Simon Leeson
email: simon.leeson@wales.nhs.uk

Remi Alalade

Neelam Singh

Pekka Nieminen

Margaret Cruickshank

Xavier Carcopino

Christine Bergeron

Options for triage and implications for colposcopists within European HPV-based cervical screening programmes

Keywords:

The development of human papillomavirus (HPV)-based screening should detect more pre-cancerous changes and so reduce the incidence and mortality from cervical squamous carcinoma and cervical adenocarcinoma. However, many more women are high risk HPV (hrHPV) screen positive compared to cytology-based screening, especially in younger age-women. A variety of tests have become available which may triage into those hrHPV test-positive women who need immediate referral to colposcopy from those who need early repeat HPV tests or recall on the basis of their disease status.

We performed a literature review of publications and a manual search from 2010, reporting cytology, HPV partial genotyping, dual-staining and DNA methylation for triage of hrHPV positive tests, including their comparative performance between these methods as well as the effectiveness of some triage combinations with reference to HPV-based screening services in Europe. Cost effectiveness and the structure of triage algorithms for colposcopists also have been considered. From one report evaluating four options for triage as single options or as combined algorithms, partial genotyping for HPV 16 and 18 with dual-staining yielded the highest risk of cervical intraepithelial neoplasia grade three or worse within an HPV positive population and with an acceptable colposcopy rate. From a separate paper, this option appeared cost effective. However, publications were difficult to compare objectively.

All options have their merits but a combination triage involving any two of cytology, HPV partial genotyping or dual-staining seems most efficient at present. HPV vaccination may impact upon the performance of future partial genotyping. DNA Methylation may become an acceptable future option.

Introduction

Several national programmes have converted from a cytologybased cervical cancer screening programme to one based upon human papillomavirus (HPV) testing. Other European states are considering this change. Four European prospective randomised screening studies have shown a 70% reduction in cervical cancer rates for those with negative tests including adenocarcinoma [START_REF] Ronco | Efficacy of HPV-based screening for prevention of invasive cervical cancer: follow-up of four European randomised controlled trials[END_REF]. Only a minority of all positive high-risk HPV (hrHPV) cases have pre-cancerous lesions requiring treatment. There are various triage tests available to prioritise those women requiring colposcopic assessment as opposed to surveillance in the community based on the risk of underlying cervical intraepithelial neoplasia grade two or worse (CIN2+) or high grade squamous intraepithelial lesions or worse (HSIL+). A plethora of published data has compared the performance of triage options in a range of scenarios. As screening programmes vary in a number of ways such as target populations, screening intervals and the level of risk to recommend referral, it is difficult to determine which may be the ideal triage for each programme. Time and resources may be wasted as a consequence in attempting to improve colposcopic diagnostic performance. Furthermore, the colposcopic identification of HSIL from within the group of women referred with low grade squamous intraepithelial lesion (LSIL) cytology is often difficult with a poor sensitivity in part due to low prevalence of pre-cancerous disease in this group [START_REF] Budithi | Evaluating colposcopy with dynamic spectral imaging during routine practice at five colposcopy clinics in Wales: clinical performance[END_REF]. A Eurogin Roadmap examining triage strategies has stressed the importance of a clear triage strategy of each national screening programme either as a single or as a combination option. Choices are dependent upon the complexity of the consequent management algorithms and cost [START_REF] Cuschieri | Eurogin roadmap 2017: triage strategies for the management of HPV-positive women in cervical screening programs[END_REF].

Turkey was the first European state to introduce an HPV-based cervical screening programme in 2014 [START_REF] Gultekin | Initial results of population based cervical cancer screening program using HPV testing in one million Turkish women[END_REF]. The Netherlands followed in 2017 with various regions in Finland, Italy and Sweden converting from cytology [START_REF] Aitken | Introduction of primary screening using high-risk HPV DNA detection in the Dutch cervical cancer screening programme: a populationbased cohort study[END_REF][START_REF] Maver | Primary HPV-based cervical cancer screening in Europe: implementation status, challenges, and future plans[END_REF] with France converting in 2020. In the UK, Wales converted in 2018 with the remaining UK nations converting in 2020. It is important to recognize that there are service issues impacting upon newly developed HPV-based screening programmes. The HPV positive test rate varies in differing screening programmes with a range of five to 16% [START_REF] Munoz | Epidemiologic classification of human papillomavirus types associated with cervical cancer[END_REF][START_REF] Arbyn | Trials comparing cytology with human papillomavirus screening[END_REF][START_REF] Howell-Jones | Multi-site study of HPV type-specific prevalence in women with cervical cancer, intraepithelial neoplasia and normal cytology, in England[END_REF][START_REF] Katki | Cervical cancer risk for 330,000 women undergoing concurrent HPV testing and cervical cytology in routine clinical practice at a large managed care organisation[END_REF] with 13% in the English Sentinel Sites pilot [START_REF] Rebolj | 16/18 genotyping in triage of persistent human papillomavirus infections with negative cytology in the English cervical screening pilot[END_REF]. However, precancerous lesions defined as CIN2+ or HSIL+ are present consistently in less than 2% of each HPV screening round [START_REF] Ogilvie | Effect of screening with primary cervical HPV testing vs cytology testing on highgrade cervical intraepithelial neoplasia at 48 months. The HPV FOCAL randomized clinical trial[END_REF]. Triage of those women within the hrHPV positive group is required to avoid overwhelming colposcopy services with referrals who will mostly have a low risk of pre-invasive or invasive disease and avoid unnecessary colposcopy with associated physical and psychological impacts on women themselves. Suitable triage also reduces lead time delay to diagnosis of significant lesions [START_REF] Ronco | HPV16 and HPV18 genotyping in cervical cancer screening[END_REF]. A switch of cytology from the primary screen to become the triage test of hrHPV positive cases is a logical choice but there are other options.

We aim to describe the updated performance of triage tests which are currently validated and commercially available since the publication of the 2017 Eurogin Roadmap to develop recommendations for practice. To achieve this, a literature search was performed, using MEDLINE requesting articles published since 2010 written in English from Europe, North America and Australia. We used a combination of Mesh Subject Headings and free text search terms Papillomaviridae, HPV, human papilloma virus, triage, triage test, DNA methylation, dual stain, cytology triage, partial genotyping, human papillomavirus 16/ or type 16 and human papillomavirus 18/ or type 18 for female human studies. Seventy-four papers were obtained on 21.05.2020, of which 13 were relevant and in addition to a manual search.

The performance of cytology, HPV partial genotyping, p16 and Ki-67 dual-staining, DNA methylation and combinations have been reviewed to see what effects they have on detecting CIN2+ or HSIL+ as well as keeping women at low risk in the community so reducing the need for unnecessary colposcopy and associated interventions. These triage options were rated according to the reproducibility of published findings as detailed in the latest and earlier editions of the American Society for Colposcopy and Cervical Pathology (ASCCP) consensus guidelines (see Table 1).

Options for triage of HPV positive cases

Cytology

Compared to cytology-based screening, in an unblinded crosssectional and longitudinal randomized controlled trial of 58,076 women in a population screening programme in Finland, HPV testing with cytology as triage was more sensitive for the detection of CIN3+ for all age groups (relative rate in HPV arm for CIN3+ 1.44 (95% confidence interval, CI 1.01-2.05) for all women as the intention to treat group and 1.77 (1.16-2.74) for those that attended (per protocol). For those with negative hrHPV testing the relative rate was 0.28 (0.04-1.17) [START_REF] Anttila | Rate of cervical cancer, severe intraepithelial neoplasia, and adenocarcinoma in situ in primary HPV DNA screening with cytology triage: randomized study within organized screening programme[END_REF]. These findings have been reported in other cross-sectional and longitudinal studies [START_REF] Elfgren | Colposcopic and histopathologic evaluation of women participating in population-based screening for human papillomavirus deoxyribonucleic acid persistence[END_REF][START_REF] Kitchener | HPV testing in combination with liquid-based cytology in primary cervical screening (ARTISTIC): a randomised controlled trial[END_REF][START_REF] Ronco | Efficacy of human papillomavirus testing for the detection of invasive cervical cancers and cervical intraepithelial neoplasia: a randomised controlled trial[END_REF][START_REF] Leinonen | Detection rates of precancerous and cancerous cervical lesions within one screening round of primary human papillomavirus DNA testing: prospective randomised trial in Finland[END_REF][START_REF] Rijkaart | Human papillomavirus testing for the detection of high-grade cervical intraepithelial neoplasia and cancer: final results of the POBASCAM randomised controlled trial[END_REF].

