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Abstract

Human papillomavirus testing has been shown to be far more sensitive and robust in detecting cervical intraepithelial neoplasia 2 and above (and cervical intraepithelial neoplasia 3 and above) for cervical screening than approaches based on either cytology or visual inspection; however, there are a number of issues that need to be overcome if it is to substantially reduce the morbidity and mortality associated with cervical cancer at the population level. The two main issues are coverage (increasing the number of women who participate in screening) and the management of women who test positive for high-risk human papillomavirus. This article will review the potential for vaginal self-collection to improve coverage and the options for triage of high-risk human papillomavirus-positive women in high-resource and low-resource settings.

Keywords

cancer prevention human papillomavirus testing screening for cervical cancer triage of hrHPV-positive women vaginal self-sampling

Cervical screening works by detecting and treating high-grade cervical intraepithelial neoplasia (CIN), typically CIN2 and above (CIN2₊), or in some settings CIN3 and above (CIN3₊). In the absence of treatment some, but not all, CIN3 would progress to cervical cancer [1,2]. Cervical cancer is rare in women previously treated adequately for CIN [3,4].

For many years cervical screening was carried out by conventional cytology; a sample was collected using a spatula and smeared onto a glass slide before being analyzed under a microscope in a laboratory. Today many screening programs use liquid-based cytology instead; samples are now collected using a small plastic broom and stored in a liquid transport medium. Glass slides are prepared from the cells in this transport medium in the laboratory. Liquid-based cytology is considered to be better than conventional cytology, in that it is easier and quicker to see what is on the slide, but research studies have not shown that it is better than conventional cytology at detecting high-grade CIN [5].

The success of effective screening programs stem from their good coverage (e.g., in the UK approximately 80% of the target population has been screened in the last 5 years) and the high-level of quality assurance. Cervical cytology depends on the collection of good samples and the skilled interpretation of cells under the microscope. Both activities require extensive training and continuous quality assurance. Examining cells under a microscope is a skilled job that relies on concentration (so as not to miss something of significance) and human judgment to interpret what is seen. Cervical screening has been ineffective in most low-resource countries because of an inability to obtain high coverage with a reliable screening test.

Cervical cancer is caused by approximately 15 high-risk types of the human papilloma virus (HPV), notably types 16 and 18, which together give rise to around 70% of all cases. Although infection of the cervix with HPV is very common, there would be no (or virtually no) cervical cancer without HPV infection [6]. In most cases, the body's immune system clears a cervical HPV infection within a year or two [7]. High-risk HPV (hrHPV) infection may lead to high-grade CIN, which sometimes progresses to cervical cancer. The time it takes for an hrHPV infection to develop into cervical cancer varies hugely, but it is very rare for cancer to develop in less than 7 years and it is not uncommon for cancer to develop more than 30 years after infection.

HPV testing

Since hrHPV infection is needed for cancer to develop and because hrHPV DNA is still present during the development of cancer, looking for hrHPV DNA should be a good approach for identifying women with high-grade CIN. In addition, as the time from hrHPV infection to cancer is long, a woman who is HPV-negative is extremely unlikely to develop cervical cancer over the next 5–10 years and infrequent screening would be safe. Therefore, there are two potential uses for HPV testing: to identify those likely to have disease now (and would benefit from treatment); and to identify those who might develop disease in the next few years (or by elimination of those who are extremely unlikely to develop disease over the next several years, and therefore do not need to be screened again for several years). However, any HPV assay used for screening purposes will need to be clinically validated for the detection of CIN2+. Most commercial HPV tests are based on DNA, but two are based on RNA. One of these tests is restricted to five HPV types and has lower sensitivity, and the other has similar sensitivity to the DNA-based test [8].

There have been many studies comparing HPV testing to cytology for the detection of high-grade CIN. All large studies have found that HPV testing is at least as good as cytology at detecting high-grade CIN and most studies find HPV testing to be better (i.e., it is more sensitive) [9 –12]. The disadvantage of HPV testing is that it is less specific than cytology. That is, among women without high-grade CIN more will test positive for HPV than will have abnormal cytology.

Another advantage of HPV testing is that it does not depend on the preservation of good quality morphology. In stark contrast to cytology, reasonable results can be obtained by using HPV testing on samples that women collect themselves [13]. Furthermore, HPV testing does not rely on human judgment and the results are reproducible in different laboratories. In studies comparing cytology to HPV testing in Europe and North America [11], the sensitivity of cytology varied from less than 40% to approximately 80% (average 53%), whereas the sensitivity of clinician-based HPV testing was consistently above 85% (average 96%). The specificity of both tests increased with age; however, cytology was on average more specific (96%) than HPV testing (91%).

Longitudinal studies show that the rate of high-grade CIN after a negative HPV test is considerably less than the rate after negative cytology [14 –17]. In a joint European study, the cumulative risk of CIN3₊ 6 years after a negative HPV test was significantly less (0.27%) than the cumulative risk 3 years after a negative cytology (0.51%) [14]. One study of cervical cancer incidence in the 5 years following screening with both cytology and HPV testing reported half the rate of cervical cancer following a negative HPV test compared with following a negative cytology test [18].

