double-stranded DNA genome that can be accessed easily from exfoliated cells, and natural sources of infectious virus, such as wart scrapings, were used to develop early molecular nucleic acid tests almost 30 years ago. The first clinically applied HPV detection methods in the 1980s included in situ hybridization (ISH) with cloned or synthetic oligonucleotide probes. 8,9 These had the advantage of maintaining cellular structure and localization, but were not sufficiently sensitive for routine use. 10 The earliest solution hybridization assays included Southern blotting to detect the hybrids, but the results were variable and not sufficiently robust for clinical application. The first reliable, quality standardized HPV DNA test was developed by the Digene Corporation (Gaithersburg, MD, USA) and gained United States Food and Drug Administration (FDA) approval in 1999 for reflex testing of patients with atypical squamous cells of undetermined significance (ASC-US) cytology. 11 This was the aptly named Hybrid Capture assay which was based on scientifically sound principles of hybridization in solution of one strand of template Table 1. Classification of a-human papillomavirus (a-hpv) genotypes by carcinogenic potential Group 1 Carcinogenic to humans HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 Group 2A Probably carcinogenic to humans HPV 68 Group 2B Possibly carcinogenic to humans HPV 26, 53, 64, 65, 66, 67, 69, 70, 73, 82 Group 3 Not classifiable as to carcinogenicity in humans HPV 6, 11

Understanding HPV tests 291 DNA to complementary RNA probes of 13 different HPV types. In 2003, FDA approval was extended to the use of Hybrid Capture 2 alongside routine Papanicolaou (Pap) testing for women over the age of 30 years. 12 The technology has stood the test of time over 20 years and the second-generation Hybrid Capture 2 High-Risk HPV DNA Test (hc2) remains widely applied today and is associated with the largest evidence base for application (see next section). However, as with any test, hc2 is not perfect and can be associated with cross-reactivity of the probe cocktail with untargeted HPV types, especially HPV 11, 53, 54, 55 and 66. In addition, hc2 does not contain an internal cellular control, which can be helpful for the adjudication of false negatives. hc2 also has an additional 5% false-positive rate in samples which apparently contain no HPV DNA according to the application of highly sensitive broad-range polymerase chain reaction (PCR) tests. 13 Current HPV technologies The emergence of nucleic acid amplification techniques (NATs) has allowed for the development of alternative HPV detection assays beyond hc2. These are broadly divided into signal and target amplification assays. The latter are heterogeneous with respect to detection range (type specific or broad spectrum), the molecule amplified (DNA or mrna), the amplification chemistry and the particular HPV gene targeted. These differing technologies allow further subdivision within the target amplification set and some have sufficient sensitivity to be clinically useful in a range of biospecimens. Comprehensive expert reviews of the more established HPV tests can be found in Poljak and Kocjan 14 and Cuschieri. 15 The principal categories and subdivisions of HPV detection assays are summarized in Table 2a,b, together with examples of signal amplification and target amplification respectively, by no means exhaustive, of the available HPV tests. 16 38 Indirect tests of HPV infection, such as p16 ink4a, Ki67 and MCM, which are involved in risk stratification and confirmation of disease status, 39 are not included in Table 2a,b, and are described in the Emerging biomarkers section below. Although many HPV detection methods have been used successfully in research laboratories for years, only five are currently approved by the FDA for clinical use, including four DNA-based assays [digene HC2 (Qiagen NV, Venlo, Netherlands), Cervista HPV HR (Hologic, WI, USA), Cervista HPV 16/18 tests (Hologic Inc., Bedford, MA, USA), Cobas 4800 HPV (Roche Molecular Diagnostics, Pleasanton, CA, USA)] and one RNA assay [APTIMA HPV assay (formerly GenProbe Inc., San Diego, CA, USA)] for the detection of E6 and E7 HR-HPV mrna. Several additional tests have fulfilled the simpler Conformite Europeenne (CE) marking process in Europe. In this rapidly changing market, new companies and mergers are not infrequent, with hc2 now being owned by Qiagen and GenProbe having recently merged with Hologic. Performance measurement, validation and quality assurance of HPV tests in an increasingly competitive market The burgeoning market for HPV tests makes standardization and comparison of performance difficult and causes considerable confusion for those new to HPV testing. In 2009, Meijer et al. 40 published a sentinel paper providing guidelines on how to assess the performance of a new HPV assay in the setting of cervical screening. Underpinning the Meijer criteria was the premise that a new test should show demonstrable non-inferiority with respect to clinical sensitivity and specificity when compared with the clinically validated hc2 assay. The guidelines also incorporated an assessment of the technical robustness of new assays through the measurement of intra- and inter-laboratory reproducibility. Meijer et al. 41 then applied these standards to their own well-known GP5+/6+ HPV assay, thus producing the first of many comparative validation studies to show non-inferiority to hc2. Although these criteria have been useful in setting a common reference point, it is important to remember that they were designed to assess the performance of HPV tests as a screen in women over 30 years of age. The prevalence of infection and associated disease will vary according to context (primary screening, triage, test of cure), as will the priority for performance in terms of sensitivity and specificity. Stoler et al. 42 presented a slightly different metric for the validation of new HPV screening assays, recommending a clinical sensitivity of 92 3% for the detection of cervical intraepithelial neoplasia or worse (CIN3+) and a specificity of at least 85% to achieve an adequate positive predictive value (PPV) for CIN3, with no link to a reference test. Both Meijer s and Stoler s recommendations have the crucial aim of achieving a practical and clinically relevant