Cytology as triage leads to an acceptable colposcopy referral rate (36.4% of hrHPV positive tests) in the Kaiser Permanente cytology and HPV co-testing screening programme [START_REF] Castle | Five-year experience of human papillomavirus DNA and Papanicolaou test contesting[END_REF], 38.6% in the Finnish population-based screening programme [START_REF] Leinonen | Type-and age-specific distribution of human papillomavirus in women attending cervical cancer screening in Finland[END_REF] and 33.1% in the English Sentinel Sites Pilot [START_REF] Rebolj | Primary cervical screening with high risk human papillomavirus testing: observational study[END_REF]. The importance of negative cytology as triage is that approximately eight percent of each screening round is returned to early recall. Women with hrHPV positive and cytology negative testing overall accounts of 7.1-8.6% of each round of cervical screening in the Sentinel Sites Pilot and in Wales. The abnormal cytology rate has decreased in Wales following conversion to HPV-based screening in 2018 as hrHPV negative cases do not have cytology triage. Moreover, delayed referral of those with negative cytology potentially prevents overtreatment of regressive SIL as approximately 40% of these women clear HPV in 12 months [START_REF] Macdonald | Prevalence of high-grade cervical intraepithelial neoplasia in women with persistent high-risk HPV genotypes and negative cytology[END_REF]. In the UK, women would be referred to colposcopy with HPV persistency at 12 or 24 months in the absence of any cytological abnormality. This group added 54% extra referrals to the ongoing colposcopy workload of the in the Sentinel Sites Pilot [START_REF] Rebolj | Primary cervical screening with high risk human papillomavirus testing: observational study[END_REF] and 36% of all colposcopies in Italy [START_REF] Mistro | Cervical cancer screening by high risk HPV testing in routine practice: results at one year recall of high risk HPV-positive and cytology-negative women[END_REF]. The same effect has been reported in France (Carcopino; personal communication). Despite this significant increase in colposcopy workload, the risk of CIN2+ in this group is only three to seven percent after a single test and is below the agreed threshold for colposcopy referral of 10-20% [START_REF] Mistro | Cervical cancer screening by high risk HPV testing in routine practice: results at one year recall of high risk HPV-positive and cytology-negative women[END_REF][START_REF] Petry | Inclusion of HPV testing in routine cervical cancer screening in women above 29 years in Germany: results for 8466 patients[END_REF][START_REF] Thrall | High-risk HPV testing in women 30 years or older with negative Papanicolaou tests: initial clinical experience with 18-month follow-up[END_REF].

The value of cytology as triage was reported in a Dutch substudy comparing 14 triage strategies for hrHPV positive women from the VUSA-screen study. Cytological triage and follow-up with cytology for initial cytology negative women at 12 months was recommended. This provided a high negative predictive value (NPV 99.3%) but the lowest colposcopy referral rate of 33.4% compared to other strategies including HPV partial genotyping in more than 25,600 women aged 30-60 years [START_REF] Rijkaart | Evaluation of 14 triage strategies for HPV DNA-positive women in population-based cervical screening[END_REF]. Co-testing as recommended in the US with HPV and cytology both as a primary screen does not improve performance [START_REF] Katki | Cervical cancer risk for 330,000 women undergoing concurrent HPV testing and cervical cytology in routine clinical practice at a large managed care organisation[END_REF], albeit with slight (4.7%) increase in sensitivity but a 35.2% increase of screen positives compared to hrHPV testing alone [START_REF] Castle | Performance of carcinogenic human papillomavirus (HPV) testing and HPV16 or HPV18 genotyping for cervical cancer screening of women aged 25 years and older: a subanalysis of the ATHENA study[END_REF].

Cytology provides grading of risk that current colposcopists are familiar with from previous cytology-based screening. HPVbased screening using cytology as triage is effective in reducing the incidence of cervical adenocarcinoma [START_REF] Ronco | Efficacy of HPV-based screening for prevention of invasive cervical cancer: follow-up of four European randomised controlled trials[END_REF] where women who were hrHPV positive and had abnormal cervical cytology, as well as those who showed hrHPV persistency were referred for colposcopy [START_REF] Leeson | HPV testing and vaccination in Europe[END_REF]. However, cytology is subject to inter-observer variation particularly with atypical squamous cells of undetermined significance (ASCUS) [START_REF] Stoler | Interobserver reproducibility of cervical cytologic and histologic interpretations. Realistic estimates from the ASCUS-LSIL Triage Study[END_REF][START_REF] Sherman | The Bethesda Interobserver Reproducibility Study (BIRST): a web-based assessment of the Bethesda 2001 System for classifying cervical cytology[END_REF] and prior HPV status may influence cytology interpretation by cytologists [START_REF] Rijkaart | Evaluation of 14 triage strategies for HPV DNA-positive women in population-based cervical screening[END_REF][START_REF] Leinonen | Age-specific evaluation of primary human papillomavirus screening vs conventional cytology in a randomized setting[END_REF]. This has not been the universal view [START_REF] Bergeron | Informed cytology for triaging HPV-Positive women: substudy nested in the NTCC randomized controlled trial[END_REF]. Variations in referral practice to colposcopy amongst women who were HPV positive ranging from 20-57% have also been reported in Italy, although histological endpoints were not assessed [START_REF] Ronco | Impact of variations in triage cytology interpretation on human papillomavirus-based cervical screening and implications for screening algorithms[END_REF]. Laboratories which had a high proportion of low grade samples with cytology-based screening appeared to continue with a high rate of low grade samples after converting to HPV-based screening in England. This behaviour can be closely scrutinized with robust quality assurance (Tidy, personal communication on behalf of the Sentinel Sites Pilot). Cytology as a triage with referral to colposcopy with any cytological abnormality has a large volume of evidence validating its role. Despite cytological examination being a subjective assessment producing variations in referral rates to colposcopy and detection rates of CIN2+, it is the favoured single triage option (preferred: ASCCP strength A; quality II).

HPV 16/18 typing HPV types 16 or 18 has been detected in 63% of CIN3 and 91% of cervical glandular intraepithelial neoplasia [START_REF] Howell-Jones | Multi-site study of HPV type-specific prevalence in women with cervical cancer, intraepithelial neoplasia and normal cytology, in England[END_REF]. Genotyping for HPV 16 and 18 may allow for modified management depending upon genotype status [START_REF] Tidy | NHS cervical screening programme colposcopy and programme management[END_REF] and is not dependent on cytotechnician interpretation of findings unlike cytology [START_REF] Ronco | HPV16 and HPV18 genotyping in cervical cancer screening[END_REF]. The ATHENA study of 47,208 women aged 21 years or older having routine screening with cytology and HPV testing reported on 4,219 who were hrHPV positive and cytology negative in women older than 30 years of age (including a subset of 886 hrHPV negative women). The prevalent CIN2+ rate of HPV 16/18 positive cases was 11.4%; the rate for those hrHPV positive for all types was 6.1 % and 0.8% for those who were hrHPV negative. The prevalent rate for CIN2+ for the 12 non-16/18 oncogenic genotypes was 4.6% [START_REF] Wright | Evaluation of HPV-16 and HPV-18 genotyping for the triage of women with high-risk HPV+ cytology-negative results[END_REF]. From the same study of 41,955 women older than 25 years of age, HPV 16 or 18 positive or LSIL cytology positive women had an increased sensitivity for detection of CIN3+ (p < 0.0001) compared to ASCUS + cytology only [START_REF] Castle | Performance of carcinogenic human papillomavirus (HPV) testing and HPV16 or HPV18 genotyping for cervical cancer screening of women aged 25 years and older: a subanalysis of the ATHENA study[END_REF]. Typing for HPV 16/18 in addition to cytology led to the detection of around 25% of hrHPV positive CIN3+ one year earlier than with repeat testing at expense of 1.5% drop in positive predictive value (PPV) [START_REF] Ronco | HPV16 and HPV18 genotyping in cervical cancer screening[END_REF]. Other hrHPV positive types could be managed with early recall rather than with colposcopy.

There is an increased risk of CIN2+ if same virus type persists for two years (odds ratio or OR of 813.0, CI 168.2À3,229.2) in prospective study of 10,758 women 20-29 years [START_REF] Kjaer | Type specific persistence of high risk human papillomavirus (HPV) as indicator of high grade cervical squamous intraepithelial lesions in young women: population based prospective follow up study[END_REF]. Immediate referral to colposcopy of HPV types 16, 18, 31, 33 and 45 with no further triage will possibly lead to a high colposcopy referral rate [START_REF] Rijkaart | Evaluation of 14 triage strategies for HPV DNA-positive women in population-based cervical screening[END_REF].

There appears to be age trend in the prevalence of HPV 16 and 18. HPV 16/18 associated CIN3+ appears to be more common in women under 30 years of age compared to women older than 45

Strength of recommendation

Descriptor

A.

Good evidence for efficacy and substantial clinical benefit support recommendation for use.

B.

Moderate evidence for efficacy or only limited clinical benefit supports recommendation for use.

C.

Evidence for efficacy is insufficient to support a recommendation for or against use, but recommendations may be made on other grounds.

D.

Moderate evidence for lack of efficacy or for adverse outcome supports a recommendation against use.

E.

