Alexandra Sargent

HPV testing in combination with liquid-based cytology in primary cervical screening (ARTISTIC): a randomised controlled trial.

BACKGROUND Testing for human papillomavirus (HPV) DNA is reportedly more sensitive than cytology for the detection of high-grade cervical intraepithelial neoplasia (CIN). The effectiveness of HPV testing in primary cervical screening was assessed in the ARTISTIC trial, which was done over two screening rounds approximately 3 years apart (2001-03 and 2004-07) by comparing liquid-based cytology (LBC) combined with HPV testing against LBC alone. METHODS Women aged 20-64 years who were undergoing routine screening as part of the English National Health Service Cervical Screening Programme in Greater Manchester were randomly assigned (between July, 2001, and September, 2003) in a ratio of 3:1 to either combined LBC and HPV testing in which the results were revealed and acted on, or to combined LBC and HPV testing where the HPV result was concealed from the patient and investigator. The primary outcome was the detection rate of cervical intraepithelial neoplasia grade 3 or worse (CIN3+) in the second screening round, analysed by intention to treat. This trial is registered with the International Standard Randomised Controlled Trial Number ISRCTN25417821. FINDINGS There were 24 510 eligible women at entry (18 386 in the revealed group, 6124 in the concealed group). In the first round of screening 233 women (1.27%) in the revealed group had CIN3+, compared with 80 (1.31%) women in the concealed group (odds ratio [OR] 0.97, 95% CI 0.75-1.25; p>0.2). There was an unexpectedly large drop in the proportion of women with CIN3+ between the first and second rounds of screening in both groups, at 0.25% (29 of 11 676) in the revealed group and 0.47% (18 of 3866 women) in the concealed group (OR 0.53, 95% CI 0.30-0.96; p=0.042). For both rounds combined, the proportion of women with CIN3+ were 1.51% (revealed) and 1.77% (concealed) (OR 0.85, 95% CI 0.67-1.08; p>0.2). INTERPRETATION LBC combined with HPV testing resulted in a significantly lower detection rate of CIN3+ in the second round of screening compared with LBC screening alone, but the effect was small. Over the two screening rounds combined, co-testing did not detect a higher rate of CIN3+ or CIN2+ than LBC alone. Potential changes in screening methodology should be assessed over at least two screening rounds. FUNDING National Institute of Health Research Health Technology Assessment Programme.

HPV testing in routine cervical screening: cross sectional data from the ARTISTIC trial

To evaluate the effectiveness of human papillomavirus (HPV) testing in primary cervical screening. This was a cross-sectional study from the recruitment phase of a prospective randomised trial. Women were screened for HPV in addition to routine cervical cytology testing. Greater Manchester, attendees at routine NHS Cervical Screening Programme. In all, 24 510 women aged 20–64 screened with liquid-based cytology (LBC) and HPV testing at entry. HPV testing in primary cervical screening. Type-specific HPV prevalence rates are presented in relation to age as well as cytological and histological findings at entry. In all, 24 510 women had adequate cytology and HPV results. Cytology results at entry were: 87% normal, 11% borderline or mild, 1.1% moderate and 0.6% severe dyskaryosis or worse. Prevalence of HPV decreased sharply with age, from 40% at age 20–24 to 12% at 35–39 and 7% or less above age 50. It increased with cytological grade, from 10% of normal cytology and 31% of borderline to 70% mild, 86% moderate, and 96% of severe dyskaryosis or worse. HPV 16 or HPV 18 accounted for 64% of infections in women with severe or worse cytology, and one or both were found in 61% of women with severe dyskaryosis but in only 2.2% of those with normal cytology. The majority of young women in Greater Manchester have been infected with a high-risk HPV by the age of 30. HPV testing is practicable as a primary routine screening test, but in women aged under 30 years, this would lead to a substantial increase in retesting and referral rates. HPV 16 and HPV 18 are more predictive of underlying disease, but other HPV types account for 30% of high-grade disease.

ARTISTIC: A randomised trial of human papillomavirus (HPV) testing in primary cervical screening