292 Table 2. Overview of HPV tests: (a) signal and (b) target amplification Sub-division Technology Examples (suppliers) Comments/applications References (a) Hybridisation (b) Amplification Consensus DNA PCR Consensus DNA real-time PCR with limited genotyping Consensus RNA amplification RNA amplification with limited typing Full genotyping In situ hybridisation INFORM HPV III (Roche) Kits with cocktails of HPV Family 6 or HPV Family 16 probes for both cytology and tissue applications Solution hybridisation and capture of RNA probes complimentary to L1 DNA sequences hc2 (Qiagen) Detects 13HR HPV types in aggregate. Well established assay for cervical screening and disease management As above care HPV (Qiagen) Simplified version of hc2 suitable for field testing in low resourced countries Solution Cervista HPV HR (Hologic) Novel approach using cleavase enzyme; hybridisation with detects 14 HR HPV types across three probe oligo and species-specific wells. Approved for Invader oligo cervical screening an disease management Cervista HPV 16/18 Reflex test giving additional typing information Degenerate/ multiplex/ consensus primers As above, but also Incorporating limited type specific amplification Transcription mediated Amplification (RNA) NASBA and type specific resolution using molecular beacons for 5 HR HPV types PCR with hybridisation using enzyme immunoassay (EIA) PCR with reverse hybridisation of amplicons on nylon strips with immobilised probes MY09/11; PGMY GP5+/6+ RealTime HR HPV (Abbott Molecular, IL, USA); Cobas 4800 HPV (Roche) Aptima HPV (GenProbe now Hologic) HPV Proofer (Norchip, Klokkarstua, Norway) Nuclisens HPV (Biomerieux, Marcy L Etoile, France) GP5+/6+-PCR-EIA Generalised amplification against L1 sequences GP5+/6+ used extensively clinically for cervical screening especially in Netherlands Detect HPV 16 and 18 individually and other HR-HPVs in aggregate; suitable for cervical screening with risk stratification beyond presence/absence of HPV Detection of E6/E7 HPV mrna (14 HR HPV types); evidence for increased specificity, particularly in triage contexts High specificity but lower sensitivity due to limited type range (HPV 16, 18, 31, 33, 45) N.B. HPV proofer is available in Scandinavia & UK; Nuclisens HPV is similar test in mainland Europe As with all full genotyping assays suitable for epidemiology and surveillance; R&D including detection in new conditions Linear Array (Roche) Line blot based on PGMY primers 33 InnoLiPA (Innogenetics, Gent Belgium) Line blot based on SPF10 primers; validated on FFPE sections and can be automated 34 16 17 18 19 20 22 23 26 27 29 30 31 32

Understanding HPV tests 293 Table 2 (continued) Sub-division Technology Examples (suppliers) Comments/applications References Mid range typing Microarray Luminex technology Multiplex real time PCR Papillocheck HPV (Greiner Bio-one, Frickenhausen, Germany) CLART â HPV2 (Genomica, Coslada, Spain) Multimetrix HPV Genotyping Test (DiaMex, Heidelberg, Germany) BD Viper Assay (BD, NJ, USA) PCR with Microarray reverse hybridisation; targets 1 gene and involves simultaneous detection and genotyping of 24 LR and HR types Hybridiastion to each probe in array in triplicate; detecting up to 35 types with visualisation using low density arrays Sensitive, can be used to detect up to 100 different targets Recently developed by BD; offers consensus test result plus individual typing of 16, 18, 45, 31, 51 52, 33/58, 59/56/66, 35/39/68 35 36 37 38 balance between excessive referral and the risk of missing high-grade disease. This said, there are many HPV assay comparison studies using disparate populations and varied outcome measures, from studies of technical concordance in a cross-sectional sample to the measurement of clinical performance in longitudinal populations. 43 45 Many comparison studies have adopted the test A versus test B approach, but the frequently cited Predictors suite of studies incorporates multi-test comparisons. 46,47 These studies assessed the comparative performance of cytology and over 10 HPV assays, including DNA and RNA amplification tests, genotyping tests and p16 ink4a (p16), within a population of c. 1000 women referred to colposcopy. Although the associated data are undoubtedly of value (discussed in more detail in section HPV Triage of Low grade Disease ), given the high prevalence of disease in this setting, caution should be exercised before extrapolating test performance to other settings in which the prevalence of disease is lower. Notwithstanding the challenges with regard to validation, the choice of an appropriate test for a service is not the end of the story. Ongoing quality assurance programmes (QAPs) should be in place to ensure that the right test is carried out on the right specimen, and the right result with the right interpretation is delivered to the right person at the right time. The programme should include regular use of internal controls, both internal and external quality assessment or proficiency testing of the assay(s) used and regular competency assessment of the staff involved. A QAP was developed for the English NHS cervical screening programme (CSP) HPV/liquid-based cytology (LBC) pilots in 2003. 48 This acted as the spur to develop a national external quality assessment (EQA) scheme through the UK National External Quality Assessment Scheme (UKNEQAS). 49 HPV distributions consisting of four unknown samples every quarter are sent to around 60 European laboratories delivering HPV tests as part of their cervical screening service. The reports generated by UKNEQAS allow participants to assess their proficiency against other laboratories. Other national EQA schemes exist, such as in Australia, 50 as do international schemes, such as the World Health Organization (WHO) HPV Labnet HPV proficiency panel 51 and the European Quality Control for Molecular Diagnostics (see www.qcmd.org). In the UK, where HPV testing has just begun to be incorporated into service provision, a set of quality assurance standards has been produced and compliance is essential for any laboratory to be permitted to deliver clinically applied tests (Table 3). 52 Challenges of HPV testing for disease control and management There are a number of contexts in which HPV testing might be applied, and different tests may be more suited to one context than another according

294 Table 3. Adapted from quality assurance standards for human papillomavirus (HPV) testing in NHS Cervical Screening Programme (CSP), UK 2012 52 Number Standard 1 Accreditation through CPA or equivalent; HPV tests listed in their repertoire of services 2 Participation in accredited external quality assurance scheme for HPV, such as UKNEQAS Molecular Detection of HPV scheme 3 Each member of staff undertaking HPV testing must first pass company training procedures, with training recorded in an individual s training log 4 Competency of staff/laboratory should be demonstrated through testing of an HPV Validation Panel 5 Competency should be assessed regularly for as long as staff have responsibility for the function 6 Laboratories undertaking HPV testing must include in every run known positive and negative IQC samples validated by repeated testing, in addition to required kit controls 7 Laboratories undertaking HPV testing should check regularly (e.g. weekly; 1% of samples; every 100th sample) for systemic errors and environmental testing of the laboratory areas used CPA, Clinical Laboratory Accreditation (UK) Ltd; IQC, internal quality control; UKNEQAS, UK National External Quality Assessment Scheme. to the prevalence of infection and the nature of the population with respect to risk and location that is screening or clinic setting, high- or low-income country. The vast majority of data on HPV testing relate to its use for the detection and prevention of cervical cancer through screening programmes. However, as our understanding of HPV and its association with other cancers develops and immunization programmes embed, we also discuss suitable tools for management and measurement in such settings for completeness. HPV testing in uterine cervical settings There are four main clinical applications for HPV testing in cervical screening programmes primary cervical screening (sometimes referred to as HPV first); HPV triage of low-grade cytological abnormalities; test of cure following treatment; and resolution of uncertainties. 