Good evidence for lack of efficacy or for adverse outcome supports a recommendation against use.

Quality of evidence Descriptor

I.

Evidence from at least one randomized, controlled trial.

II.

Evidence from at least one clinical trial without randomization, from cohort or case-controlled analytic studies (preferably from more than one center), or from multiple time-series studies, or dramatic results from uncontrolled experiments.

III.

Evidence from opinions of respected authorities based on clinical experience, descriptive studies, or reports of expert committees.

Terminology used for recommendations

Descriptor

Recommended.

Good data to support use when only one option is available.

Preferred.

Option is the best (or one of the best) when there are multiple options. Acceptable.

One of multiple options when there is either data indicating that another approach is superior or when there are no data to favor any single option.

Not recommended.

Weak evidence against use and marginal risk for adverse consequences. Unacceptable.

Good evidence against use. For women over 45 years the risk ratio of having HPV16/18+ associated CIN3+ was 0.61 (95% CI 0.43-0.88) compared to women under 30 [START_REF] Aro | Agespecific HPV type distribution in high-grade cervical disease in screened and unvaccinated women[END_REF]. Importantly, reported data has largely been from unvaccinated populations. The performance of hrHPV typing can be only speculative when the vaccinated population against hrHPV types enters the screening age range. Typing performance may be different for the recently introduced nonavalent vaccine as opposed to the older bi-and quadrivalent vaccines. However, an effect may be seen shortly in the younger colposcopy referrals. The prevalence of hrHPV types 16/18 in all assays in SHEVa study of women up to 21 years of age in the Scottish Cervical Screening Programme (where screening of women from 20 years continued to 2016) dropped by more than 75% in vaccinated cohorts with no change in non-16/18 types compared to the non-vaccinated cohort [START_REF] Bhatia | Use of HPV testing for cervical screening in vaccinated women -insights from the SHEVa (Scottish HPV prevalence in vaccinated women) study[END_REF]. There is a more than 90% uptake of the three dose vaccine schedule in Scotland. The current two dose vaccination rate from 2015 to 2017 was up to 86% in England and Wales for 12 and 13year old girls but uptake in other European states is inconsistent, for instance with less than 50% in France and Germany [START_REF] Bruni | Global estimates of human papillomavirus vaccination coverage by region and income level: a pooled analysis[END_REF]. Whilst Scotland has reported reduction in HPV16/18 infections, CIN2+ detection and in colposcopy referrals and treatments, this must be seen in light of high vaccine uptake. Vaccination may not have an impact upon colposcopy referral rates for 10 years or more until a vaccinated cohort predominates in the under 40-year old referrals. Furthermore, the Sentinel Sites Pilot reported a 1.2% (CI 0.6-2.0) improved detection rate of CIN2+ at the expense of 5.9% extra colposcopy referrals with the use of HPV 16/18 partial typing. The conclusion was that within a quality assured screening programme and with women reliably returning for early recall with low grade cytology as triage, partial genotyping with HPV 16/18 did not significantly improve detection of pre-cancerous lesions [START_REF] Rebolj | 16/18 genotyping in triage of persistent human papillomavirus infections with negative cytology in the English cervical screening pilot[END_REF]. Nonetheless, partial genotyping would be a suitable triage option for populations where vaccination coverage is below an acceptable threshold (such as less than 80%) for an effect in preventing CIN2+ [START_REF] Cuzick | Predicted impact of vaccination against human papillomavirus 16/18 on cancer incidence and cervical abnormalities in women aged 20-29 in the UK[END_REF]. In the unvaccinated population partial genotyping appears more sensitive than cytology and testing is automated (acceptable: ASCCP strength A; quality II).

Reproduced with permission from

p16/Ki-67

Differential expression host cell cycle regulatory proteins may have a role as biomarkers for dysplasia. A cyclin-dependent kinase inhibitor, p16 is upregulated with expression of HPV E7 protein. Levels of p16 correlate with the grade of abnormal cytology and the degree of CIN [START_REF] Tsoumpou | p16INK4a immunostaining in cytological and histological specimens from the uterine cervix: a systematic review and meta-analysis[END_REF]. Over-expression is seen in 12% of normal cytology and two per cent of normal biopsies from a systematic review of 61 studies [START_REF] Tsoumpou | p16INK4a immunostaining in cytological and histological specimens from the uterine cervix: a systematic review and meta-analysis[END_REF].

Triage of hrHPV tests with p16 had a significantly higher relative sensitivity for detection CIN2+ compared to cytology as triage but especially for the 25 to 34-year age group with over three years of follow-up (3.01; CI 1.82À5.17) without a significant change of colposcopy referral rate as part of the Italian NTCC HPV-based screening trial [START_REF] Carozzi | Use of p16-INK4A overexpression to increase the specificity of human papillomavirus testing: a nested substudy of the NTCC randomised controlled trial[END_REF]. This was also reported in a prospective US study of triage of 3,225 hrHPV positive women having co-testing as screening [START_REF] Wentzensen | Clinical evaluation of human papillomavirus screening with p16/Ki-67 dual stain triage in a large organized cervical cancer screening program[END_REF] meaning that short term retesting could be avoided [START_REF] Ronco | HPV16 and HPV18 genotyping in cervical cancer screening[END_REF][START_REF] Stoler | Approaches to triage optimization in HPV primary screening: extended genotyping and p16/ Ki-67 dual-stained cytologyretrospective insights from ATHENA[END_REF]. For triage of ASCUS and LSIL smears in cytology-based screening programmes, the sensitivity of p16 was equivalent to hrHPV triage but with a higher specificity [START_REF] Ridder R For The | The sensitivity and specificity of p16INK4a cytology vs HPV testing for detecting high-grade cervical disease in the triage of ASC-US and LSIL pap cytology results[END_REF].

Dual-staining with p16 and Ki-67, a proliferation marker, may also have a higher specificity compared to p16 alone [START_REF] Schmidt | Cytology Study Group. p16/Ki-67 dual-stain cytology in the triage of ASCUS and LSIL papanicolou cytology[END_REF]. Dualstaining had a sensitivity for CIN2+ 91.9% and a specificity of 82.1% in the Wolfsburg HPV-based screening trial, a study of 425 women older than 30 years of age who were hrHPV positive and cytology negative [START_REF] Petry | Triaging pap cytology negative, HPV positive cervical cancer screening results with p16/Ki-67 dual-stained cytology[END_REF]. The performance of dual-staining as triage was also investigated for hrHPV positive women in a longitudinal study of 1549 women with mean age of 42.2 years at enrolment. This study reported a significantly higher cumulative risk of CIN2+ (31.0 v 25.0%) if dual-stain positive and a significantly lower risk of CIN2+ (8.5 v 12.3%) if negative compared to cytology with a median follow-up of 3.7 years [START_REF] Clarke | Five-year risk of cervical precancer following p16/Ki-67 dual-stain triage of HPV-positive women[END_REF]. Alternatively, a longer retesting interval at three years is an option for negative dual-staining which was the same as the risk for negative cytology at one year, so potentially reducing referral to colposcopy. This provides reassurance following earlier concern [START_REF] Ronco | HPV16 and HPV18 genotyping in cervical cancer screening[END_REF] that negative dual-staining in the context of HPV infection can predict those at low risk of developing significant disease. Dual-staining may be a triage option in the HPV vaccinated population [START_REF] Wentzensen | Clinical evaluation of human papillomavirus screening with p16/Ki-67 dual stain triage in a large organized cervical cancer screening program[END_REF][START_REF] Stoler | Approaches to triage optimization in HPV primary screening: extended genotyping and p16/ Ki-67 dual-stained cytologyretrospective insights from ATHENA[END_REF].

Like cytology, dual-staining requires visual interpretation of the degree of staining which is dependent upon subjective assessment and may be merely the presence of a single dual-stained cell. This technique may vary depending on the precise method used and reproducibility is a key issue. Quality assurance of laboratory testing is needed as standardization of the reliability of testing is variable especially for p16 staining for low grade cytology [START_REF] Tsoumpou | p16INK4a immunostaining in cytological and histological specimens from the uterine cervix: a systematic review and meta-analysis[END_REF]. As opposed to immunohistochemistry which is traditionally used, insitu hybridisation for p16 measures gene copy numbers and may have greater accuracy and sensitivity with equivalent specificity compared to cytology for predicting CIN2+ for hrHPV positive women [START_REF] Zeng | The value of p16ink4a expression by fluorescence in situ hybridization in triage for high risk HPV positive in cervical cancer screening[END_REF]. Other techniques for standardisation of dual-staining have been described including cytotechnician training and automated slide reading [START_REF] Wentzensen | Clinical evaluation of human papillomavirus screening with p16/Ki-67 dual stain triage in a large organized cervical cancer screening program[END_REF][START_REF] Grabe | A virtual microscopy system to scan, evaluate and archive biomarker enhanced cervical cytology slides[END_REF][START_REF] Wentzensen | Interobserver reproducibility and accuracy of p16/Ki-67 dual-stain cytology in cervical cancer screening[END_REF].

