Title: Extended cytological follow-up and prospective collection of cervical samples for HPV testing in ARTISTIC Trial cohort
Funder: Department of Health and Social Care

Abstract: Existing data and proposed follow-up In the ARTISTIC Trial 24,510 women attending for routine cervical cytology in Greater Manchester in 2001-2003 were recruited. Their LBC samples at entry and at rounds 2 and 3 were tested for HPV and the residual LBC samples were stored. The women were randomly allocated between revealing or concealing their HPV test results. During the trial women were recalled 3-yearly and histology results were obtained from local laboratories. After 2009 histological follow-up and sample collection ended and the women returned to routine cytological screening with recall 3-yearly below age 50 and 5-yearly at age 50-64. We now propose to extend follow-up to 2021 by obtaining histology results from local laboratories from 2009 to 2021. These three labs (Manchester, Stockport and Pennine) process ~88% of current (2016) cytology and histology in the cohort. The whole trial cohort is followed up for CIN3 and cancer through linkage to national cancer registration, but CIN2, which is not registered and constitutes approximately half of all high-grade histology, is managed clinically in the same way as CIN3. We shall also have lifetime cytology records for the whole cohort by linkage to the cervical screening call-recall system. This extension to 2021 will provide 10-year follow-up for high-grade CIN from the second HPV test in the trial, when persistent and newly acquired HPV infection can be distinguished and women with two consecutive negative tests can be identified. This will provide a framework for estimating the delay in diagnosing CIN2 and CIN3+ under different HPV testing and triage policies in primary screening. Options include extending the screening interval up to 10 years for HPV negative women and delaying recall by up to 5 years for HPV positive women with normal cytology whose infections are usually transient. Further sample collection and storage We also propose to collect residual LBC samples for HPV testing from the ~10,000 ARTISTIC Trial cohort women whose cytology will be processed by the Manchester Cytology Centre (which covers the Greater Manchester area) over the 5-year period 2017-2021. Together with the samples collected in 2001-09 these further samples collected in the context of routine screening within the NHSCSP will provide direct evidence on a 10-year HPV screening interval for women who were HPV negative in 2001-2009. We shall observe age-specific CIN2+ detection rates at their 2017-2021 HPV tests among ~9,100 women who were HPV negative at their last test in 2001-2009, which in most cases will be 10 or more years earlier. When added to their cumulative CIN2+ rate over the intervening period this will provide an upper bound for the CIN2+ detection rate with a 10-year interval. (A proportion of CIN2+ cases diagnosed at intervening cytology tests would have regressed by 10 years.) We shall also observe cumulative CIN2+ rates over ~10 years and final HPV status for ~700 women who were HPV positive with normal cytology at their last test in 2001-2009. A substantial reduction in triage costs and procedures will be achieved if a longer delay in their recall is shown to be safe. Samples will be obtained for ~300 women who were diagnosed and treated for CIN2+ and had a subsequent HPV test (250 HPV- and 50 HPV+) in 2001-2009. Cumulative CIN2+ recurrence and final HPV status in 2017-2021 for these women will provide data on short-term HPV clearance as a long-term test of cure. Relevance to national screening policy The National Screening Committee (NSC) based its recommendation that HPV testing should replace cytology in primary screening largely on the 2009 follow-up results of the ARTISTIC trial. The NSC must now decide on screening intervals in time for national roll out of primary HPV screening, currently scheduled for April 2019. Our first priority will be to update cytology through call-recall linkage and and high-grade histology through local laboratories from 2009 to the end of 2016. An important early milestone is to provide the NSC with this analysis by the end of 2017. The NSC will presumably reconsider screening intervals from second and subsequent rounds of national HPV screening when follow-up to 2021 is available. Median follow-up for women aged <50 at entry to the trial will increase from 11.9 years in Dec 2016 to 15.9 years in Dec 2021. Important further evidence will be provided by follow-up from round 2, when persistent and newly acquired infections can be distinguished and the high prevalence of accumulated CIN2/CIN3 missed by earlier screening had been eliminated at entry. The proportion screened 10 or more years after round 2 will increase from 18% in 2016 to 72% in 2021 for women aged <50 and from 37% to 54% in women aged 50-54. (Women aged 55 or more at round 2 had already had their final test by 2016.) A resource for other researchers The further samples collected in 2017-2021 will constitute a substantial addition to the existing collection of LBC samples taken in 2001-2009, which is already being used to evaluate novel DNA resequencing assays (methylation in somatic and HPV genes) for triage of HPV-positive women to identify those who should be recalled earlier or referred immediately for colposcopy. The data on cytology, histology and type-specific HPV acquisition, clearance and persistence spanning up to 20 years will also enable the natural history models that underlie formal cost-benefit analysis of screening and HPV vaccination policy to be recalibrated and perhaps substantially amended. Screening intervals of up to 20 years and earlier age at last test may be considered, particularly when polyvalent vaccination covering the 7 highest risk HPV types is introduced. Approximately half of the 10,000 women from whom further samples will be obtained, including about 200 HPV positives, will be aged 50-64 when they are tested in 2017-2021.