Randomized controlled trials (RCTs) have shown that the additional cases of high-grade CIN, picked up by HPV testing but missed by cytology, are not simply indolent diseases that would not have progressed to cancer in the short term. In all four RCTs of cytology versus HPV testing within established screening programs with published results for the second round of screening, the rate of high-grade CIN detected at round two (i.e., between 1 and 5 years after enrolment) was substantially lower (~half) in those individuals originally screened by HPV testing compared with those originally tested by cytology alone [19 –22]. Thus, by treating more women in the first round, the trials found that there was less detected in the second round. Furthermore, the limited data on the incidence of cervical cancer in RCTs suggest that HPV testing does indeed result in fewer cases of cervical cancer on follow-up. In the Italian trial, there were nine cancers in the cytology arm compared with none in the HPV arm after the initial screen [22] and in the Dutch trial there were 14 versus four [21]. Results in the Swedish trial including those detected at first screen, found five squamous cell carcinomas in the cytology arm and one in the HPV arm [20].

Initially, many people believed that HPV testing was too expensive to be used in middle- and low-income countries, and that cytology or simply looking at the cervix would be adequate to greatly reduce the burden of cervical cancer. There was much interest in visual inspection after the application of dilute acetic acid (VIA). Indeed, for many years VIA was the favored approach of the Alliance for Cervical Cancer Prevention; one of the main organizations working to prevent cervical cancer in developing countries. However, a trial in the San Martin region of Peru that compared conventional cytology, VIA and HPV testing to screen over 5000 women found that the sensitivity of HPV testing was far superior to that of cytology and VIA, and that the specificity of VIA was considerably lower [23]. Furthermore, a cluster randomized trial in India, reported “34 deaths from cervical cancer in the HPV-testing group, compared with 64 in the control group (hazard ratio: 0.52; 95% CI: 0.33–0.83). No significant reductions in the numbers of advanced cancers or deaths were observed in either the cytology group or the VIA group, as compared with the control group” [24].

Management of women positive for HPV

The great advantage of HPV testing for most women is that HPV-negative women can have their screening interval extended to over 3 years [14]. By extending the screening interval to 6 or even 10 years, the proportion of women who will test positive on at least one HPV screen by the age of 65 years will be similar to the proportion who test positive on at least one cytology screen in current programs. Since the rate of disease following an abnormal HPV test is half the rate of disease following an abnormal cytology, where the screening interval is 5 years, extending the screening interval to 10 years should roughly result in the same number of women testing positive as they currently do.

The greater proportion of women testing HPV-positive on a single screen (compared with the proportion with abnormal cytology) need not be a problem as demonstrated by the very large trials of primary HPV testing that have been run successfully in several countries (including England) [19]. The first solution is good management of HPV-positive women. A positive HPV test in primary screening should only trigger a triage test, not automatic referral to colposcopy. The most widely studied triage test in this situation is cytology. Triage with cytology can be carried out on the same sample as the HPV test without the need for women to attend another clinic visit (when the sample is taken using liquid-based cytology transport medium). Effectively, the same women (those positive on both cytology and HPV testing) are immediately referred to colposcopy regardless of whether one screens using cytology, triaged by HPV testing, or HPV testing, triaged by cytology [25]. In practice, because cytology is subjective, more women may be considered morphologically abnormal when they are known to be HPV-positive. Evidence of this was observed in the Finnish trial, where women were referred to colposcopy based on their cytology result, in particular women under the age of 35 years [12]. Introduction of primary HPV testing should be closely monitored to assess whether the increase in morphologically abnormal cytology is associated with the learning curve or whether it will affect referral rates in the long term. Conversely, a very small number of women who would be referred to colposcopy with high-grade cytology, without HPV triage, will test negative for HPV and will not have cytology triage. HPV-positive women with normal cytology will be rescreened at a shorter interval (e.g., every 12 months). In this way, the sensitivity of HPV testing will be maintained, but the numbers referred for colposcopy need not be excessively increased.

Current proposed strategies to deal with HPV-positive/cytology-negative women suggest they should be retested 12–24 months later (with HPV and cytology). Women who are still HPV-positive or those who develop cytological abnormalities upon retesting are referred to colposcopy [26 –28]. Support for this strategy comes from cohort study evidence that suggests that 90% of HPV infections will be self-clearing in a period of 1–2 years [15,16]. Nevertheless, referring all women who test HPV-positive/cytology-negative twice, 12 months apart, leads to higher colposcopy referral rates than those observed in cytology screening programs.

Genotyping for HPV16 & HPV18

Genotyping for HPV16 and HPV18 has been proposed as a good alternative for women who test HPV-positive/cytology-negative because the risk of CIN3₊ is genotype-dependent [25,29,30] and HPV types 16 or 18 are found in over 70% of cervical cancer [31]. HPV16 (but not HPV18) is associated with a high cross-sectional rate of CIN2₊ and CIN3₊ [8]. Women with HPV18 and HPV45 are more likely to develop cervical intraepithelial neoplasia (particularly adenocarcinomas in the endocervical canal) than women with other high-risk types [31,32]. The risk of CIN3 reaches 10% over 1–4 years for HPV16-positivity and over 2–5 years for HPV18 positivity [30,33]. By contrast, the 12-month risk of CIN3₊ after a HPV-positive/cytology-negative test ranges from 0.8 to 4.1% [26].