HPV testing provides a long term risk of CIN2+ but dual-staining within the same cell provides identification of E6 and E7 mediated inactivation of tumour suppressor proteins and cell cycle deregulation relevant for immediate outcomes [START_REF] Ridder R For The | The sensitivity and specificity of p16INK4a cytology vs HPV testing for detecting high-grade cervical disease in the triage of ASC-US and LSIL pap cytology results[END_REF][START_REF] Peeters | Meta-analysis of the accuracy of p16 or p16/Ki-67 immunocytochemistry versus HPV testing for the detection of CIN2+/CIN3+ in triage of women with minor abnormal cytology[END_REF]. Dualstaining, like cytology is a subjective assessment but appears a reasonable option for triage of hrHPV positive women. Several small studies report improved sensitivity and specificity compared to triage with cytology (acceptable: ASCCP strength C; quality I).

Methylation testing

DNA methylation is an epigenetic mechanism for the control of gene expression for many cellular functions. Identification of specific sites may identify those that have an altered risk of HSIL+ as opposed to those that possess oncogenic virus. High methylation can equate with reduced risk of CIN3 and invasion at some sites [START_REF] Piyathilake | A higher degree of methylation of the HPV 16 E6 gene is associated with a lower likelihood of being diagnosed with cervical intraepithelial neoplasia[END_REF] but other sites confer a high risk via the nuclear gene promoters in the host or L1 and L2 genes in hrHPV [START_REF] Kelly | Performance of DNA methylation assays for detection of high-grade cervical intraepithelial neoplasia (CIN2+): a systematic review and meta-analysis[END_REF]. At present it is unclear if methylation of the viral genome or that of the host is more important but a combination of test sites may improve test accuracy [START_REF] Kelly | Performance of DNA methylation assays for detection of high-grade cervical intraepithelial neoplasia (CIN2+): a systematic review and meta-analysis[END_REF]. Methylation at certain sites usually of the L1 open reading frame generally identifies HPV infection with progression to CIN3+ [START_REF] Sun | Methylation of HPV16 genome CpG sites is associated with cervix precancer and cancer[END_REF][START_REF] Kottaridi | Quantitative measurement of L1 human papillomavirus type 16 methylation for the prediction of preinvasive and invasive cervical disease[END_REF] as well for apparent longer lasting CIN3.

Host microRNAs (miRs) with methylation are responsible for silencing target genes such as tumour suppressor activity. Kocsis et al. [START_REF] Kocsis | Performance of a new HPV and biomarker assay in the management of hrHPV positive women: subanalysis of the ongoing multicenter TRACE clinical trial (n > 6,000) to evaluate POU4F3 methylation as a potential biomarker of cervical precancer and cancer[END_REF] reported improved sensitivity at a similar specificity using POU4F3 as a host methylation marker to detect CIN3+ compared to cytology for triage of HPV positive samples. Aberrant methylation of the host gene site miR-124 provides a biomarker for cervical cancer [START_REF] Wilting | Methylation-mediated silencing and tumour suppressive function of hsa-miR-124 in cervical cancer[END_REF]. MAL/ miR-124-2 genes on hrHPV positive selfsamples had a sensitivity non-inferior to cytology triage for detection of CIN2+ [START_REF] Verhoef | Methylation marker analysis and HPV16/18 genotyping in high-risk HPV positive self-sampled specimens to identify women with high grade CIN or cervical cancer[END_REF]. A test kit identifying hyper-methylation at FAM19A4/ miR-124-2 is now commercially available. The high sensitivity recorded for pre-cancer detection is independent of genotype and appears to show a sensitivity and specificity for the detection of CIN2+ similar to that of HPV testing [START_REF] Rogeri | Methylation of the hsa-miR-124, SOX1, TERT, and LMX1Agenes as biomarkers for precursor lesions in cervical cancer[END_REF]. This study shown to be a prognostic marker to identify women with progressive CIN2 [START_REF] Louvanto | Methylation in predicting progression of untreated high-grade cervical intraepithelial neoplasia[END_REF]. Methylation testing can be used as a triage test for hrHPV positive women to identify CIN2+ at a sensitivity of 90% equivalent to the referral population to colposcopy amongst HPV positive women and abnormal cytology [START_REF] Brentnall | A DNA methylation classifier of cervical precancer based on human papillomavirus and human genes[END_REF]. A meta-analysis of pooled data from 43 studies reported that DNA methylation testing had a marginally lower relative sensitivity of 0.81 (CI: 0.63-1.04) but a higher specificity than cytology at a cut off of ASCUS+ (1.25 (0.99-1.59)) but a higher relative sensitivity of 1.22 (1.05-1.42) with equivalent specificity to HPV 16/18 genotyping (1.03 (0.94-1.13)) amongst hrHPV positive women [START_REF] Kelly | Performance of DNA methylation assays for detection of high-grade cervical intraepithelial neoplasia (CIN2+): a systematic review and meta-analysis[END_REF]. A lower sensitivity compared to cytology (at ASCUS + or LSIL+) in women under 30 years of age is possibly due to more regressive lesions in this age group. From the Dutch POBASCAM trial of HPV-based screening in 1,040 women with 14 years of follow-up, negative methylation testing with FAM19A4 and mir-124-2 genes provides a low cervical cancer risk in women over 30 years of age [START_REF] Strooper | Cervical cancer risk in HPV-positive women after a negative FAM19A4/mir124-2 methylation test: a post hoc analysis in the POBASCAM trial with 14 year follow-up[END_REF]. Overall, there are presently less studies evaluating methylation as a triage option when compared to other methods [START_REF] Kelly | Performance of DNA methylation assays for detection of high-grade cervical intraepithelial neoplasia (CIN2+): a systematic review and meta-analysis[END_REF]. The current body of evidence to support methylation is poor (acceptable: ASCCP strength C; quality I). Table 2 is a list of the cited reports of the performance of single triage tests.

Combined triage tests

Triage combinations may be more accurate than cytology alone for predicting HSIL+ in a triage setting. Sensitivity is not the priority here for women already hrHPV positive and considering referral to colposcopy due to the high sensitivity of HPV testing. Triage combinations may be more effective at providing reassurance for those at low risk by improving specificity. For instance, negative p16 and Ki-67 dual-stain with HPVO for hrHPV positive women could be retested in three years [START_REF] Wentzensen | Clinical evaluation of human papillomavirus screening with p16/Ki-67 dual stain triage in a large organized cervical cancer screening program[END_REF]. Various triage strategies were explored for 520 self-sampled hrHPV positive women from the Dutch PROHTECT-3B trial, and of 18 options considered, three were considered acceptable with an NPV for CIN3+ being at least 98%, combined with maintenance or improvement of sensitivity and an increase in specificity compared to triage with cytology alone [START_REF] Ebisch | The clinical value of HPV genotyping in triage of women with high-risk-HPV-positive self-samples[END_REF]. They were HPV16+ and/ or at least LSIL cytology; HPV16+ and/ or at least HSIL; HPV16+ and/ or HPV18+ and/ or at least HSIL. The second option resulted in a decrease in referral rate to colposcopy from 52.2-39.5%. HPV typing in addition to low grade or normal cytology allows delayed referral to colposcopy for women if HPVO and hrHPV positive.

Macdonald et al. [START_REF] Macdonald | Prevalence of high-grade cervical intraepithelial neoplasia in women with persistent high-risk HPV genotypes and negative cytology[END_REF] reported retrospective data from 1076 women who were hrHPV positive and cytology negative and having further triage with HPV 16/18 typing as part of the Sentinel Sites Pilot. Management depended upon typing with HPV 16/18 seen in colposcopy after first repeat if unchanged at 12 months and with HPVO seen in colposcopy if still unchanged 12 months later. Amongst the HPV 16 and 18 cases, 10% had CIN2+ when seen at one year and amongst the HPVO cases, four percent had CIN2+ at two years. Ten-year follow-up of cases from the ARTISTIC trial cohort also reported that one year repeat of testing was safe for HPV 16/18 positive women with low grade cytology and two-year follow-up was safe for HPVO genotypes [START_REF] Gilham | Triaging women with human papillomavirus infection and normal cytology or low-grade dyskaryosis: evidence from 10year follow up of the ARTISTIC trial cohort[END_REF].