Lay abstract: Background Cervical cancer is caused by the human papillomavirus (HPV). HPV is a very common infection, particularly in young women, but it usually clears without any symptoms or long-lasting effects. There are 13 “high-risk” HPV types that can cause pre-cancer or cancer, but more than 70 percent of cervical cancers are caused by two types of the virus, HPV16 and HPV18. In England and Wales the national cervical cancer screening programme became an organised call-recall programme in 1988 and invites women aged 25-49 to attend for a smear test (known as cytology) every 3 years and every 5 years for women aged 50-64. The programme has been very successful at preventing cervical cancer by diagnosing and treating pre-cancer. The smear test detects abnormal cells from the cervix. Abnormal smear results range from CIN1, a mild abnormaility that is usually caused by a transient HPV infection that will soon disappear, to CIN3 which usually persists and should be treated. Primary HPV screening The latest scientific evidence (including results from the ARTISTIC trial) shows that screening for HPV infection (known as “primary HPV screening”) is better than screening for abnormal cytology. This method is currently being tested in six pilot areas in England, and there are plans to roll it out nationally by April 2019. It’s not as simple as swapping one test for another, however, and there are still decisions to be made on how best to go about it. Testing for HPV detects more cases of abnormality than cytology which means that if a woman tests negative for HPV she can be more sure that she isn’t at risk of pre-cancer or cancer than if she tests negative by cytology. This means that women testing negative for HPV do not need to return for another test for at least 5 years. It could also mean that women who are negative in their fifties may not need to return for another test in their sixties. The disadvantage of primary HPV testing is that more women, particularly young women, will test positive for HPV. It is not sensible to refer a large number of women for further tests when their infection is likely to clear on its own and so a second test has to be used to identify the women who are at highest risk (called a triage test). A balance must be achieved so that most of the women with abnormalities are identified but the overall number of referrals is minimised. This makes it cost-effective for the NHS and also reduces unnecessary anxiety to women. At the moment women who are positive for HPV will be referred only if they also have some abnormal cells (from a cytology test). But the cytology test is not perfect and there will be women with disease who stay positive for HPV but do not appear to have abnormalities on their cytology test. It is important that these women are also identified. The ARTISTIC trial The ARTISTIC Trial recruited 24,510 women attending for routine cervical screening in Greater Manchester in 2001-2003. All the cervical smears taken from the women were also tested for HPV. Laboratory records with cytology results and also any histology records were collected. (If a woman was referred to colposcopy because of an abnormal smear and biopsy taken, the result of the biopsy would be recorded in a histology laboratory). Women were invited to return for further routine smears after 3 and 6 years which are defined as round 2 and round 3. The aim of the trial was to see whether HPV testing could improve on the already successful cytology screening. Data from this trial was put together with 3 other large European trials and showed that testing for HPV prevented more cervical cancers in the long run. This joint study provided evidence for many screening programmes around the world to change over to primary HPV screening. We cannot know how long women have been infected when they test HPV positive at the beginning of the trial as they will be a mix of new infections and persisting infections. By comparing the HPV results taken at entry to the trial to those collected at the second round of screening (approximately 3 years after entry), we can categorise women into those who have persisting infections, new infections or no infections. We found a much higher proportion of women with severe abnormalities (CIN2, CIN3 or cancer, referred to as CIN2+ hereafter) in the first round of ARTISTIC compared to the later two rounds (2.4% compared with 1%). This was because better screening in the trial picked up these abnormalities which had been missed by cytology screening previously. Similar proportions of abnormalities have been seen in the pilot sites (where primary HPV screening is being currently tested). This means that the first round of ARTISTIC can tell us a lot about what happens when HPV testing is introduced and the second round of ARTISTIC can tell us a lot about what happens to women in an established HPV primary screening programme. Since the trial finished in 2009, the women have returned to routine screening. We have been following