Studies have shown that HPV DNA testing followed by triage with cytology and screening for persistent HPV type-specific infection (genotyping) has a considerably higher sensitivity for detecting CIN3₊ than screening by cytology alone, with a modest increase in screening tests and referrals [25,28]. These results have been validated as part of the ATHENA trial in a scenario where HPV was the primary test [9]. Being hrHPV-positive but HPV16/18-negative, carries an increased risk of CIN3 in the following 2 years of 2.9 % [21]. For this reason, the lack of HPV16/18 cannot be used to return women to routine screening.

Genotyping for triage has been primarily investigated in women over the age of 30 years. In younger women, the proportion testing HPV16/18 positive may be too high to justify referring them all; however, the proportion of CIN3₊ harboring HPV16 also decreases in older women, indicating the need for careful follow-up of hrHPV women regardless of HPV type [9].

Immunocytochemical detection of p16INK4a

Triage using other biomarkers has been less comprehensively evaluated. One particularly promising test is the immunocytochemical detection of overexpression of the p16INK4a tumor suppressor gene as a surrogate marker for the transforming activity of hrHPV oncoproteins, which are essential for the initiation and maintenance of the neoplastic process. A number of studies looking at p16INK4a staining of cervical cytology showed that women with CIN1 who are positive for p16 were more likely to be subsequently diagnosed with CIN2₊ [34 –36]. Unfortunately, the sensitivity of p16 varies hugely in the literature owing to difficulties in reading the slides, lack of standardized reporting and a need for substantial expert interpretation. In general, p16 is less sensitive for CIN2₊ or CIN3₊ than HPV testing, but most studies have found it to be more specific at identifying women with high-grade disease [8,37 –39].

A more recent development has been double-staining for p16INK4a and Ki-67. Several studies investigating the performance of the paired markers p16INK4a/Ki-67 found that the results were quite similar to p16INK4a alone. The main difference between the single and the paired markers is that p16INK4a should be read by an expert and requires considerable interpretative skills to rule out false positives due to staining of endocervical and other cells. By contrast, the p16INK4a/Ki-67 marker pair is read differently and can be more easily interpreted correctly by a less experienced cytopathologist [38,40 –42]. A study by Carozzi et al. is of particular interest since good results for p16INK4a were demonstrated in women aged 25–34 years, showing that p16INK4a may be considered as a triage marker for these hrHPV-positive women [38]. A recent paper from the same study with additional follow-up showed that p16INK4a positivity in hrHPV-positive women had a cumulative 3-year absolute risk for CIN2+ of 19.5% with a longitudinal sensitivity of 75.6% [43]. The data suggest that p16INK4a may be considered as an option for triage of hrHPV-positive women.

DNA methylation

DNA methylation (DNAme) of host-cell DNA has been proposed as an alternative triage for hrHPV-positive women because DNAme plays an essential role in gene transcription and genomic stability. Aberrant methylation leads to cell immortality and malignant transformation [44]. The use of DNAme for triage would have the advantage of being an automated, objective test that could be run on the same sample as the HPV assay.

Of the more than 50 human genes tested so far in cervical tissue, 15 have been reported in five or more studies and three genes (CADM1, DAPK1 and RARE) have been repeatedly shown to have elevated methylation in cervical cancers [45]. Hesselink et al. evaluated CAD1/MAL methylation to triage HPV-positive women in a large population-based screening study [46]. They found it to be as effective at detecting CIN3₊ in HPV-positive women as cytology with HPV16/18 genotyping. Studies of host-cell methylation use different clinical specimens, methylation assays, assay thresholds and/or selected promoter regions, making consistency among results difficult [44].

The methylation of HPV genes, especially HPV16, in disease progression has been a major focus of research. Elevated methylation of the HPV16 L1 and L2 open reading frames in particular is associated with high-grade CIN and invasive cancer [47 –49]. Recently the association between specific patterns of DNAme in HPV16 L1 and L2 and high-grade CIN has been validated in a small prospective study [50]. Further validation in larger studies is required. The potential utility of methylation of other regions of the HPV16 genome as a biomarker for CIN2₊ is less clear because studies have been small and the results highly variable. To expand the potential utility of HPV16 methylation assays for predicting the risk of cancer additional research needs to be directed to other HPV types, such as 18, 31, 33 and 45.

DNAme of a panel of both human and HPV gene markers is an exciting area of current research.

Vaginal self-collection for HPV testing

For years conventional cervical screening has relied on clinician-collected cervical specimens; however, vaginal self-collection for HPV testing is likely to become an integral part of screening in both the developed and developing world. This is because self-collection has the potential to overcome two major problems inherent in current screening strategies: cost and coverage. It is difficult to maintain the large numbers of clinics required for mass population screening, especially in resource-constrained regions. Even in countries that manage to do this well, compliance of 80% with the recommended screening intervals is considered extremely good. Women who are not screened or attend infrequently are at the highest risk of developing cervical cancer [51]. Studies have shown that self-collection for HPV testing is a practical and effective way to collect exfoliated cell specimens from the vaginal tract and cervix that is broadly acceptable to women and eliminates the cost of visiting a clinician [52 –55].