The three-year risk of CIN3+ for all hrHPV positive women with ASCUS was 5.2% in women over 21 years of age [START_REF] Schiffman | A study of HPV typing for the management of HPV-positive ASC-US cervical cytologic results[END_REF], risk being highest for HPV type 16 (16.0%), 7.4% for HPV18; 7.1% for HPV33 or 58 and 7.0% for HPV31. The remaining 40% of hrHPV cases may not need immediate referral for colposcopy but would encompass 10% of all CIN3+ cases. HPVO cases would be recommended for short term recall to avoid missing a subpopulation of non 16/18 CIN2+ cases. Similar outcomes were seen for those over 25 and over 30 years of age [START_REF] Schiffman | A study of HPV typing for the management of HPV-positive ASC-US cervical cytologic results[END_REF]. However, in the US, women who are over 30 years with an hrHPV positive test and are cytology negative with positive HPV 16/18 typing have a PPV of 11.4 % for CIN2+ (9.8 % for CIN3+) with immediate referral to colposcopy recommended [START_REF] Massad | updated consensus guidelines for the management of abnormal cervical cancer screening tests and cancer precursors[END_REF]. Retesting is suggested in 12 months for those who were HPVO from the ATHENA study [START_REF] Wright | Evaluation of HPV-16 and HPV-18 genotyping for the triage of women with high-risk HPV+ cytology-negative results[END_REF]. A slight variation of this algorithm was suggested from the Kaiser Permanente screening programme cotesting with cytology and HPV-based screening, those with ASCUS and are HPV 16/18 positive could be referred for immediate colposcopy and types 31/33/58 may also benefit from this. Other hrHPV types could be delayed for retesting at 12 months [START_REF] Schiffman | A study of HPV typing for the management of HPV-positive ASC-US cervical cytologic results[END_REF].

Wentzensen et al. [START_REF] Wentzensen | Clinical evaluation of human papillomavirus screening with p16/Ki-67 dual stain triage in a large organized cervical cancer screening program[END_REF] compared the performance of triage combinations of HPV 16/18 typing and cytology (at ASCUS+), typing and dual-staining, but also single triage options of cytology alone and dual-staining alone in a prospective study of hrHPV positive women. Both the combination triage strategies were superior to the single triage tests. Dual-staining was significantly superior to cytology when both were combined with HPV typing (p=0.005), reduced unnecessary colposcopy referral by 16.1% and so reducing unnecessary biopsies and treatments.

Combined triage by MAL/miR-124-2 methylation analysis with HPV16/18 typing as a sub-study of the PROHTECT-3 trial had a sensitivity equivalent to methylation alone of 77.6% but with an increased specificity of 54.8 versus 47.2% (p=0.001) compared to methylation alone to identify women with CIN3+ among selfsampled HPV positive women. The referral rate to colposcopy was also 5.8% lower for the triage combination (p=0.03) [START_REF] Verhoef | Methylation marker analysis and HPV16/18 genotyping in high-risk HPV positive self-sampled specimens to identify women with high grade CIN or cervical cancer[END_REF]. Furthermore, this combined strategy may be objective as it is fully molecular [START_REF] Kelly | Performance of DNA methylation assays for detection of high-grade cervical intraepithelial neoplasia (CIN2+): a systematic review and meta-analysis[END_REF][START_REF] Verhoef | Methylation marker analysis and HPV16/18 genotyping in high-risk HPV positive self-sampled specimens to identify women with high grade CIN or cervical cancer[END_REF]. Automatable cytology reading is also feasible using FocalPoint (BD Burlington, NC) with partial genotyping and may be comparable to standard cytology and genotyping reported in 1839 women who were HPV positive [START_REF] Yu | Automated cervical screening and triage, based on HPV testing and computerinterpreted cytology[END_REF]. See Table 3 for the performance of triage test combinations.

Apart from the triage tools already discussed, further important modifiers of cervical cancer risk can be included to develop a personal risk assessment. For instance, screening history, HPV vaccination status and nicotine use can be incorporated with current screening status as part of prediction modelling to triage to early repeat testing, referral to colposcopy or biopsy based on risk. Five clinical responses to screening and triage have been described by Schiffman et al. [START_REF] Schiffman | A study of HPV typing for the management of HPV-positive ASC-US cervical cytologic results[END_REF] with treatment for extremely high risk of CIN3+, colposcopic biopsy for substantial risk, intensified retesting with HPV and no pre-cancer, routine screening if no HPV and finally, exit from screening at virtually no risk. A risk-based management algorithm can be devised according to the level of risk [START_REF] Wentzensen | ASCCP colposcopy standards: risk-based colposcopy practice[END_REF] and an example of colposcopic management according to combined triage tests is shown in Fig. 1.

A threshold for colposcopy has been suggested in the US at 11.1% as equivalent to the one-year risk of CIN2+ with an hrHPV positive test and ASCUS cytology and that for one-year retesting as 6.2% as equivalent to the one-year risk of CIN2+ with an hrHPV positive test and negative cytology [START_REF] Clarke | Five-year risk of cervical precancer following p16/Ki-67 dual-stain triage of HPV-positive women[END_REF]. Such thresholds appear to vary as a recommended threshold for colposcopy in Europe may be a 10% risk of CIN3+ [START_REF] Arbyn | Genotyping for HPV16 and HPV18 in women with minor cervical lesions: a systematic review and meta-analysis[END_REF]. Immediate testing with a combined triage can be designed so that both tests are offered simultaneously and appears preferable to sequential testing to reduce anxiety and avoid default of a second attendance [START_REF] Leeson | The future role for colposcopy in Europe[END_REF]. Combinations of triage can be evaluated with a neural network and can still be compared if datasets are incomplete as triage outcomes can be concluded as a 'best effort basis' [START_REF] Kyrgiou | Personalised management of women with cervical abnormalities using a clinical decision support scoring system[END_REF][START_REF] Karakitsos | Identification of women for referral to colposcopy by neural networks: a preliminary study based on LBC and molecular markers[END_REF]. In a study of 2267 women prospectively assessed with cytology, DNA HPV typing, HPV RNA and dualstaining compared to cytology alone in a screening setting, a neural network developed by the research group and incorporating all the listed triage options had a higher accuracy in predicting CIN2+. All had colposcopy but biopsy was not performed in every case [START_REF] Kyrgiou | Personalised management of women with cervical abnormalities using a clinical decision support scoring system[END_REF]. A more complex test structure combining test outcomes with clinical parameters may provide an improved risk of cervical pre-cancer. A personalized risk-based approach to predict present or future CIN3 + based upon current test results, screening history and previous treatment is a new addition for the 2019 Consensus Guidelines from the ASCCP [START_REF] Perkins | ASCCP risk-based management consensus guidelines for abnormal cervical cancer screening tests and cancer precursors[END_REF].

Cost-effectiveness of various options

Combinations that reduce colposcopy referral yet maintain a high PPV for HSIL/ CIN2+ and a high specificity to provide strong reassurance with negative triage testing would appear to be potentially the best use of healthcare resources. Triage with HPV 16/18 typing and dual-staining with p16 and Ki-67 in the context of an HPV-based screening programme appears cost effective compared to cytology-based screening in Belgium [START_REF] Tjalma | The impact on women's health and the cervical cancer screening budget of primary HPV screening with dual-stain cytology triage in Belgium[END_REF] and in Thailand [START_REF] Termrungruanglert | Cost effectiveness analysis of HPV primary screening and dual stain cytology triage compared with cervical cytology[END_REF]. In a further study HPV 16/18 typing with dualstaining and cytology appeared ideal in comparison to three alternatives including cytology as triage and also with co-testing and triage with partial genotyping and dual-staining in Mexico [START_REF] Beal | Cost analysis of different cervical cancer screening strategies in Mexico[END_REF]. Yet co-testing with HPV 16/18 typing as triage appeared most cost effective of six options in the US with the lowest incremental cost-effectiveness ratio per quality-adjusted life year saved [START_REF] Vijayaraghavan | Costeffectiveness of using human papillomavirus 16/18 genotype triage in cervical cancer screening[END_REF]. HPVO and LSIL cytology for HPV positive women appears most cost effective if re-tested in 12 months rather than with a direct referral DNA methylation as a triage test has higher specificity than cytology (ASCUS+) and higher sensitivity than genotyping.

[44] Cytology, genotyping, p16/Ki-67

Prospective, observational study.

3,225 HPV +ve women.

Combinations better at predicting risk of CIN3+ than single triage and p16/Ki-67 combined with genotyping better than cytology with genotyping.

Women -ve for genotyping and dual-staining can be re-tested in 3 yrs.

[ to colposcopy [START_REF] Simms | Optimal management strategies for primary HPV testing for cervical screening: costeffectiveness evaluation for the national cervical screening program in Australia[END_REF] for the national screening programme in Australia. Cost effectiveness calculations would vary for differing populations depending on local HPV vaccination uptake, local care costs and what would be considered a suitable cost-effectiveness threshold per year of life saved for that population. Overall, there is a lack of consistent data to clarify cost effectiveness for triage strategies. Despite these caveats, a contemporary cost benefit analysis of various triage options within a national HPV-based screening programme and additional calculations for the impact of HPV vaccination would be a welcome addition to current published clinical data. See Table 4 for details of the reported cost effectiveness studies. 12-month follow-up for women with HPVO and low-grade cytology associated with low risk of developing cervical cancer.