53 Whether HPV primary screening or HPV triage is implemented within national screening programmes, balancing the sensitivity and specificity in screening is challenging and, assuming that only clinically validated tests are permitted, service providers also need to consider throughput, location, time management and cost. 54 Furthermore, there are additional practical challenges surrounding the particular collection medium used for liquid-based cellular samples. Although many laboratories have developed their own collection media for cervical screening samples, two commercial types predominate those designed for optimal cellular preservation, such as PreservCyt (Hologic) and SurePath â (BD, Franklin Lakes, NJ, USA), and those designed for optimal nucleic acid preservation and HPV detection, including Specimen Transport Medium, STM TM (Qiagen Ltd, Hilden, Germany), Specimen Transport Buffer (Abbott Molecular, Des Plaines, IL, USA) and Cobas â PCR Cell Collection Media (Roche Molecular Diagnostics). One of the advantages of using LBC medium is being able to perform both cytology and HPV testing on the same specimen. ThinPrep â -collected specimens provide sufficient material for molecular tests with limited processing dependent on the HPV test selected. Rather more processing is required for SurePath, where the density gradient centrifugation process used to remove blood and mucus leaves only a small volume for other tests. Unlike ThinPrep, the SurePath vial does not have FDA approval for HPV testing, and recent media coverage suggested that this was because of the possibility of false-negative results, 55 although the evidence base for this is poorly defined. Current evidence suggests that HPV nucleic acid (particularly RNA) is less well preserved in SurePath medium compared with ThinPrep. This has been shown in time course experiments in which the yield and degradation of HPV nucleic acid have been quantified. 56,57 In addition, certain mrna assays, such as the PreTect HPV Proofer (NorChip, Klokkarstua, Norway), are still not validated for this medium. Consequently, more stringent criteria in terms of sample processing, transit, storage and assay turnaround have been developed for SurePath. By way of an example, with the APTIMA assay (using the TIGRIS DTS system, Hologic/GenProbe, San Diego, CA, USA),

Understanding HPV tests 295 ThinPrep samples can be stored for up to 105 days before being added to the assay medium, compared with 7 days for SurePath samples. Provided that the criteria are observed, and the clinical performance is unaffected, shortfalls in analytical performance between the two media may be largely academic. However, the logistical challenges associated with satisfying such criteria can be non-trivial; thus, it is important that they are revisited and evidenced-based to be shown to be no stricter than necessary. 58,59 Concern has been expressed that the treatment of samples with glacial acetic acid (GAA) may affect the performance of HPV testing. With ThinPrep samples, this can be avoided by the use of the Pre-quot step, which allows the removal of an aliquot before cytological processing. However, recent studies published in this issue of Cytopathology have suggested that GAA treatment has little impact on the performance of both target and signal amplification assays, at least at the qualitative level. 60,61 HPV primary screening There is now strong evidence to suggest that primary screening with HPV may be effective in women over 30 years of age. The recent meta-analysis of Arbyn et al., 53 which incorporated the analysis of a total of 49 studies (including eight randomized controlled trials), confirmed that HPV testing showed a higher cross-sectional sensitivity for CIN2+ and CIN3+ relative to cytology, and that HPV-negative women had a significantly lower cumulative incidence of CIN3+ in the second round of screening compared with cytology-negative women. 6,53,62 65 From a UK perspective, data from the ARTISTIC screening trial indicated that a negative HPV test provides the same degree of protection over two screening rounds as negative cytology for one screening round, allowing the screening interval to be increased to 5 6 years or longer in women over 30 years of age. 66 At the time of preparation of the manuscript, around five assays 40,53 had been demonstrated to fulfil the 2009 Meijer criteria and could be used for primary screening, and it is likely that others will follow suit. Many of the challenges of the introduction of primary HPV testing are technical and/or logistical. The workload is high so only tests amenable to high throughput with minimal hands-on time are practical. Automated platforms generally have a large footprint and physical location of the instrumentation may require laboratory rearrangement. As a first-line screening test, it would also be beneficial if the assay could be used with samples collected at home (see section Self-sampling for more detail). This is pertinent when considering that the added value of performing co-testing with cytology is questionable: in an assessment of over 300 000 women within the Kaiser Permanante system, HPV testing alone showed similar performance for the detection of CIN3+ at 5 years relative to co-testing with cytology, although co-testing remains the current recommendation in the USA. 62 Although many manufacturers will embed the high cost of the testing platforms in kit prices or cost per reportable results, validated HPV screening assays are beyond the means of many low-income countries. This is unfortunate, as it has been shown that a single HPV test at around 35 years of age is the most cost-effective intervention in reducing the burden of cervical cancer in countries with low or no screening coverage. 67 However, low-cost, lowtech HPV tests have been developed and trialled, with success the Care HPV test (Qiagen), which is modelled on hc2 chemistry but requires no electricity or running water, has been shown to yield equivalent clinical performance to conventional hc2 in a screening population in rural China. 68 It is also notable that, as a consequence of the MARCH study (Mexican appraisal of routine cytology versus vaginal HPV screening), Mexico is incorporating HPV testing into a screening strategy which includes selfsampling (for HPV testing) to improve population coverage and sensitivity of the screening test. 69 In relation to the introduction of HPV DNA-based primary screening, Castle et al. 70 offer a global perspective and update in their recent review. One of the biggest challenges relating to HPV primary screening is undoubtedly how to manage women who are HPV positive, and the assessment of optimal triage modalities is currently a dynamic area of research and debate. Having demonstrated in a cohort of over 10 000 Costa Rican women that about 20% of those with HPV persistence and 40% of those with HPV 16 persistence (for at least a year) had a significantly higher risk of developing highgrade CIN in the subsequent 3 5 years, Castle et al. 71 recommended repeat HPV testing after 1 year, and suggested that an assay that could detect at least HPV 16 and HPV 18 would be useful in identifying those at greatest risk. In contrast, in a recent assessment of 14 theoretical triage strategies for the screening of HPV DNA-positive women, which incorporated different combinations of cytology plus