Acceptability of vaginal self-collection

Numerous studies in a wide variety of countries and ethnic groups over the past decade have reported that self-collection is well accepted by women and in some more educated groups may be preferred to speculum-based cervical sampling [56 –61]. Virtually all measures of acceptability, including pain, embarrassment and anxiety favor the self-collection approach. However, a consistent negative theme across the studies is that 50–70% of women worry they will not take the sample properly [56 –58,62]. This is actually a misconception, it is easy for a woman to take a self-collected sample correctly and the samples are almost always good. There do not appear to be any specific cultural or religious barriers to self-sampling [62,63], although one study in the UK found that Muslim women were reluctant to use the self-collection approach [52].

The main problem in high-income countries with well-established screening programs is low coverage and falling participation rates. A well-recognized barrier to cervical screening attendance is the need to undergo a pelvic examination, which some women find to be uncomfortable, invasive or unacceptable (for emotional or cultural reasons) [54]. However, practical barriers may be more predictive of nonattendance behavior [64]. The self-collection approach allows the women to obtain samples in privacy and comfort, with the additional benefit of being able to choose the time and place of sampling.

The devices most commonly used for self-collection are swabs, which may be made of spun polyester fiber or treated cotton on a plastic shaft or special flocked swabs with a somewhat rough surface; untreated cotton swabs should not be used. Another device is a small soft nylon brush similar in shape to an artist's brush, but with much wider diameter bristles (Rovers® Viba-Brush, Rovers Medical Devices, Oss, The Netherlands), or a small soft nylon brush similar to a Christmas tree-shaped mascara brush. Yet other approaches use plunger-like devices (Delphi device; Delphi Devices, Scherpenzeel, The Netherlands) to carry out a vaginal lavage. Women may be reluctant to use devices that look uncomfortable or appear like medical implements; to some extent even simple swabs may be resisted by some women. There are now several companies focused on delivering elegant self-collection devices, with a look and feel that is more appealing to women (e.g., the Evalyn® sampler, Rovers Medical Devices). While such devices may cost more, they may be more acceptable, more readily used and returned at higher rates to the clinicians, thus promoting greater compliance with screening and saving costs in the long run.

Sensitivity & specificity of vaginal self-sampling

Overall, the reviews that compared self-collection versus clinician-collected samples found comparable values for HPV prevalence [65 –67], although self-collection may detect more low-risk HPV types [65,68]. The performance of the HPV test on self-collected sample, in terms of clinical sensitivity and specificity for CIN2₊ in the screening setting, is not as good as HPV testing on clinician-taken cervical specimens. Of the ten studies comparing self-collected samples versus clinician-collected samples reported by Snijders et al., nine found the clinician-collected sample to be more sensitive and one found it to be as sensitive [13]. In most studies, the sensitivity or specificity of self-collected samples is quite variable, showing values between 60 and 90% [13]. Best results, in terms of clinical sensitivity and specificity, depend on the combination of the sampler, transport conditions (medium and conduits) and the assay. Clinical validation of a combination, rather than just its individual components, should be required before it is used in routine practice [13].

It is fairly clear that HPV testing on self-collected samples is as sensitive, and sometimes more sensitive, than developed-country cytology-based screening of cervical specimens; however, cytology is the more specific test [13]. By contrast, in resource constrained regions where routine cytology is often quite poor, HPV testing on self-collected samples is usually more sensitive than cytology-based screening. A large randomized clinical trial in Mexico [69] and several smaller observational studies in China [70] have shown that HPV testing of self-collected specimens is a much more sensitive approach than local routine cytology screening and may address issues related to the lack of access of poor women to screening. The study by Lazcano-Ponce et al. in Morelos State (Mexico) randomized 25,061 women living in 540 poor communities to either routine clinic-based cytology or self-collection in their homes followed by HPV testing. Women who showed positive results on either test were referred to routine colposcopy and biopsy of any abnormal areas of the cervix [69]. Among the 11,054 participants of the cytology arm, there were 38 CIN2₊ discovered, of which eight were invasive cancers, while among the 9292 participants in the HPV arm there were 108 CIN2₊ detected, of which 28 were cancers; overall 3.4-times (95% CI: 2.4–4.9) more high-grade CIN were detected by HPV testing on self-collected samples than by routine cytology. A report from China presents a pooled analysis of the diagnostic accuracy of self-collection in five small-to-medium population-based studies of mostly poor, rural women [70]. The reference standard for these studies was colposcopy (with biopsy when appropriate) on women who tested positive on any of the screening tests, including liquid-based cytology, visual inspection by acetic acid, self-collection and physician-based cervical HPV testing (using the same HPV assay as in Mexico). Of 13,004 women in the pooled analysis, 507 were diagnosed with CIN2₊. Self-collection with HPV testing had a sensitivity of 86.2% and a specificity of 80.7%, compared with a sensitivity of 97% and a specificity of 82.7% for physician-based HPV testing. Liquid-based cytology was significantly less sensitive than either HPV test.