Abbreviations: ICER -incremental cost-effectiveness ratio, QALY -quality-adjusted life year.

Implications for colposcopy

After a positive screen, a single positive triage prompts referral to colposcopy, and those negative to early recall; with an option of colposcopy if HPV positivity persists for at least 12 months. Colposcopy appears to perform well when the referral group is accurately selected on basis of risk of underlying HSIL. With a low rate of disease, the risk is of over-investigating and treating women with all the associated adverse effects. However, it is difficult to assess the comparative performance of cytology, partial genotyping, dual-staining and methylation as triage options as no published studies have compared all four options. The sensitivity for CIN3+ may be better for partial genotyping than for cytology [START_REF] Castle | Performance of carcinogenic human papillomavirus (HPV) testing and HPV16 or HPV18 genotyping for cervical cancer screening of women aged 25 years and older: a subanalysis of the ATHENA study[END_REF]. The five-year cumulative risk for CIN2+ and CIN3+ may be higher for dual-stain positive cases than for cytology (threshold ASCUS+) and lower if dual-stain negative than for negative cytology [START_REF] Clarke | Five-year risk of cervical precancer following p16/Ki-67 dual-stain triage of HPV-positive women[END_REF]. Methylation may have a lower sensitivity for CIN2 + than cytology (ASCUS+) but a higher sensitivity than partial genotyping [START_REF] Kelly | Performance of DNA methylation assays for detection of high-grade cervical intraepithelial neoplasia (CIN2+): a systematic review and meta-analysis[END_REF].

Combination triages can be performed sequentially and would recommend short term recall if the first and second triages are negative (see first example in Fig. 2). Assuming the triages are performed sequentially and assuming the first triage is partial genotyping, then HPV 16/18 positive women would be referred to colposcopy. HPVO cases would be offered a second triage with cytology [START_REF] Huh | Use of primary high-risk human papillomavirus testing for cervical cancer screening: interim clinical guidance[END_REF], dual-staining or methylation. Colposcopy would be recommended also if the second triage is positive. But for cytology as the second triage, the grade may be relevant with colposcopy recommended for ASCUS+, LSIL+ or for HSIL+. Alternatively, tests can be performed simultaneously, for example, with the first triage as partial genotyping being HPV 16 or 18 positive; the second triage can be negative leading to short term recall rather than referral to colposcopy (see second example in Fig. 2). If either partial genotyping or cytology is positive then sensitivity is improved compared to LSIL+ only for triage [START_REF] Castle | Performance of carcinogenic human papillomavirus (HPV) testing and HPV16 or HPV18 genotyping for cervical cancer screening of women aged 25 years and older: a subanalysis of the ATHENA study[END_REF] as two triage tests are used instead of one. The risk of CIN2+ has been reported as greater than 20% for HPV 16/18 positive and with dual-stain or cytology positive as the second triage [START_REF] Wentzensen | Clinical evaluation of human papillomavirus screening with p16/Ki-67 dual stain triage in a large organized cervical cancer screening program[END_REF] and the combination of cytology (ASCUS+) and dual-stain positive cases [START_REF] Clarke | Five-year risk of cervical precancer following p16/Ki-67 dual-stain triage of HPV-positive women[END_REF]. Specificity was improved with an equivalent sensitivity for combined partial genotyping and methylation [START_REF] Verhoef | Methylation marker analysis and HPV16/18 genotyping in high-risk HPV positive self-sampled specimens to identify women with high grade CIN or cervical cancer[END_REF].

A sequential combined triage would refer more cases to colposcopy than a single triage and simultaneous combined triage (see Fig. 2). Simultaneous combined triage requires more tests so is more expensive but may reduce colposcopy referrals and so may be cost effective. Overall a simultaneous combined triage algorithm with any two of cytology, partial genotyping or dualstaining is preferred. Combined triage options would seem ideal from the available evidence as they have improved performance compared to single triage tests (preferred: ASCCP strength A; quality I).

Conclusions

We have explored different options for triage of HPV positive women in the context of a primary HPV-based screening programme. With HPV-based screening compared to cytology-based screening more women will be referred with only HPV infection or LSIL. A lower prevalence of HSIL+/CIN2+ in the low grade group will result in a drop in the PPV for the colposcopic impression of high grade lesions with the risk of over investigation of women. Effective triage should improve selection of women at high risk of HSIL+/CIN2 + to colposcopy thereby maintaining colposcopy performance and avoidance of excessive referral rates. Data will be needed from vaccinated populations to guide practice which is expected to alter with an anticipated reduced incidence of HPV types 16, 18 and possibly other oncogenic genotypes over several years. Triage combinations appear more accurate than algorithms using a single triage test. The ideal option or combination of options is likely to differ for different national programmes. This will be dependent upon national HPV prevalence and relative importance of high risk HPV types, particularly of types 16 and 18 with reference to differing national vaccination uptake rates, as well as the referral rate to colposcopy with a positive triage. Quality assurance of screening programmes, including colposcopy and laboratory services need to guide and validate programme change. Cost effectiveness for the local population as well as acceptability of triage algorithms for colposcopists are also important considerations. Our conclusions are consensus opinion only because, except for a few studies, the reported outcomes are difficult to compare as they differ in terms of risk of high grade cervical disease, cost effectiveness, or other outcomes. This highlights a need to benchmark various HPV positive outcomes within each cervical screening programme to use as a template to modify triage schedules and guide future practice.

Patient consent for publication

Not required.

  Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333 Options for triage of HPV positive cases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333 Cytology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333 HPV 16/18 typing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334 p16/Ki-67 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335 Methylation testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335 Combined triage tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337 Cost-effectiveness of various options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338 Implications for colposcopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340 Patient consent for publication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340

Fig. 1 .

 1 Fig.1. Risk of CIN2+ combining colposcopic opinion, HPV partial genotyping and cytology triage for HPV + ve women. Abbreviation: TZ -transformation zone. Modified with permission from Wentzensen et al.[START_REF] Wentzensen | ASCCP colposcopy standards: risk-based colposcopy practice[END_REF].

Fig. 2 .

 2 Fig. 2. Examples of triage test combinations. *If cytology is triage b, management may be graded: e.g. colposcopy for ASCUS+ with early recall for NILM or alternatively colposcopy for HSIL+ with early recall for LSIL. For other methods of triage then triage b +ve are all referred to colposcopy. Abbreviation: NILM -negative for intraepithelial lesion or malignancy.

Table 1

 1 American Society for Colposcopy and Cervical Pathology rating of published evidence.

  of age as reported in a Finnish population-based cohort study of 1279 women referred to colposcopy for abnormal cytology. Other hrHPV types than HPV16/18 (HPVO) accounted for 19.3% (11/ 57) of CIN3+ in women under 30 years and 44.7%(17/38) in women over 45 years of age.

Perkins RB, Guido RS, Castle PE, Chelmow D, Einstein MH, Garcia F et al. 2019 ASCCP risk-based management consensus guidelines for abnormal cervical cancer screening tests and cancer precursors. J Low Genit Tract Dis 2020; 24: 102-131. years