OBJECTIVES Primary cervical screening uses cytology to detect cancer precursor lesions [cervical intraepithelial neoplasia stage 3 or beyond (CIN3+)]. Human papillomavirus (HPV) testing could add sensitivity as an adjunct to cytology or as a first test, reserving cytology for HPV-positive women. This study addresses the questions: Does the combination of cytology and HPV testing achieve a reduction in incident CIN3+?; Is HPV testing cost-effective in primary cervical screening?; Is its use associated with adverse psychosocial or psychosexual effects?; and How would it perform as an initial screening test followed by cytology for HPV positivity? DESIGN ARTISTIC was a randomised trial of cervical cytology versus cervical cytology plus HPV testing, evaluated over two screening rounds, 3 years apart. Round 1 would detect prevalent disease and round 2 a combination of incident and undetected disease from round 1. SETTING Women undergoing routine cervical screening in the NHS programme in Greater Manchester. PARTICIPANTS In total 24,510 women aged 20-64 years were enrolled between July 2001 and September 2003. INTERVENTIONS HPV testing was performed on the liquid-based cytology (LBC) sample obtained at screening. Women were randomised in a ratio of 3:1 to have the HPV test result revealed and acted upon if persistently positive in cytology-negative cases or concealed. A detailed health economic evaluation and a psychosocial and psychosexual assessment were also performed. MAIN OUTCOME MEASURES The primary outcome was CIN3+ in round 2. Secondary outcomes included an economic assessment and psychosocial effects. A large HPV genotyping study was also conducted. RESULTS In round 1 there were 313 CIN3+ lesions, representing a prevalence in the revealed and concealed arms of 1.27% and 1.31% respectively (p = 0.81). Round 2 (30-48 months) involved 14,230 (58.1%) of the women screened in round 1 and only 31 CIN3+ were detected; the CIN3 rate was not significantly different between the revealed and concealed arms. A less restrictive definition of round 2 (26-54 months) increased CIN3+ to 45 and CIN3+ incidence in the arms was significantly different (p = 0.05). There was no difference in CIN3+ between the arms when rounds 1 and 2 were combined. Prevalence of high-risk HPV types was age-dependent. Overall prevalence of HPV16/18 increased with severity of dyskaryosis. Mean costs per woman in round 1 were 72 pounds and 56 pounds for the revealed and concealed arms (p < 0.001); an age-adjustment reduced these mean costs to 65 pounds and 52 pounds. Incremental cost-effectiveness ratio for detecting additional CIN3+ by adding HPV testing to LBC screening in round 1 was 38,771 pounds. Age-adjusted mean cost for LBC primary screening with HPV triage was 39 pounds compared with 48 pounds for HPV primary screening with LBC triage. HPV testing did not appear to cause significant psychosocial distress. CONCLUSIONS Routine HPV testing did not add significantly to the effectiveness of LBC in this study. No significant adverse psychosocial effects were detected. It would not be cost-effective to screen with cytology and HPV combined but HPV testing, as either triage or initial test triaged by cytology, would be cheaper than cytology without HPV testing. LBC would not benefit from combination with HPV; it is highly effective as primary screening but HPV testing has twin advantages of high negative predictive value and automated platforms enabling high throughput. HPV primary screening would require major contraction and reconfiguration of laboratory services. Follow-up continues in ARTISTIC while maintaining concealment for a further 3-year round of screening, which will help in screening protocol development for the post-vaccination era.

A comparison of HPV DNA testing and liquid based cytology over three rounds of primary cervical screening: Extended follow up in the ARTISTIC trial

BACKGROUND The additional sensitivity of HPV testing compared with cytology could permit extended cervical screening intervals. We wished to determine, through a further (third) round of screening in the ARTISTIC trial, the protection provided by a negative baseline HPV screen compared with that of cytology over a 6 year period. METHODS Cumulative rates of CIN2 or worse (CIN2+) and CIN3 or worse (CIN3+) were correlated with baseline HPV status and cytology. HPV was detected using the Hybrid Capture 2 (Qiagen) assay for high risk types and genotyped using the Linear Array (Roche) and Papillocheck (Greiner) assays. LBC was performed using ThinPrep (Hologic). FINDINGS Round 3 included 8,873 women of whom 6,337 had been screened in both rounds 1 and 2 and 2,536 had not been screened since round 1. The median duration of follow-up was 72.7 months. The cumulative rate of CIN2+ over three rounds was 3.88% (95%CI 3.59%, 4.17%) overall; 2.39% in round 1, 0.78% in round 2 and 0.74% in round 3. Cumulative rates by baseline status were 20.53% (95%CI 19.04%, 22.08%) for abnormal cytology, 20.12% (95%CI 18.68%, 21.61%) for HPV detection, 1.41% (95%CI 1.19%, 1.65%) for negative cytology and 0.87% (95%CI 0.70%, 1.06%) for a negative HPV test. In HPV negative women aged over 50 the cumulative rate was 0.16% (95%CI 0.07%, 0.34%). Women who were HPV positive/cytology negative at entry had a cumulative CIN2+ rate of 7.73% (95%CI 6.29%, 9.36%) over 6 years, twice the overall rate. INTERPRETATION A negative HPV test was significantly more protective than normal cytology over three rounds. The findings of this extension of ARTISTIC suggest that the screening interval could be extended to 6 years if HPV testing replaced cytology as the primary screening test.

Epigenetic variability in cells of normal cytology is associated with the risk of future morphological transformation.