Cervical screening using vaginal self-sampling for HPV testing

Conventional cervical screening has never been successfully transferred to developing or low-income countries because of the need for infrastructure and the associated costs. Unsurprisingly, 85% of the global burden of cervical cancer arises from these areas [101]. However, self-collection is comparatively resource-efficient and has the potential to provide effective, widely available screening to these largely unscreened populations [69,71,72].

Studies mainly in The Netherlands and Scandinavia have shown a way to implement self-collection in developed countries that is complementary to current screening systems, regardless of whether the primary screening is based on cytology or HPV testing. The main problem in these countries is a lack of adherence to screening guidelines. Several studies have shown that offering self-collection to noncompliant women was superior to a recall invitation for cytology, in attracting them to participate again in the screening program [13,58]. Nine studies reporting response rates to self-sampling amongst nonattendees showed that between 8 and 39% of nonattendees of the cervical screening program provided self-collection samples to the laboratory [73 –81]. Giorgi-Rossi et al. found a substantial decrease in participation among Italian women when self-sampling was available as an opt-in versus providing self-collection devices directly to the nonattendees (8.7 vs 19.6%) [73]. An interesting study of self-collection in The Netherlands used the national postal service to deliver and return the devices [77]. The research team studied 26,409 noncompliant women of whom 26,145 were offered self-collection, while a control group of 264 women were offered a re-invitation to cytology. They found a 30.8% return of the self-collection devices versus 6.5% of the control group who attended cytology screening. Subsequently 89% of the HPV-positive women took a triage cytology test, with 95.8% of those with abnormal cytology going to colposcopy. The rate of CIN2₊ and invasive cancer discovered on colposcopy in the self-collection group was 1.5 and 0.1%, respectively, rates that are similar to those seen in the developing world. The next challenge for self-collection in countries with an established screening program will be to see whether the increased coverage observed in research settings will translate into population screening. Achieving high rates of women attending triage following an HPV-positive self-sampling test (reflex cytology is not possible from the self-sample), presents a particular challenge. This is why a molecular test (such as DNA methylation or genotyping) from the self-collected sample is attractive because it can be carried out without requesting another sample or an examination. Currently the only commercially available molecular triage is genotyping for HPV16 and perhaps HPV18, although the latter has questionable specificity; however, in the future methylation may provide a suitable triage assay for vaginal self-collected samples.

Overall, the studies support the use of self-collection to improve cervical cancer prevention in noncompliant and poor women worldwide. It appears that the next logical step is large-scale pilot implementation of HPV testing on self-collected samples in developed countries, as well as in resource-constrained regions and poor countries that have sufficient economic capacity and provider skills, with appropriate infrastructure. Prior to implementation, there should be a thoughtful analysis of the program including: the screening groups and means of contact; the primary testing and triage approach; rendering of accurate final diagnoses; and treatment, follow-up and documentation.

Conclusion

HPV testing is seen to be the preferred approach to primary cervical screening in both high- and low-resource settings [82]. Testing for HPV in vaginal self-collected samples offers a relatively cheap and effective way of improving cervical screening coverage in many settings. Currently the best well-validated option for triage of HPV-positive women is cytology, but that requires a clinician-collected sample from the cervix. Genotyping for HPV16 and some other HPV types, or alternatively p16 alone or in combination with Ki-67, are also currently under evaluation as possible triage options for hrHPV-positive women. In the future, DNA methylation may provide a suitable triage assay for vaginal self-collected samples. Even with affordable HPV testing, middle- and low-income countries will have to overcome the infrastructure challenges needed to ensure good follow-up, triage and treatment of HPV-positive women.

Future perspective

HPV cotesting has become the predominant method of screening in the USA. Eventually, HPV testing is likely to become the primary screen in the USA, with cytology used as the triage test for hrHPV-positive women. In the next 5–10 years, HPV testing will be introduced as the primary screening test in organized screening programs in many countries in Europe. HPV screening in Asia and Latin America is also taking hold; there will be a diversity of tests and approaches and, in general, adoption will be quite slow and region-specific. Triage of HPV-positive women, using various combinations of genotyping, immunochemistry and molecular markers, will begin to be introduced as part of some population-based screening programs. Vaginal self-collection will be used to boost coverage wherever HPV testing is used.

Executive summary

Human papillomavirus testing

High-risk human papillomavirus (hrHPV) infection is needed for cervical cancer to develop, and since hrHPV DNA is still present during the development of cancer, looking for hrHPV DNA should be a good approach to identifying women with high-grade cervical intraepithelial neoplasia.

The advantages of hrHPV testing over cytology (currently the most widely used screening method) are that HPV-negative women are extremely unlikely to develop cervical cancer over the next 5–10 years and infrequent screening would be safe. Additionally, HPV testing does not depend on the collection of samples with good morphology.

Screening for cervical cancer using HPV testing is more sensitive than screening using cytology, but considerably less specific. A greater proportion of women will test HPV-positive on a single screen compared with the proportion found to have abnormal cytology. Therefore, a good way of managing HPV-positive individuals is essential.