296 limited and extensive genotyping, Rijkaart et al. 72 concluded that the referral of such women for cytology, followed by repeat cytology, led to an acceptable colposcopy referral rate (33.4% of positives) and a high negative predictive value (99.3%). Indirect tests, such as p16, may also have value in delineating significant HPV-positive tests. In a subanalysis of the new technologies for cervical cancer screening (NTCC) primary screening trial, Carozzi et al. 73 showed that the sensitivity of p16 at 3 years of follow-up for the detection of CIN3+ was higher than that of conventional cytology (77.8% at all ages). The authors also concluded that HPV-positive/ p16-negative women were at significantly lower risk of developing CIN2+ compared with double-positive women and could be returned to routine recall, whereas double-positive women should be referred to colposcopy, particularly those aged over 35 years. This is one of the first studies to directly address the utility of a biomarker in a primary screening context, and it will be interesting to see whether the results are confirmed in other countries, particularly those that have organized screening programmes. The high positive rates for HPV testing in young women limit the clinical effectiveness of primary HPV screening under the age of 30 years, at least in un-immunized women, 53 and the introduction of HPV primary screening will bring unique challenges according to setting. In countries in which there is little screening, there is clearly less to change and dismantle compared with countries with organized screening programmes or opportunistic programmes that achieve high coverage, such as the USA. Furthermore, countries which already start screening at 30 years perhaps face fewer challenges than those that start earlier. Undoubtedly, high-grade lesions and cancer can be detected in young women. National data from Scotland (which has an organized programme that starts at 20 years of age) showed that, of the c. 13 500 women diagnosed with CIN3 over the period 2006 2010, c. 7000 were between the ages of 20 and 29 years, and 163 of 1553 cancers diagnosed in this period were from women aged 20 29 years. 74 Although cytologybased screening has been shown to be diminished in young women, 75 the exclusion of the younger age groups from any form of screening could be controversial, at least in countries in which it was previously offered. Data from the Netherlands, which has an organized programme that initiates at 30 years of age, are reassuring, in that cancer rates are no worse [European Age Standardized (EAS) incidence rate of 7.9 per 100 000 years in 2007] 76 than in settings which start screening earlier, such as Scotland, which had an EAS incidence rate of 11.2 per 100 000 years in 2009. 74 Certainly, insights from other countries will be helpful in informing modern systems. Habbema et al. 77 applied a very interesting approach by comparing objectively the performance of two very different screening programmes, albeit in affluent countries (USA and the Netherlands), and concluded that, despite starting later and taking fewer smears, the system in the Netherlands was at least as effective as that in the USA. What is certain is that the provision of clear and appropriate information for women is also crucial when a change from current cytology screening to one based on HPV first is proposed. HPV triage of low-grade disease HPV testing for the triage of low-grade abnormalities has been used for several years in various countries. The sensitivity of this approach for the detection of CIN2+ has been found to exceed repeat cytology when using a test which has at least equivalent performance to hc2. With respect to the validation of tests for ASC-US/borderline triage, Arbyn et al. 53 recently proposed as a rule of thumb that a test should be at least 90% sensitive for CIN2+, with a relative sensitivity not significantly lower than that of hc2 and a lower CI of 0.90. According to these criteria, the authors concluded that the Abbott RT PCR, Papillocheck (Greiner Bio-one, Frickenhausen, Germany), Cervista and APTIMA tests had been demonstrated to show such performance and, again, it is likely that others will follow. The UK has been trialling HPV testing for triage for a number of years, and successively demonstrated that HPV triage for women with borderline cytological abnormalities and mild dyskaryosis was feasible and cost-effective, 78 allowing c. 30% to be returned immediately to routine recall and for a substantial proportion to be referred for colposcopy without repeat cytology. 79 The hc2 assay was used in the demonstration projects, but with a value of 2 relative light units/ cut-off (RLU/CO) to improve specificity in line with results from the ARTISTIC trial. 66 With more quality assured HPV tests becoming available, an ambitious project was undertaken to compare the clinical performance of four other tests against hc2 in line with the Meijer guidelines. The tests selected were Abbott