Table 2

 2 Triage studies reporting single triage methods in this review.

	Author	Triage type	Design	Study size	Findings	Conclusion
	[14]	Cytology	Prospective	58,076 women with	76 CIN3+ in HPV arm, 53 cases CIN3+ in	Cytology as triage with HPV
			population-based	95,600yrs follow-up in	cytology arm.	screening better than cytology-
			screening data.	HPV arm; 95,700yrs		based screening.
				follow-up in cytology
				arm.
	[20]	Cytology	>30 yr HPV, cytology	580,289 women.	36.4% of HPV+ve tests had ASCUS+	Acceptable colposcopy referral rate
			co-testing.		cytology.	for co-testing.
	[22]	Cytology	Observational.	578,547 women.	2015 HPV+ve/ cytology+ve @ 12 months &	HPV+ve/ cytology-ve represent 54%
					2186 HPV+ ve/ cytology-ve @ 24 months.	of extra colposcopy referrals over
						2yrs.
	[24]	Cytology	Retrospective,	46,694 women.	Rate of persistent hrHPV+ve was 58%	Only small number of high-grade
			longitudinal		(830/1,435).	lesions were detected among the
					A high-grade lesion was detected in 42	group of hrHPV+ve/ cytology-ve
					(5.1%) at 1-yr repeat.	women who repeated testing 1-yr
	[27]	Cytology,	30À60yr retrospective.	25,658 women.	Cytology most efficient triage of 9	Lowest colposcopy referral rate
		genotyping			strategies with repeat testing with 33.4%	with cytology triage and repeat
					colposcopy referral rate & NPV 98.9%.	cytology @12 months.
	[34]	Cytology	35À64 yr,	72,869 women.	Proportion of HPV+ women referred to	Repeat HPV testing limits
			retrospective.		colposcopy varied across programmes	subjectivity of cytology
					(20À57 %; average 37 %) and so did CIN2+	interpretation on overall
					detection (49À94 %; average 77%).	colposcopy referral and sensitivity.
	[36]	Genotyping	HPV+ ve, cytology-ve,	4,219 HPV+ve & 886	Prevalence CIN2 + 11.4% HPV 16/18, 6.1%	Genotyping useful for cytology -ve
			>30yr.	HPV-ve women.	HPV + ve, 0.8% if HPV-ve.	women.
	[28]	Genotyping	>25yr. Prospective sub-	41,955 women.	Cobas HPV test more sensitive than LBC	Improved sensitivity to detect CIN3
		vs cytology	study of ATHENA.		cytology for detection of CIN3+ (92.0% vs	+ compared to cytology (p <
					53.3%).	0.0001).
	[37]	Genotyping	20À29yr prospective.	10,758 women.	Increased risk CIN2+ if persistent same	Risk of CIN2+ highest with same
					hrHPV type (OR 813; CI 168.2-3,229.2).	HPV type on repeat testing.
	[43]	p16 vs cytology	Randomised controlled	1,137 HPV+ve women.	Improved relative sensitivity for p16 to	Improved sensitivity as triage with
			trial.		detect CIN2+ 3.01 (CI 1.82À5.17).	p16 compared to cytology.
	[46]	p16 vs	Retrospective, triage of	810 slides.	Specificity better for p16 compared to	p16 sensitivity = to HPV testing
		genotyping	ASCUS & LSIL.		HPV for ASCUS or LSIL (both p <.001).	with improved specificity. Efficient
						for triage of ASCUS and LSIL.
	[47]	p16/Ki-67	Retrospective, triage of	776 women.	Dual-staining a similar sensitivity but	Dual-staining improved specificity
			ASCUS & LSIL.		higher specificity compared to hrHPV	to p16 alone.
					testing.
	[48]	p16/Ki-67	HPV+ve, cytology-ve,	425 women.	Dual-staining sensitivity for CIN2+ 91.9%	Dual-staining useful triage for HPV
			>30yr prospective.		and specificity of 82.1%.	+ve, cytology-ve cases.
	[49]	p16/Ki-67	Prospective cohort,	1549 HPV+ve women.	Higher 5 yr cumulative risk of CIN2+ (31.0	Risk of CIN2+ in dual-stain -ve
		vs cytology	HPV/ cytology co-		v 25.0%) if dual-stain +ve and significantly	means that recall can be @ 3yrs.
			testing >30yr.		lower risk of CIN2+ (8.5 v 12.3%) if Àve
					compared to abnormal cytology.
	[57]	Methylation	Prospective, 21À67yr.	145 women.	L1 HPV16 mean methylation significantly	Elevated methylation level
			HPV16+ve.		increased with disease severity (CIN3,	associated with increased disease
					17.9% vs CIN2, 11.6%, p <.001 or vs CIN1,	severity and has good ability to
					9.0 %, p <.001).	discriminate HPV16+ve women
						that have CIN3+.
	[58]	Methylation	Prospective, 25-65	5,384 samples from	For CIN3+ histological endpoint	Quantitative triage tool for
			years.	HPV+ve women.	methylation test of POU4F3 achieved	identification of CIN3+.
					relative sensitivities of 1.74 (95% CI:
					1.25À2.33) and 1.64 (1.08À2.27),
					respectively, after verification bias
					adjustment.
	[61]	Methylation	Retrospective	447 cytology samples.	hsa-miR-124-2 had best performance of 6	Sensitivity/ specificity for detection
					candidate methylation genes for	of CIN2+ similar to HPV testing.
					identifying CIN2+ (OR 5.1).	Performance independent of
						genotype.
	[62]	Methylation	Prospective cohort.	149 women with	Highest area under the curve (AUC) was	S5-classifier shows high potential
				untreated CIN2.	0.735 (95% CI 0.621À0.849) in regression	to identify women with progressive
					vs. progression outcome with	CIN2.
					combination of S5 and cytology. HPV16/
					18 or HPV16/18/31/33 genotyping did not
					provide additional prognostic
					information.
	[63]	Methylation	Retrospective HPV+ve,	1,493 women.	Methylation of viral and host genes may	Methylation testing can be used as a
			HG or persistent LG		improve PPV.	triage test for HPV+ve to identify
			cytology.			CIN2+.
	[64]	Methylation	Retrospective,	1,040 women,	Kaplan-Meier estimate of 14-year	Negative FAM19A4/mir-124-2
			longitudinal.	29À61yrs.	cumulative cervical cancer incidence 1.7%	methylation test provides low
					(95 % CI: 0.66À3.0) among baseline	cervical cancer risk in HPV+ve
					methylation -ve and 2.4% (1.4À3.6)	women of !30 yrs.
					among baseline cytology-ve women (risk
					difference: 0.71% [0.16À1.4]).
	Abbreviations: yrs -years; NPV -negative predictive value.

Table 3

 3 Triage studies reporting triage combinations in this review.

	Author	Triage type	Design	Study size Findings	Conclusion
	[60]	Genotyping,	Sub-study of PROHTECT-3	1019 HPV	Triage of methylation & genotyping sensitivity	Methylation testing can be used as triage test for
		methylation	trial of self-sampled HPV	+ ve	equivalent to methylation alone of 77.6% with	HPV+ve with genotyping to identify cervical
			tests.	women	specificity of 54.8 vs 47.2% (p=0.001) compared to	precancer.
				33À63yrs.	methylation alone for CIN3+. Referral rate to
					colposcopy 5.8% lower for triage combination
					(p=0.03).
	[55]	Methylation	Meta-analysis 43 studies	16,336	DNA methylation testing lower relative
		vs cytology	including 8 host and 2 viral	women.	sensitivity of 0.81 (CI: 0.63-1.04); higher
		vs	genes.		specificity than cytology at cut off ASCUS (1.25
		genotyping			(CI: 0.99-1.59)) or LSIL; higher relative sensitivity
					of 1.22 (CI: 1.05-1.42) with equivalent specificity
					to genotyping (1.03 (CI: 0.94-1.13)).

Table 4

 4 Triage studies reporting cost effectiveness in this review.

	Author	Triage type	Design	Study	Findings	Conclusion
	[76]	Genotyping,	25À65yrs	Cytology-based vs HPV-based screening.	After 2 cycles of screening 21% costing	Combination appears cost
		p16/Ki-67 in	Budget-based		saving due to drop of prevalence of CIN.	effective.
		HPV arm	impact model.
	[77]	Genotyping,	30À65 yrs	Cytology-based vs HPV-based screening.	Comparison with cytology and HPV-	Combination with dual-staining
		p16/Ki-67 in	Budget-based		based screening with genotyping and	and genotyping appears more
		HPV arm	impact model.		dual-staining.	cost effective than cytology-
						based screening.
	[78]	Genotyping,	Calculation of	Cytology vs HPV as primary screening with	Triage with genotyping, cytology with	Combining multiple tests may be
		cytology,	total cost of each	cytology triage vs HPV as primary screening	p16/Ki67 best alternative because is	cost-effective.
		p16/Ki67	option to treat	with genotyping, cytology and p16/Ki67	least expensive with acceptable
			each case of CIN2	triage vs co-testing with hrHPV and cytology	number of missed cases.
			+.	with genotyping and p16/Ki67 triage.
	[79]	Genotyping,	Lifetime model	Cytology vs cytology + HPV triage vs HPV	HPV genotyping with co-testing most	Addition of HPV-16 and -18
		cytology	for 6 screening	cytology triage vs co-testing vs co-testing +	effective strategy with ICER of $33,807/	genotype triage to co-testing was
			strategies.	HPV genotyping, vs HPV only + HPV	QALY compared to HPV genotyping for	cost-effective.
				genotyping.	all hrHPV-positive women.
	[80]	Cytology,	Dynamic model.	Referral to colposcopy vs 12-month follow-up	Immediate colposcopy referral
		genotyping		for HPVO and low grade cytology.	increases the number of colposcopies/
					cancer.

Acknowledgements

Pamela Jones, Library Services at Betsi Cadwaladr, University Health Board, Charles Redman, European Federation for Colposcopy, Rue de la Presse, 4, 1000 Brussels, Belgium.

Data sharing

Data available from corresponding author upon request.

Author contributions

SL devised and wrote the draft manuscript, corresponding author. RA, NS, PN, MS, XC, CB reviewed and modified the draft text.

Source of support grant

None.

Declaration of Competing Interest

None.