Management of women positive for HPV

A positive HPV test in primary screening should only trigger a triage test, not automatic referral to colposcopy. The most widely studied triage test in this situation is cytology. Triage with cytology can be carried out on the same sample as the HPV test without the need for women to attend the clinic again.

HPV-positive women with normal cytology do not need to be retested at a short interval, but neither should they be rescreened only every 3 or 5 years. A repeat test after an interval of 12–24 months seems reasonable.

Genotyping for HPV16 and HPV18 has been proposed as a good alternative for women who test HPV-positive and cytology-negative because the risk of cervical intraepithelial neoplasia 3₊ is genotype-dependent and HPV16 or HPV18 are found in over 70% of cervical cancer. However, being HPV-positive, but HPV16- and 18-negative, confers an increased risk of cervical disease and these women cannot be returned to routine screening.

One particularly promising triage test is the immunocytochemical detection of overexpression of the p16INK4a tumor suppressor gene as a surrogate marker for the transforming activity of hrHPV oncoproteins, which are essential for the initiation and maintenance of the neoplastic process. Currently the potential of this test is limited by the lack of standardized reporting and a need for substantial expert interpretation.

DNA methylation of a panel of both human and HPV gene markers to be used as triage tests is an exciting area of current research. Vaginal self-collection for HPV-testing

Testing for HPV in vaginal self-collected samples offers a relatively cheap and effective way of improving cervical screening coverage in many settings.

The performance of the HPV test on self-collected samples, in terms of clinical sensitivity and specificity for cervical intraepithelial neoplasia 2 and above in the screening setting, is not as good as HPV testing on clinician-taken cervical specimens; in most studies the sensitivity or specificity are quite variable, showing values between 60 and 90%. However, it is fairly clear that HPV testing on self-collected samples is as sensitive, and sometimes more sensitive, than developed-country cytology-based screening of cervical specimens, although cytology is the more specific test.

Conclusion

HPV testing is the preferred approach to primary cervical screening in both high- and low-resource settings.

The best well-validated option for triage of HPV-positive women is cytology. Genotyping for HPV16 and some other HPV types, or alternatively p16 alone or in combination with Ki-67, are also currently under evaluation as possible triage options for hrHPV-positive women.

Testing for HPV in vaginal self-collected samples offers a relatively cheap and effective way of improving cervical screening coverage in many settings.

In the future, DNA methylation may provide a suitable triage assay for vaginal self-collected samples.

The impact of HPV vaccination on women of HPV screening age (those over 30 years of age) is still 5–10 years away and more data on the population-based efficacy of the vaccine for preventing cervical cancer will be needed before recommendations on altering current screening algorithms can be made. Rational algorithms will distinguish women known to have been vaccinated (three doses) while under the age of 15 years from those not known to have been vaccinated or known to have received less than three doses of the vaccine against HPV16 and HPV18. Nevertheless, a combination of HPV vaccination and HPV testing will be the cornerstone of cervical cancer control globally.

Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

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Overview of human papillomavirus-based and other novel options for cervical cancer screening in developed and developing countries. Vaccine 26(Suppl. 10), K29–K41 (2008).

15.

Cuzick

Clavel

Petry

Overview of the European and North American studies on HPV testing in primary cervical cancer screening. Int. J. Cancer 119(5), 1095–1101 (2006).

16.

Overview of the sensitivity and specificity of cytology and HPV testing. First to show that HPV testing is uniformly more sensitive for the detection of high-grade cervical intraepithelial neoplasia.

17.

Leinonen

Nieminen

Kotaniemi-Talonen

Age-specific evaluation of primary human papillomavirus screening vs conventional cytology in a randomized setting. J. Natl Cancer Inst. 101(23), 1612–1623 (2009).

18.

Snijders

Verhoef

Arbyn

High-risk HPV testing on self-sampled versus clinician-collected specimens: a review on the clinical accuracy and impact on population attendance in cervical cancer screening. Int. J. Cancer 132(10), 2223–2236 (2013).

19.

Good paper for those wanting to know more about HPV self-sampling.

20.

Dillner

Rebolj

Birembaut

Long-term predictive values of cytology and human papillomavirus testing in cervical cancer screening: joint European cohort study. BMJ 337, a1754 (2008).

21.

Good paper on the longer-term predictive value of cytology and HPV testing.

22.

Mesher

Szarewski

Cadman

Long-term follow-up of cervical disease in women screened by cytology and HPV testing: results from the HART study. Br. J. Cancer 102(9), 1405–1410 (2010).

23.

Sherman

Lorincz

Scott

Baseline cytology, human papillomavirus testing, and risk for cervical neoplasia: a 10-year cohort analysis. J. Natl Cancer Inst. 95(1), 46–52 (2003).

24.

Kjaer

Hogdall

Frederiksen

The absolute risk of cervical abnormalities in high-risk human papillomavirus-positive, cytologically normal women over a 10-year period. Cancer Res. 66(21), 10630–10636 (2006).

25.

Katki

Kinney

Fetterman

Cervical cancer risk for women undergoing concurrent testing for human papillomavirus and cervical cytology: a population-based study in routine clinical practice. Lancet Oncol. 12(7), 663–672 (2011).

26.