Understanding HPV tests 297 rthpv, APTIMA, Cervista HR HPV and Roche Cobas 4800, all of which were shown to be comparable with hc2 in terms of performance, with all showing a specificity relative to hc2 of greater than 1 and a relative sensitivity for detection of CIN3+ ranging from 0.97 to 1.06. 80 Although few would dispute the sensitivity of HPV testing in the triage context, specificity is less optimal, particularly for low-grade squamous intraepithelial lesion (LSIL) or mild dyskaryosis, and especially in young women in whom the high prevalence of transient infection can result in excessive colposcopy referrals. RNA-based, restricted typing or biomarker assays may have the potential to improve specificity compared with DNA consensus tests, and it is notable that, in a recent meta-analysis, the AP- TIMA assay was shown to have equivalent sensitivity, but higher specificity, than hc2, including in LSIL. 81 Furthermore in the Predictors suite of studies 46,47 the three most specific tests for CIN2 and CIN3+ were the HPV PreTect Proofer, p16 (CINtec) and the APTIMA test all either direct or indirect markers of oncogene expression. 46,47 In a subanalysis of the Predictors population, positivity for HPV 16 was also shown to confer a greater risk with the PPV for CIN2+ in women over the age of 30 years being 62.4% compared with 34.3% in women infected with other types. 23 A similar excess risk for HPV 16 within a triage context has also been observed in women recruited to the ATHENA trial. 25 Another way to enhance specificity is to alter the assay cut-off. Although the use of test cut-offs that do not match with manufacturer s recommendations can alter the number of referrals, its practice is contentious. This problem relates principally to hc2, where the manufacturer recommends a cut-off of 1 RLU/CO. Setting a higher cut-off can improve specificity, 82,83 and England has retained a cut-off of 2 RLU/CO for HPV triage as used in earlier English trials. Recently, an issue has arisen with the Cervista assay, in which the manufacturer s cut-off in the presence of low positivity in all three wells relating to a single sample increases sensitivity, but may represent a non-specific reactivity. The cut-off here might need to be increased to improve specificity. This is currently under study. 84 HPV in post-treatment follow-up/test of cure Women who have had cervical lesions removed at colposcopy remain at risk of subsequent CIN. In a meta-analysis of 28 studies, the extent of treatment failures was estimated to range from 7.1% to 11.3%. 53 Furthermore, women who have had complete excision of their lesion are thought to remain at increased risk of recurrence for up to 20 years. 85 This reconciles with the recent elegant analysis of Rebolj et al., 86 where the authors identified an excess (approximately fourfold) risk of cancer in Dutch women who had been treated for any lesion (irrespective of grade) and who had three subsequent normal smears as part of standard follow-up. The perceived excess risk of this population has often led to intense and prolonged post-treatment follow-up schedules and raised anxiety in women. Given the high sensitivity and negative predictive value of HPV testing, its incorporation into posttreatment algorithms can reduce follow-up significantly. In the Dutch study from Kocken et al., 87 one HPV-negative test at 6 months post-treatment was associated with a 10-year risk of CIN3+ of 2.1% and, if performed as a co-test with cytology, this risk was reduced to 1.4% if both were negative. Conversely, if a woman was HPV positive at 6 months post-treatment, her risk of CIN3 over the 10-year period was 29%. As the test of cure population is at greater risk, it is reasonable to apply a test with high sensitivity. As with other patient-management settings, only validated tests should be used, but which one? It is notable that most HPV multi-test comparison studies have been performed in colposcopy referral/triage populations in which the HPV prevalence is higher than in the test of cure group, as is the distribution and prevalence of disease. To address this, a recent Scottish study of c. 1200 women was set up to assess the performance of five commercial HPV assays in the post-treatment context. Interim data presented at the 2012 Eurogin meeting showed variation in HPV prevalence at 6 months post-treatment from 17% to 27% depending on assay, with all assays showing a high clinical sensitivity (of over 90%) for the cases of residual CIN2+ (Cuschieri K, Canham M, Moore C, Pedraza J, Graham C, Cubie H, manuscript in preparation). 88 Despite the requirement for high sensitivity, the attainment of a perfect threshold is challenging, particularly when considering how to manage women who are referred back to colposcopy because of their HPV-positive status, but who have no conspicuous evidence of any disease. More studies are required to ascertain appropriate and safe pathways for such women, and it will be of interest to determine

298 whether new technologies, such as type-specific management and biomarkers (see section on Emerging Biomarkers ), may have an adjunctive role in this endeavour. HPV in the resolution of uncertainties The resolution of uncertainties is sometimes described as clinical testing and is used to aid the management of individual patients who have already entered the colposcopy referral process. A prospective study (published in this issue of Cytopathology) was set up in London to address the value of HPV testing in difficult cases. 89 In this study, two categories of women accounted for 90.3% of the resolution of uncertainties. These were women with persistent CIN1 and low-grade cytology for more than 12 months (55.9%; 51.7% HPV negative) and those with recurrent lowgrade cytology post-treatment (34.4%; 60.2% HPV negative). The rates of biopsy-proven CIN2+ across these two groups were 0.8% for HR-HPV-negative and 7.0% for HR-HPV-positive women, using hc2 at an RLU/CO of 2, showing the ability of HPV testing to identify women at greatest risk. 89 From 2008, the remit of the Scottish HPV Reference Laboratory included a limited number of funded HPV tests to be used in specific settings following case discussion at multidisciplinary team (MDT) meetings. 90 This has resulted in around 100 such requests each year. In Scotland, the two most significant types of request are for older females who have not exited the Scottish CSP at 60 years because of continuing low-grade disease and younger women who have persistent low-grade abnormality after more than two treatments and where fertility is an issue. The issue in this scenario is the value of HPV genotyping. The absence of HPV is definitely helpful in limiting follow-up, and the detection of HPV 16 might prompt regular surveillance until the virus is cleared. However, the detection of HPV types within Groups 2A/2B (Table 1) raises additional dilemmas, particularly for women who are not keen to have further appointments if the risk is low. These individual cases need informed discussion between the colposcopy team and the patient. Some would therefore argue for a screening assay, such as hc2, rather than genotyping, but a screening assay with restricted genotyping could well be suitable in this diagnostic situation. Because there will always be small numbers of tests within this category, considerations of throughput and footprint of equipment are less relevant. HPV testing in non-cervical screening/ management settings Other anogenital cancers It is well established that there are other anogenital cancers with an HPV aetiology, including anal, vulval, vaginal and penile cancers. 91 Given that there is a less well-established precursor phase and no comprehensive screening programme for these cancers, the justification and opportunity for the use of HPV testing in their associated management are not currently established. Some investigators have assessed the performance and value of HPV genotyping within opportunistic anal cytology screening for high-risk groups (men who have sex with men, MSM) to guide referral to anoscopy. 