Antoine Netter

Raphael Niddam

Aubert Agostini

Patrice Crochet
email: patrice.crochet@ap-hm.fr

Vaginal NOTES approach for risk-reducing salpingo-oophorectomy in BRCA mutation carriers: A video demonstration

Keywords:

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

International guidelines state that women with BRCA1/2 mutations should be offered risk-reducing bilateral salpingo-oophorectomy (BSO) by the age of 40 years or when childbearing is complete [1]. The current gold standard approach is laparoscopy. Transvaginal Natural Orifice Transluminal Endoscopic Surgery (vNOTES) is a technique recently described in gynecology, either for adnexal or hysterectomy procedures [START_REF] Li | Transvaginal natural orifice transluminal endoscopic surgery (vNOTES) in gynecologic surgeries: a systematic review[END_REF]. The vNOTES approach allows access to the peritoneal cavity through the vagina without skin incisions.

This paper aims to present the vNOTES technique as an alternative for risk-reducing BSO and includes a step-by-step video demonstration of the entire procedure for educational purposes [START_REF] Celentano | Development and validation of a recommended checklist for assessment of surgical videos quality: the LAParoscopic surgery Video Educational GuidelineS (LAP-VEGaS) video assessment tool[END_REF].

Case presentation

The patient selected was a fifty two years old woman, gravida 2 para 2, and a carrier of the BRCA2 mutation. Her past-surgical history was unremarkable. Her ASA score was 1 and her body mass index was 27. According to French recommendations, she underwent a preoperative evaluation via pelvic ultrasound and CA-125 biomarker dosage [START_REF] Baekelandt | Adnexectomy by vaginal natural orifice transluminal endoscopic Surgery versus laparoscopy: results of a first randomised controlled trial (NOTABLE trial)[END_REF]. The operative time was 55 min. Four hours after surgery, the patient had a VAS pain score of 0. She described "absolutely no pain" from the recovery room onwards. She was discharged at day one after surgery and the post-operative course was uneventful. The histology report on the specimen was benign. The patient gave her consent to the publication of anonymized video footage of the procedure.

Surgical technique

Operative settings

This procedure is performed under general anesthesia. The patient is placed in a lithotomy position with buttocks slightly over the edge of the operating table. The patient's abdominal wall and vagina are disinfected before a Foley catheter is placed. The patient is draped as for a classic vaginal approach. Additionally, classic drapes are set on the abdomen, in case of conversion to conventional laparoscopy or laparotomy during the procedure. The surgeon is situated between the legs of the patient as for a classic vaginal approach. The laparoscopic tower is placed on the right side of the patient to provide a screen in front of the surgeon over the abdomen of the patient. The surgeon has two assistants. During the laparoscopic part of the procedure, the first assistant is placed on the right side of the surgeon, holding the camera. The second assistant stands on the left side of the patient to provide the surgeon with laparoscopic instruments. An antibiotic prophylaxis is administered intravenously.

The four steps of the procedure

The four consecutive steps of the procedure will be presented; these are the posterior colpotomy, the placement of the vNOTES port, the adnexectomies, and vaginal closure.

1. The posterior colpotomy. The colpotomy is performed using a classic vaginal approach. The cervix is exposed using an anterior and posterior vaginal retractor. The cervix is grasped by two Pozzi forceps and pulled upwards to better expose the colpotomy site. Before starting the incision, a 10 mL solution of ropivacaine (20 mg) + adrenaline (0.1 mg) is injected into the posterior fornix to facilitate the dissection, to decrease intraoperative hemorrhage and to reduce postoperative pain. A 3 cm colpotomy is performed using a cold steel scalpel 2 mm posterior to the cervical margin, between the 4-5 o'clock and 7-8 o'clock position. The peritoneum of the Douglas cul-de-sac is subsequently cut to access the peritoneal cavity. The opening is large enough to allow the insertion of the vNOTES port when an index finger can be easily inserted. 2. The introduction of the vNOTES port into the peritoneal cavity.

The vNOTES port used was the GelPOINT Ò Advanced Access Platform (Applied Medical, Rancho Santa Margarita, CA, USA). It has three components: a GelSeal cap, three trocars individually placed in the GelSeal cap, and an Alexis Ò wound retractor. The Alexis Ò wound retractor has two 7-cm diameter rings: a purple-colored inner ring to be placed inside the abdominal cavity beyond the posterior fornix, and a white-colored outer ring, which adheres to the vaginal entrance. A fourth device is an inserter that helps with folding the inner ring of the Alexis Ò retractor and inserting it through the colpotomy incision into the peritoneal cavity.

To facilitate the insertion of the inner ring inside the abdominal cavity, the posterior fornix is exposed using a 4-cm wide vaginal retractor, allowing the placement of a 2-cm wide vaginal retractor into the colpotomy. Then, the inserter is pushed upwards at a 45°a ngle and the inner ring can be deployed in the abdominal cavity. We recommend sweeping the inner ring with a finger to ensure that the ring is appropriately placed in the abdominal cavity and that no tissue is trapped between the and the Douglas culde-sac. The Alexis Ò retractor is moderately tensioned by winding the outer ring two turns. The three trocars are positioned on the GelSeal cap as laterally as possible to benefit from maximum triangulation and to preserve the surgical ergonomics. The GelSeal cap is then fixed on the outer ring of the Alexis Ò retractor and the sealing valve is closed. CO2 gas insufflation starts at a 8 -10 mmHg pressure while the patient is positioned in a 15°to 20°Trendelenburg. The 0°camera in inserted in the trocars at 6 h, and the adnexectomies step can start. 3. The adnexectomies. Before starting adnexectomies, exploration of the abdominal cavity should be undertaken by moving the camera above the pelvic area. Each adnexectomy is composed of 3 consecutives sub-steps: i) the utero-ovarian ligament division, ii) the salpingotomy, iii) the infundibulopelvic ligament division.

The camera looks at the adnexa from below. Consequently, visualization of anatomical landmarks is specific to this vNOTES approach and driven by gravity. One instrument holds the uterine fundus upwards to facilitate exposure. Before starting the adnexectomy, the adnexa is mobilized to identify the three anatomical landmarks: the utero-ovarian ligament, the proximal origin of the fallopian tube, and the infudibulopelvic ligament. Additionally, the ureter must be visualized. A bipolar electrosurgery device is used to coagulate and cut the utero-ovarian ligament and the proximal origin of the fallopian tube successively. Then, the ovary is grasped and rolled around the grasper. This grasper is positioned inwards and holds the uterine fundus upwards; this maneuver creates enough space for the infudibulopelvic ligament to be safely coagulated by the bipolar energy instrument, away from the ureter path.

A glove filed with gas can be placed in the peritoneal cavity over the adnexa to protect the digestive tract from the coagulation device. An example is shown in the video on the left side of the patient. The surgical specimens are extracted by opening the Gel-POINT platform and then sampled for final histological examination. The absence of bleeding is checked and the GelPOINT Ò platform is then removed. 4. Vaginal closure. Vaginal retractors are placed in the vagina, and the Pozzi forceps are again positioned on the posterior lip of the cervix to facilitate exposure. The vagina is closed with a running suture using an absorbable 0-type monofilament thread. Attention must be paid to including the peritoneum in the suture to avoid any intra-peritoneal bleeding into the cavity. The vaginal retractors and the Foley catheter are then removed.

Discussion

vNOTES is an innovative, minimally invasive technique that limits surgical trauma and potentially reduces postoperative pain. Thus, vNOTES may be a desirable approach to BSO then compared to conventional laparoscopy [5]. This technique undeniably provides a new option for the gynecological surgeon, as BSO without hysterectomy cannot be safely performed using a classic vaginal approach.

This presentation illustrates that the technique requires both vaginal surgical skills (i.e., colpotomy, vaginal closure) and laparoscopic skills specific to vNOTES (i.e., a view of the pelvic anatomy from below, ergonomic constraints inherent to the monotrocar vNOTES port). A prerequisite is good access to the peritoneal cavity. Indeed, the procedure must be avoided in patients with cul-de-sac disease such as endometriosis or suspected adhesions [START_REF] Kapurubandara | Consensus on safe implementation of vaginal natural orifice transluminal endoscopic surgery (vNOTES)[END_REF].

Risk-reducing BSO usually presents as a simple case because normal anatomy is preserved. A potential drawback of this technique is that the peritoneal cavity cannot be fully explored using a classical 0°e ndoscope, especially around the prevesical peritoneum. The rate of occult ovarian cancer identified following risk-reducing BSO is estimated at 2.5% [START_REF] Domchek | Occult ovarian cancers identified at risk-reducing salpingo-oophorectomy in a prospective cohort of BRCA1/2 mutation carriers[END_REF]. Thus, the risk of undiagnosed peritoneal cancer due to the vNOTES approach is likely to be very low.

Conclusion

The vNOTES technique appears to be particularly interesting in the context of prophylactic surgery for risk-reducing BSO. We provide a step-by-step description of the technique and illustrate its feasibility. This technique needs to be evaluated and compared to the conventional laparoscopic approach.

Acknowledgment

We thank the Karl Storz society for kindly lending us the VITOM 90°camera to take the video footage of the colpotomy and vaginal closure.

Funding

This study received no funding.

Declaration of Competing Interest

The authors declare no conflict of interest.

Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.jogoh.2021.102212.