Kitchener

Almonte

Thomson

HPV testing in combination with liquid-based cytology in primary cervical screening (ARTISTIC): a randomised controlled trial. Lancet Oncol. 10(7), 672–682 (2009).

27.

Naucler

Ryd

Tornberg

Human papillomavirus and Papanicolaou tests to screen for cervical cancer. N. Engl. J. Med. 357(16), 1589–1597 (2007).

28.

Rijkaart

Berkhof

Rozendaal

Human papillomavirus testing for the detection of high-grade cervical intraepithelial neoplasia and cancer: final results of the POBASCAM randomised controlled trial. Lancet Oncol. 13(1), 78–88 (2012).

29.

Ronco

Giorgi-Rossi

Carozzi

Efficacy of human papillomavirus testing for the detection of invasive cervical cancers and cervical intraepithelial neoplasia: a randomised controlled trial. Lancet Oncol. 11(3), 249–257 (2010).

30.

Almonte

Ferreccio

Winkler

Cervical screening by visual inspection, HPV testing, liquid-based and conventional cytology in Amazonian Peru. Int. J. Cancer 121(4), 796–802 (2007).

31.

Sankaranarayanan

Nene

Shastri

HPV screening for cervical cancer in rural India. N. Engl. J. Med. 360(14), 1385–1394 (2009).

32.

Although there are many good trials of HPV screening, this is the only one assessing its impact on mortality from cervical cancer.

33.

Naucler

Ryd

Tornberg

Efficacy of HPV DNA testing with cytology triage and/or repeat HPV DNA testing in primary cervical cancer screening. J. Natl Cancer Inst. 101(2), 88–99 (2009).

34.

Saslow

Solomon

Lawson

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35.

Sasieni

Cuzick

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36.

Rijkaart

Berkhof

van Kemenade

Evaluation of 14 triage strategies for HPV DNA-positive women in population-based cervical screening. Int. J. Cancer 130(3), 602–610 (2012).

37.

Schiffman

Herrero

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The carcinogenicity of human papillomavirus types reflects viral evolution. Virology 337(1), 76–84 (2005).

38.

Kjaer

Frederiksen

Munk

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39.

de Sanjose

Quint

Alemany

Human papillomavirus genotype attribution in invasive cervical cancer: a retrospective cross-sectional worldwide study. Lancet Oncol. 11(11), 1048–1056 (2010).

40.

Bosch

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41.

Khan

Castle

Lorincz

The elevated 10-year risk of cervical precancer and cancer in women with human papillomavirus (HPV) type 16 or 18 and the possible utility of type-specific HPV testing in clinical practice. J. Natl Cancer Inst. 97(14), 1072–1079 (2005).

42.

Addresses the increased risk of cervical cancer associated with certain HPV subtypes.

43.

Negri

Bellisano

Zannoni

p16 ink4a and HPV L1 immunohistochemistry is helpful for estimating the behavior of low-grade dysplastic lesions of the cervix uteri. Am. J. Surg. Pathol. 32(11), 1715–1720 (2008).

44.

Ozaki

Zen

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. Biomarker expression in cervical intraepithelial neoplasia: potential progression predictive factors for low-grade lesions. Hum. Pathol. 42(7), 1007–1012 (2011).

45.

Wang

Zheng

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Lindstrom

Wallin

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46.

Denton

Bergeron

Klement

Trunk

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47.

Carozzi

Confortini

Palma

Dalla P

Use of p16-INK4A overexpression to increase the specificity of human papillomavirus testing: a nested substudy of the NTCC randomised controlled trial. Lancet Oncol. 9(10), 937–945 (2008).

48.

For those wanting to find out more about p16, please also see the follow-up from this study [43].

49.

Bergeron

Ordi

Schmidt

Trunk

Keller

Ridder

. Conjunctive p16INK4a testing significantly increases accuracy in diagnosing high-grade cervical intraepithelial neoplasia. Am. J. Clin. Pathol. 133(3), 395–406 (2010).

50.

Petry

Schmidt

Scherbring

Triaging pap cytology negative, HPV-positive cervical cancer screening results with p16/Ki-67 dual-stained cytology. Gynecol. Oncol. 121(3), 505–509 (2011).

51.

Schmidt

Bergeron

Denton

Ridder

; European CINtec Cytology Study Group. p16/Ki-67 dual-stain cytology in the triage of ASCUS and LSIL papanicolaou cytology: results from the European equivocal or mildly abnormal Papanicolaou cytology study. Cancer Cytopathol. 119(3), 158–166 (2011).

52.

Tsoumpou

Arbyn

Kyrgiou

p16(INK4a) immunostaining in cytological and histological specimens from the uterine cervix: a systematic review and meta-analysis. Cancer Treat. Rev. 35(3), 210–220 (2009).

53.

Carozzi

Gillio-Tos

Confortini

Risk of high-grade cervical intraepithelial neoplasia during follow-up in HPV-positive women according to baseline p16-INK4A results: a prospective analysis of a nested substudy of the NTCC randomised controlled trial. Lancet Oncol. 14(2), 168–176 (2013).

54.

Lorincz

. The promise and the problems of epigenetics biomarkers in cancer. Expert Opin. Med. Diagn. 5(5), 375–379 (2011).

55.