the women (without contact with them) through linkage to screening records held by the national screening programme and to national cancer registration data. Only the highest degree of pre-cancer, CIN3, is registered nationally (along with cancers) but women with CIN2 and CIN3 are usually treated the same way (the abnormal cells are removed using a large loop excision called LLETZ). This means that it is useful to count both CIN2 and CIN3 when analysing the data since for practical purposes they will be treated the same by a doctor. We now propose to extend the follow-up to 2021 by obtaining histology results from local laboratories from 2009 to 2021. These laboratories still process samples for around 90% of the women in the study. This means that we will get diagnoses of CIN2 for this period of time, as well as CIN3. There are roughly equal numbers of each, so we shall double the number of diagnoses which will make our calculations more precise. Counting the number of years follow-up from the second test in ARTISTIC (between 2005-09, the point at which we can tell if an HPV infection is new or persistent), we will have at least 10 years of data for most of the women. In addition to collecting additional data on CIN2+ diagnoses, we are also proposing to collect further samples from these women and to test them again for HPV. About 10,000 cervical tests among ARTISTIC women will be done over the 5 year period between 2017 and 2021 in the Manchester Cytology Centre which covers the Greater Manchester area. These samples will be tested for HPV in the specialist Virology Laboratory, the positive samples will be genotyped, and the residual samples will be stored for future research on new tests. Aims of this study ARTISTIC can help the national screening committee decide on the best options to reduce the number of women being referred unnecessarily and to save money. There is no other study of women in England and Wales with such long follow-up. Health economists who build models to estimate which screening strategies are the most cost-effective rely on data from studies like ARTISTIC to give them information on how HPV behaves. Collecting further data will help make these models more accurate. Their current work is to predict what will happen when the girls who were vaccinated (currently against just HPV16 and HPV18) reach the age when cervical screening starts. Our genotyping data will be particularly useful in producing recommendations of how to screen these women as we can exclude anyone who has just types 16 or 18 as these types should not occur in vaccinated women. The specific objectives of this study are: 1. Is it safe to leave a longer interval between screening tests when a woman has a negative HPV test? It is of immediate importance that decisions on the screening interval (the time between routine tests) are made by the screening committee in 2017, ahead of the national roll-out. The CIN2 and CIN3 data that we will collect from the end of the trial in 2009 to 2017 can help inform these decisions immediately. 2. What age is it safe to stop screening? Does a woman have to be tested in her sixties if she has tested negative for HPV in her fifties? It is rare that women get new HPV infections in their sixties. We will be collecting new samples from women who are now in their sixties (but were in their fifties when we collected HPV samples during the ARTISTIC trial). Most studies throughout the world are focussed on younger women, but we will have one of the largest studies of HPV infections in older women and so we can accurately estimate how valuable HPV testing in women in their sixties actually is. 3. What follow-up (triage) tests should be done in women who test positive for HPV? We can estimate the risks of disease (CIN2+) according to different cytology results and for different HPV genotypes. We can also look at which women had transient or persisting infections. There will be around a 10 year gap between the new samples and those collected within the trial. We can use this to estimate what would happen if women were screened routinely every 10 years and then what should be done to best triage HPV positive women. 4. Is a longer screening interval also safe for women who have had treatment (such as LLETZ) for CIN2 or CIN3? It is current practice for women who have been treated for CIN2 or CIN3 to have an HPV test 6 months afterwards. This is called a “test of cure”. Women who test negative are returned to routine screening with no additional follow-up. Routine screening is currently every 3 years for women aged 25-49 and every 5 years for women aged 50-64. If screening intervals were extended when primary HPV screening is rolled out, will a longer interval also be safe for these women? We will look at subsequent results in the 775 women who were treated for CIN2 or CIN3 during the trial (2001-09)

Research activity codes: 4.2 EVALUATION OF MARKERS AND TECHNOLOGIES;4.4 POPULATION SCREENING
Health categories: CANCER AND NEOPLASMS