92 However, the high prevalence of HPV in this context (90%) limited the usefulness of this approach. 93 Whether more sophisticated biomarkers of significant HPV infection have a role in anal disease management in the future remains to be established, but preliminary evidence suggests that E6/E7 mrna detection and p16/ki67 cytology testing may have value for the risk stratification of cytologically detected anal disease, with more evidence being required. 94,95 Oropharyngeal cancers Oropharyngeal squamous cell carcinoma (OPSCC) is now considered to comprise two pathological subsets one associated with traditional risk factors of chronic alcohol and tobacco use, particularly in older men, and the other associated with HR-HPV infection and commonly arising in younger men and women. 96 Notably, the incidence of OPSCC is rising, particularly HPV-associated OPSCC. In a retrospective study of Scottish patients, Junor et al. 97 showed that the prevalence of HPV-positive OPSCC increased from 67% to 81% in men and 50% to 85% in women from two cohorts spanning 1999 2001 and 2003 2005. Similar increases have been found elsewhere, including the USA and Sweden. 98,99 Patients with HPV-associated OPSCC appear to respond better to treatment and have significantly improved disease and survival outcomes compared with those with HPV-negative OPSCC. 100 Given the overwhelming evidence that HPV-associated OPSCC has a better prognosis, there is an appetite for HPV testing of these tumours to help inform patient management. The current position of

Understanding HPV tests 299 the Scottish Intercollegiate Guidelines Network 101 acknowledges the evidence and states that HPV testing may be appropriate although this is outwith the remit of most pathology departments at present. It is technically feasible to detect HPV in OPSCC, but there is currently no commercially available HPV test validated for this purpose. Some investigators would argue that the use of p16 ink4a staining is sufficient for the categorization of HPV-associated tumours, 102 whereas others would argue that p16 is not specific for HPV and can be activated by non-hpv mechanisms. 103 The detractors of a direct HPV DNA detection approach believe that these methods are more affected by specimen quality and degradation, and simply provide a sensitive indicator of HPV presence rather than oncogenic activity. In a technical study, Smeets et al., 104 using a battery of tests and test combinations (HPV in situ, HPV DNA PCR, RT PCR and p16), concluded that p16 coupled with HPV PCR positivity was the only test combination that was 100% sensitive and specific for the diagnosis of HPVassociated OPSCC. The authors of this study have subsequently validated this approach in a contemporary series of Dutch OPSCC. 105 Another justification for a combined approach is that subclasses of OPSCC may exist that can be characterized according to differential p16 and HPV status. Weineberger et al. 106 suggested that there may be a class of HPV-inactive tumours defined by p16 /HPV+ status, where survival at 5 years is comparable with that of patients who are double negative. Other work has shown that this group (p16 /HPV+) represents a distinct clinical entity more responsive to chemoradiotherapy than the double-negative group. 97 Although this heterogeneity remains, it may be short-sighted not to perform dual HPV and p16 testing where possible. This could enable the evaluation of what is undoubtedly a growing area of both research and service. Vaccine surveillance It is essential that the impact of HPV immunization programmes is monitored over time, principally by the determination of the prevalence of HPV types in high-grade lesions and cancer. This is readily achievable in countries such as the UK and Scandinavia which have high-quality Cancer Registries. However, a significant lag time between HPV infection and cancer demands additional ways of measuring impact, such as the assessment of HPV type-specific prevalence in the population. This can be achieved through systematic, longitudinal HPV surveillance, allowing vaccine impact to be gauged more rapidly than waiting for cancer statistics it also allows for the quantification of changes in vaccine and nonvaccine types, and HPV type replacement. Although high analytical sensitivity of assays is important for epidemiology and surveillance, it is also important to use an assay which offers type-specific resolution of both high- and low-risk types. Sensitivities associated with clinically validated assays calibrated to detect disease would underestimate the true population prevalence of infection. 14 High sensitivity is also relevant when considering the diverse, often archived, biospecimens that are used for epidemiological studies. 107 For example, HPV type determination in disease cases often depends on the use of formalin-fixed, paraffin-embedded (FFPE) blocks stored in clinical pathology archives at room temperature in which nucleic acid could have degraded over time. PCR-based assays for this application often depend on the amplification of small fragments to compensate for such degradation. Urine is another biospecimen suited for surveillance, particularly relevant in younger populations. 108,109 Although it is not a clinically validated specimen, urine offers a non-invasive means to determine HPV status, but the relatively low cell count again requires the application of a highly sensitive assay. None of the assays which have gained FDA approval for use in cervical disease management are therefore optimal for use on FFPE material or urine. One of the more established approaches to genotyping for surveillance purposes involves the use of line blot assays which incorporate reverse hybridization of a PCR product to a strip which contains several immobilized HPV probes. However, other systems exist (as described in Table 2), including Luminex-based technology, 37 which utilizes type-specific HPV oligonucleotide probes coupled to beads with a unique, identifiable, spectral address. This technology can allow for the concurrent amplification and multiplex detection of up to 100 types, and has been used for HPV epidemiological surveys, including UK-based immunization surveillance. 108,109 Other assays applied for immunization surveillance include the manipulation of matrix-assisted laser desorption/ ionization-time of flight (MALDI-TOF) mass spectrometry 110 (very high throughput and low cost) and PCR followed by enzyme immunoassay (EIA). 111 All