BackgroundRecently, it has been proposed that epigenetic variation may contribute to the risk of complex genetic diseases like cancer. We aimed to demonstrate that epigenetic changes in normal cells, collected years in advance of the first signs of morphological transformation, can predict the risk of such transformation.MethodsWe analyzed DNA methylation (DNAm) profiles of over 27,000 CpGs in cytologically normal cells of the uterine cervix from 152 women in a prospective nested case-control study. We used statistics based on differential variability to identify CpGs associated with the risk of transformation and a novel statistical algorithm called EVORA (Epigenetic Variable Outliers for Risk prediction Analysis) to make predictions.ResultsWe observed many CpGs that were differentially variable between women who developed a non-invasive cervical neoplasia within 3 years of sample collection and those that remained disease-free. These CpGs exhibited heterogeneous outlier methylation profiles and overlapped strongly with CpGs undergoing age-associated DNA methylation changes in normal tissue. Using EVORA, we demonstrate that the risk of cervical neoplasia can be predicted in blind test sets (AUC = 0.66 (0.58 to 0.75)), and that assessment of DNAm variability allows more reliable identification of risk-associated CpGs than statistics based on differences in mean methylation levels. In independent data, EVORA showed high sensitivity and specificity to detect pre-invasive neoplasia and cervical cancer (AUC = 0.93 (0.86 to 1) and AUC = 1, respectively).ConclusionsWe demonstrate that the risk of neoplastic transformation can be predicted from DNA methylation profiles in the morphologically normal cell of origin of an epithelial cancer. Having profiled only 0.1% of CpGs in the human genome, studies of wider coverage are likely to yield improved predictive and diagnostic models with the accuracy needed for clinical application.Trial registrationThe ARTISTIC trial is registered with the International Standard Randomised Controlled Trial Number ISRCTN25417821.

Prevalence of type-specific HPV infection by age and grade of cervical cytology: data from the ARTISTIC trial

Human papillomavirus (HPV) infection causes cervical cancer and premalignant dysplasia. Type-specific HPV prevalence data provide a basis for assessing the impact of HPV vaccination programmes on cervical cytology. We report high-risk HPV (HR-HPV) type-specific prevalence data in relation to cervical cytology for 24 510 women (age range: 20–64; mean age 40.2 years) recruited into the ARTISTIC trial, which is being conducted within the routine NHS Cervical Screening Programme in Greater Manchester. The most common HR-HPV types were HPV16, 18, 31, 51 and 52, which accounted for 60% of all HR-HPV types detected. There was a marked decline in the prevalence of HR-HPV infection with age, but the proportion due to each HPV type did not vary greatly with age. Multiple infections were common below the age of 30 years but less so between age 30 and 64 years. Catch-up vaccination of this sexually active cohort would be expected to reduce the number of women with moderate or worse cytology by 45%, but the number with borderline or mild cytology would fall by only 7%, giving an overall reduction of 12% in the number of women with abnormal cytology and 27% in the number with any HR-HPV infection. In the absence of broader cross-protection, the large majority of low-grade and many high-grade abnormalities may still occur in sexually active vaccinated women.

The dynamics and prognostic potential of DNA methylation changes at stem cell gene loci in women's Cancer

Aberrant DNA methylation is an important cancer hallmark, yet the dynamics of DNA methylation changes in human carcinogenesis remain largely unexplored. Moreover, the role of DNA methylation for prediction of clinical outcome is still uncertain and confined to specific cancers. Here we perform the most comprehensive study of DNA methylation changes throughout human carcinogenesis, analysing 27,578 CpGs in each of 1,475 samples, ranging from normal cells in advance of non-invasive neoplastic transformation to non-invasive and invasive cancers and metastatic tissue. We demonstrate that hypermethylation at stem cell PolyComb Group Target genes (PCGTs) occurs in cytologically normal cells three years in advance of the first morphological neoplastic changes, while hypomethylation occurs preferentially at CpGs which are heavily Methylated in Embryonic Stem Cells (MESCs) and increases significantly with cancer invasion in both the epithelial and stromal tumour compartments. In contrast to PCGT hypermethylation, MESC hypomethylation progresses significantly from primary to metastatic cancer and defines a poor prognostic signature in four different gynaecological cancers. Finally, we associate expression of TET enzymes, which are involved in active DNA demethylation, to MESC hypomethylation in cancer. These findings have major implications for cancer and embryonic stem cell biology and establish the importance of systemic DNA hypomethylation for predicting prognosis in a wide range of different cancers.

HPV testing as an adjunct to cytology in the follow up of women treated for cervical intraepithelial neoplasia

Objective  To evaluate human papillomavirus (HPV) testing in combination with cytology in the follow up of treated women.