Simple overview of epigenetics including DNA methylation.

56.

Wentzensen

Sherman

Schiffman

Wang

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57.

Hesselink

Heideman

Steenbergen

Combined promoter methylation analysis of CADM1 and MAL: an objective triage tool for high-risk human papillomavirus DNA-positive women. Clin. Cancer Res. 17(8), 2459–2465 (2011).

58.

Kalantari

Calleja-Macias

Tewari

Conserved methylation patterns of human papillomavirus type 16 DNA in asymptomatic infection and cervical neoplasia. J. Virol. 78(23), 12762–12772 (2004).

59.

Brandsma

Sun

Lizardi

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60.

Mirabello

Sun

Ghosh

Methylation of human papillomavirus type 16 genome and risk of cervical precancer in a Costa Rican population. J. Natl Cancer Inst. 104(7), 556–565 (2012).

61.

Lorincz

Brentnall

Vasiljevic

HPV16 L1 and L2 DNA methylation predicts high-grade cervical intraepithelial neoplasia in women with mildly abnormal cervical cytology. Int. J. Cancer, 133(3), 637–644 (2013).

62.

Sasieni

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63.

Szarewski

Cadman

Ashdown-Barr

Waller

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64.

Igidbashian

Boveri

Spolti

Radice

Sandri

Sideri

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65.

Waller

McCaffery

Forrest

Acceptability of unsupervised HPV self-sampling using written instructions. J. Med. Screen. 13(4), 208–213 (2006).

66.

Gravitt

Belinson

Salmeron

Shah

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67.

Dzuba

Diaz

Allen

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68.

Barata

Mai

Howlett

Gagliardi

Stewart

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69.

Nobbenhuis

Helmerhorst

van den Brule

Primary screening for high risk HPV by home obtained cervicovaginal lavage is an alternative screening tool for unscreened women. J. Clin. Pathol. 55(6), 435–439 (2002).

70.

Anhang

Nelson

Telerant

Chiasson

Wright

Jr.

Acceptability of self-collection of specimens for HPV DNA testing in an urban population. J. Womens Health (Larchmt) 14(8), 721–728 (2005).

71.

Berner

Hassel

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72.

Mitchell

Ogilvie

Steinberg

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Money

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73.

Forrest

McCaffery

Waller

Attitudes to self-sampling for HPV among Indian, Pakistani, African–Caribbean and white British women in Manchester, UK. J. Med. Screen. 11(2), 85–88 (2004).

74.

Howard

Lytwyn

Lohfeld

Redwood-Campbell

Fowler

Karwalajtys

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75.

Waller

Bartoszek

Marlow

Wardle

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76.

Petignat

Faltin

Bruchim

Tramer

Franco

Coutlee

. Are self-collected samples comparable to physician-collected cervical specimens for human papillomavirus DNA testing? A systematic review and meta-analysis. Gynecol. Oncol. 105(2), 530–535 (2007).

77.

Ogilvie

Patrick

Schulzer

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78.

Stewart

Gagliardi

Johnston

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79.

Castle

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80.

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Cruz-Valdez

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Zhao

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Holanda

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83.

Qiao

Sellors

Eder

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84.

Rossi

Giorgi P

Marsili

Camilloni

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85.

Szarewski

Cadman

Mesher

HPV self-sampling as an alternative strategy in non-attenders for cervical screening – a randomised controlled trial. Br. J. Cancer 104(6), 915–920 (2011).

86.

Bais

van Kemenade

Berkhof

Human papillomavirus testing on self-sampled cervicovaginal brushes: an effective alternative to protect nonresponders in cervical screening programs. Int. J. Cancer 120(7), 1505–1510 (2007).

87.

Sanner

Wikstrom

Strand

Lindell

Wilander

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88.

Gok

Heideman

van Kemenade

HPV testing on self collected cervicovaginal lavage specimens as screening method for women who do not attend cervical screening: cohort study. BMJ 340, c1040 (2010).

89.

Gok

Heideman

van Kemenade

Offering self-sampling for human papillomavirus testing to non-attendees of the cervical screening programme: characteristics of the responders. Eur. J. Cancer 48(12), 1799–1808 (2012).

90.

Virtanen

Anttila

Luostarinen

Nieminen

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91.

Wikstrom

Lindell

Sanner

Wilander

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92.

Virtanen

Nieminen

Luostarinen

Anttila

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93.

de Kok

Van Rosmalen

Dillner

Primary screening for human papillomavirus compared with cytology screening for cervical cancer in European settings: cost effectiveness analysis based on a Dutch microsimulation model. BMJ 344, e670 (2012).

94.

Ferlay

JSH

Bray

Forman

Mathers

Parkin

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New Strategies for Human Papillomavirus-Based Cervical Screening

Abstract

Keywords

HPV testing

Management of women positive for HPV

Genotyping for HPV16 & HPV18

Immunocytochemical detection of p16INK4a

DNA methylation

Vaginal self-collection for HPV testing

Acceptability of vaginal self-collection

Sensitivity & specificity of vaginal self-sampling

Cervical screening using vaginal self-sampling for HPV testing

Conclusion

Future perspective

Financial & competing interests disclosure

References