300 four systems are predicated on type-specific detection after a broad-spectrum PCR of a relatively small sequence fragment. However, this approach can underestimate types present at low level within a mixed infection, as a result of competition for resources within the PCR. A technical gold standard would be to perform type-specific individual PCRs for all HPV types of interest, but this is impractical as epidemiological exercises frequently involve the testing of thousands of samples to determine populationlevel changes. Another issue is the need to interpret with caution comparisons of surveillance datasets which have been generated with different genotyping assays. In a recent study, Unger et al. 112 compared the prototype and subsequent Research Use Only (RUO) version of the Linear Array HPV genotyping kit (Roche Molecular Systems) on 3001 vaginal swabs collected for surveillance purposes. The RUO version detected HPV in approximately double the number of samples compared with the prototype. In a Scottish study of LBC samples and self-taken urine or swab samples, a similar impact of assay choice on observed prevalence was observed. 113 It is therefore sensible to use a particular genotyping system consistently for longitudinal surveillance exercises within a country to avoid the confounding of vaccine-induced changes. If this is not possible, a statistical correction for the impact of different assays on type-specific prevalence should be performed. There are a number of uncertainties relating to the implications of HPV immunization, including the extent of type replacement and cross-protection. Well-executed epidemiological surveys will not only provide an easy means of monitoring vaccine impact, but will also yield essential data on residual HPV infection after comprehensive immunization. Such data will undoubtedly inform the future development of HPV-associated diagnostics relevant for immunized populations. HPV testing in the future Self-sampling In clinical practice, the most relevant biospecimen for HPV testing is an LBC sample, most commonly used in cervical screening or following colposcopy referral. LBC has the advantage of allowing cytology and HPV testing to be performed on the same sample. Different LBC collection media can bring their own challenges for downstream HPV testing. More recently, much work has focused on the potential utility of self-taken samples for HPV testing. Self-taken samples usually comprise swabs placed in a suitable viral transport medium, but dry brushes, urine and Guthrie-type filter paper have also been investigated. 114,115 Self-taken samples were initially considered as tools to access hardto-reach populations as a result of financial, geographical, social and/or attitudinal barriers to clinic attendance, and a number of studies have involved the dispatch of sample collection kits to screening defaulters with varying degrees of success. In a UK study of 3000 defaulters, Szarewski et al. 116 found that sending a self-sampling kit compared with a repeat invitation to attend screening resulted in twice the response rate. However, this rate was still only around 10%. In contrast, a Swedish study of similar design yielded a response rate of around 40% to the postal testing kit. 117 These differences across settings are of interest and may reflect differences in attitudes and preferences across ethnic groups. 118 Furthermore, given the drivers for increased HPV testing, including as a primary cervical screening test, it may be short-sighted to consider that self-taken samples are only relevant as a means of accessing hard-to-reach women. Increasing choices by offering self- or physician-taken sampling for all women within an HPV-based cervical screening programme may be worthwhile. 119 Impact of vaccination In countries fortunate enough to have vaccination programmes with sustained high uptake, such as the UK, the extent and pattern of cervical disease will change dramatically. It is frequently argued that the performance of cytology-based screening will be negatively affected as a consequence of less disease. 120 The objectivity of HPV testing obviates the issues of reader fatigue and changed signal-to-noise ratio which would affect cytology. However, there is no direct evidence related to the performance of HPV tests as a primary screen in immunized women, as trials of HPV versus cytology have been performed solely in unvaccinated women. 121 It is also likely that the PPV of existing consensus HPV tests may be affected if the incidence of HPV 16 and 18 is reduced. This issue may be further compounded by an underestimation of the extent of cross-immunity generated by natural infection with HPV 16, 18, 6 and 11. 122,123 The frequently cited study of Khan et al. 124 showed

Understanding HPV tests 301 that the 10-year cumulative incidence of CIN3 in women with normal or low-grade cytology was 17.2% in women with HPV 16 infection, 13.6% in women with HPV 18 infection and only 3% in women who harboured other HR-HPV types. We can expect that consensus tests applied in well-vaccinated women will continue to give positive results through the detection of HR-HPV types that have less likelihood of being associated with significant lesions. Furthermore, although not necessarily replacing HPV 16/18 in terms of ecological niche, non-hpv 16/18 types may become increasingly unmasked in the face of less competition for assay resources. 125 In an interesting modelling study, Durham et al. 126 re-examined epidemiological data to determine the odds ratios of multiple to single infections expected in the presence or absence of crossimmunity, and concluded that the removal of HPV 16 could increase the prevalence of other HR-HPV types by up to 50%. Consequently, for screening purposes in immunized women, the application of HPV consensus tests will be insufficiently predictive in the absence of a robust triage test for screen positives. Whether this is cytology alone, cytology coupled with an associated biomarker or detection of a more subtle aspect of HPV infection, such as methylation status (as discussed in the subsequent section), is moot. However, extrapolations derived from data on un-immunized women are no longer sufficient for estimations of the impact in well-vaccinated populations. Emerging biomarkers As the search for optimal tests that can delineate significant HPV infection and/or associated disease continues, many biomarkers have been discovered and compared with HPV detection per se. It is not the remit of the present review to consider all of these in detail, but it is sensible to consider those for which evidence of relevant application is emerging. One of the most established biomarkers is p 16INK4a (p16) a cyclin-dependent kinase inhibitor and essentially a regulator of cell cycling. 127 High-level expression of the HPV E7 protein can up-regulate p16 and, generally, levels of p16 correlate with increasing grade of disease. In their 2009 meta-analysis, Tsoumpou et al. 128 showed that 2%, 38%, 68% and 82% of normal, CIN1, CIN2 and CIN3 were positive for p16, and its application has been shown to enhance the performance of histology through improved inter-observer performance and accuracy. 