Multi-site study of HPV type-specific prevalence in women with cervical cancer, intraepithelial neoplasia and normal cytology, in England

Background:Knowledge of the prevalence of type-specific human papillomavirus (HPV) infections is necessary to predict the expected, and to monitor the actual, impact of HPV immunisation and to design effective screening strategies for vaccinated populations.Methods:Residual specimens of cervical cytology (N=4719), CIN3/CGIN and cervical cancer biopsies (N=1515) were obtained from sites throughout England, anonymised and tested for HPV DNA using the Linear Array typing system (Roche).Results:The prevalence of HPV 16 and/or 18 (with or without another high-risk (HR) type) was 76% in squamous cell carcinomas, 82% in adeno/adenosquamous carcinomas and 63% and 91% in CIN3 and CGIN, respectively. Of all HR HPV-infected women undergoing cytology, non-vaccine HPV types only were found in over 60% of those with mild dyskaryosis or below, and in <20% of those with cancer. In women of all ages undergoing screening, HR HPV prevalence was 16% and HPV 16 and/or 18 prevalence was 5%.Conclusion:Pre-immunisation, high-grade cervical disease in England was predominantly associated with HPV 16 and/or 18, which promises a high impact from HPV immunisation in due course. Second-generation vaccines and screening strategies need to consider the best ways to detect and prevent disease due to the remaining HR HPV types.BACKGROUND Knowledge of the prevalence of type-specific human papillomavirus (HPV) infections is necessary to predict the expected, and to monitor the actual, impact of HPV immunisation and to design effective screening strategies for vaccinated populations. METHODS Residual specimens of cervical cytology (N=4719), CIN3/CGIN and cervical cancer biopsies (N=1515) were obtained from sites throughout England, anonymised and tested for HPV DNA using the Linear Array typing system (Roche). RESULTS The prevalence of HPV 16 and/or 18 (with or without another high-risk (HR) type) was 76% in squamous cell carcinomas, 82% in adeno/adenosquamous carcinomas and 63% and 91% in CIN3 and CGIN, respectively. Of all HR HPV-infected women undergoing cytology, non-vaccine HPV types only were found in over 60% of those with mild dyskaryosis or below, and in <20% of those with cancer. In women of all ages undergoing screening, HR HPV prevalence was 16% and HPV 16 and/or 18 prevalence was 5%. CONCLUSION Pre-immunisation, high-grade cervical disease in England was predominantly associated with HPV 16 and/or 18, which promises a high impact from HPV immunisation in due course. Second-generation vaccines and screening strategies need to consider the best ways to detect and prevent disease due to the remaining HR HPV types.

Optimal Threshold for a Positive Hybrid Capture 2 Test for Detection of Human Papillomavirus: Data from the ARTISTIC Trial

ABSTRACT We present data on the use of the Hybrid Capture 2 (HC2) test for the detection of high-risk human papillomavirus (HR HPV) with different thresholds for positivity within a primary screening setting and as a method of triage for low-grade cytology. In the ARTISTIC population-based trial, 18,386 women were screened by cytology and for HPV. Cervical intraepithelial neoplasia lesions of grade two and higher (CIN2+ lesions) were identified for 453 women within 30 months of an abnormal baseline sample. When a relative light unit/cutoff (RLU/Co) ratio of ≥1 was used as the threshold for considering an HC2 result positive, 15.6% of results were positive, and the proportion of CIN2+ lesions in this group was 14.7%. The relative sensitivity for CIN2+ lesion detection was 93.4%. When an RLU/Co ratio of ≥2 was used as the threshold, there was a 2.5% reduction in positivity, with an increase in the proportion of CIN2+ lesions detected. The relative sensitivity decreased slightly, to 90.3%. Among women with low-grade cytology, HPV prevalences were 43.7% and 40.3% at RLU/Co ratios of ≥1 and ≥2, respectively. The proportions of CIN2+ lesions detected were 17.3% and 18.0%, with relative sensitivities of 87.7% at an RLU/Co ratio of ≥1 and 84.2% at an RLU/Co ratio of ≥2. At an RLU/Co ratio of ≥1, 68.3% of HC2-positive results were confirmed by the Roche line blot assay, compared to 77.2% of those at an RLU/Co ratio of ≥2. Fewer HC2-positive results were confirmed for 35- to 64-year-olds (50.3% at an RLU/Co ratio of ≥1 and 63.2% at an RLU/Co ratio of >2) than for 20- to 34-year-olds (78.7% at an RLU/Co ratio of ≥1 and 83.7% at an RLU/Co ratio of >2). If the HC2 test is used for routine screening as an initial test or as a method of triage for low-grade cytology, we would suggest increasing the threshold for positivity from the RLU/Co ratio of ≥1, recommended by the manufacturer, to an RLU/Co ratio of ≥2, since this study has shown that a beneficial balance between relative sensitivity and the proportion of CIN2+ lesions detected is achieved at this threshold.