129,130 In addition, p16 can be used as an adjudication tool of true disease in cases in which there is uncertainty, 131 and has prognostic value in the context of low-grade lesions, p16-negative CIN1 being significantly less likely to progress relative to p16-positive lesions. In cytology, p16 has been assessed as a triage of low-grade disease and to clarify the significance of HPV infection in a primary screening context. A recent meta-analysis assessed eight studies in which both p16 and hc2 were performed for the triage of low grade disease, and concluded that p16 had a similar sensitivity compared with hc2 for the detection of CIN2+ in ASC-US, and a lower sensitivity but higher specificity for LSIL. 132 If p16 is used within a national screening programme, quality assurance across different testing sites will be crucial, as different stains can generate different positivity rates and, unlike HPV molecular testing, confirmation of a positive is more subjective. 39 Although guidelines for p16 scoring were generated a few years ago in a bid for consistency, 133 their adoption is questionable. If p16 becomes increasingly used for management, improved standardization and training will be key. The application of Ki67 in a dual stain has been proposed to enhance the analytical specificity of p16. 134 Ki67, although not a direct marker of HPV infection, is a marker of cell proliferation. Similar to p16, immunostaining with Ki67 has been shown to correlate independently with underlying disease grade and to have prognostic value. 135 Recently, a multi-site European trial of over 20 000 women attending for cervical screening assessed the performance of hc2 testing, conventional cytology and cytology with dual p16/ki67 (available as CINtec â Plus from Roche), referring all positives in women over 30 years to colposcopy. 136 Staining with p16/ Ki67 showed higher sensitivity for CIN2+ compared with conventional cytology, but was significantly less sensitive than hc2, suggesting that p16/ki67 may have more utility as a triage tool than as a primary screen. Other cellular markers indicative of HPV-associated neoplasia include Topoisomerase IIa (TOP2A), minichromosone maintenance proteins (MCMs), MYBL2 and Survivin the first two of these are included in an assay provided by Becton Dickinson (ProExC). 137 Both proteins are involved in the facilitation of cellular DNA replication, and deregulated

302 production of both has been associated with disease and disease progression. 138 Although there is less evidence on the clinical use of ProExC relative to p16, a recent study from Belgium, which examined the performance of eight primary screening strategies relative to cytology (including HR-HPV detection alone, ProExC alone, cytology with HPV triage of ASC-US and detection of HPV 16/18 status), found that HPV DNA screening with ProExC triage of positives yielded the best performance. 139 In addition to the cycling markers described above, there has been renewed interest in methylation as a biomarker of significant infection and disease. Methylation is essentially the transfer of methyl groups (CH 3 )tog C-rich areas of a genome via the action of dimethyl transferases. Methylation affects gene regulation, either through the blocking of transcription factor access to promoter and enhancer regions or by altering the conformation of nucleosomes in a manner favourable or unfavourable to transcription. Although methylation in the role of cervical and, indeed, other cancers has been examined in the context of basic science for around two decades, 140 recent developments in technology have allowed more sensitive and rapid means of detection. 141 The methylation status of both host and viral targets shows promise as a putative biomarker, as documented in recent reviews. 142,143 Increasing evidence suggests that methylation, particularly of CADM1 (host) and L1 and L2 genes (virus), is associated with a worse prognosis, but larger, longitudinal, clinical studies are required to consolidate these findings, as is a better understanding of how consistent are clinically relevant methylation patterns across different HPV types. 144 Conclusions Organized population-based cervical cytology screening has been effective for decades, but it is now widely accepted that primary HPV testing could be a more effective means to deliver cervical screening and cervical cancer prevention. The key messages are that a primary HPV test will increase sensitivity for the detection of CIN2+ compared with cytology, and could allow the screening interval to be extended with fewer lifetime tests for women. However, HPV testing is not infallible and, although it is more sensitive than cytology, cases of high-grade disease (including cancer) can be missed, even with sensitive assays. Katki et al. 62 showed that the 5-year cumulative incidence of cancer was 3.8 per 100 000 women per year in women who tested HPV negative. Defining intervals that are not overly cautious about the risk to women will be challenging, particularly if the screening age is elevated beyond 25 years. What is clear is that a growing number of commercial assays are becoming available which vary in their scope and characteristics, and it is essential that these are validated appropriately and subject to quality assurance to prevent inappropriate use and to limit harm. Equally important, validation metrics should be revisited according to the context in which an HPV test is delivered, taking into account the prevalence of infection, extent of disease and relative risk of the population, rather than there being a one-size-fits-all situation. Furthermore, the performance of HPV testing in vaccinated women is unchartered territory, and it is reasonable to expect that the PPV, at least of consensus assays, may be reduced as a consequence of a reduction in the prevalence of HPV 16 and 18. It is important that this knowledge gap is addressed through either derivation from existing longitudinal datasets for which type-specific information is available and/or through de novo studies on (well)-immunized populations. Well-characterized biobanks which can link HPV status to longitudinal screening data will be of great importance in achieving this aim. In addition, it is essential that researchers, clinicians and epidemiologists engage with screening policy makers to ensure that combined expertise is available to address the contemporary and evolving requirements of effective screening. On a positive note, great strides have been made with respect to biomarker assays that can identify more subtle aspects of HPV infection. Although some of these are still very much in the development phase, others, such as p16, are becoming increasingly evidence based with respect to their clinical performance. The advantage of such markers is that they should be detectable irrespective of HPV type, potentially obviating the vagaries of shifting HPV prevalence in a post-immunization era. The current state of the more established biomarkers indicates that they may be more effective as triage tools of HPV-positive tests in a primary screening context or for low-grade disease rather than as front-line tests. However, it will be interesting to monitor how they develop and perform over the short to mid-term, including in the management of non-cervical disease.

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