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Purpose: Response rates to treatment of vulval intraepithelial neoplasia (VIN) with imiquimod and cidofovir are approximately 57% and 61%, respectively. Treatment is associated with significant side effects and, if ineffective, risk of malignant progression. Treatment response is not predicted by clinical factors. Identification of a biomarker that could predict response is an attractive prospect. This work investigated HPV DNA methylation as a potential predictive biomarker in this setting.Experimental Design: DNA from 167 cases of VIN 3 from the RT3 VIN clinical trial was assessed. HPV-positive cases were identified using Greiner PapilloCheck and HPV 16 type-specific PCR. HPV DNA methylation status was assessed in three viral regions: E2, L1/L2, and the promoter, using pyrosequencing.Results: Methylation of the HPV E2 region was associated with response to treatment. For cidofovir (n = 30), median E2 methylation was significantly higher in patients who responded (P ≤ 0.0001); E2 methylation >4% predicted response with 88.2% sensitivity and 84.6% specificity. For imiquimod (n = 33), median E2 methylation was lower in patients who responded to treatment (P = 0.03; not significant after Bonferroni correction); E2 methylation Conclusions: These data indicate that cidofovir and imiquimod may be effective in two biologically defined groups. HPV E2 DNA methylation demonstrated potential as a predictive biomarker for the treatment of VIN with cidofovir and may warrant investigation in a biomarker-guided clinical trial. Clin Cancer Res; 23(18); 5460–8. ©2017 AACR.

Background Persistent infection with human papillomavirus (HPV) type 16 causes the majority of cervical cancers. Genital HPV infection is very common, but neoplastic progression is uncommon. There is an urgent need for biomarkers associated with cervical neoplasia, to enable triage of women who test positive for HPV. Objectives To assess the ability of quantitative measurement of HPV16 DNA methylation to separate samples of different cytological and histological grades from young women, among whom rates of HPV infection are high. Study design DNA methylation was quantified by pyrosequencing of bisulphite converted DNA from liquid based cytology samples from 234 women (mean age 20.6 years) who tested positive for HPV16 and showed varying degrees of neoplasia. Methylation was assessed at CpGs in the HPV E2 and L1/L2 regions. Results The performance of methylation-based classifiers was assessed by ROC curve analyses. The best combination of CpGs (5600 and 5609) achieved AUCs of 0.656 (95% CI = 0.520–0.792) for separation of cytologically normal and severely dyskaryotic samples, and 0.639 (95% CI = 0.547–0.731) for separation of samples with or without high-grade neoplasia (CIN2+/−). Conclusions The data are consistent with HPV L1/L2 methylation being a marker of the duration of infection in a specific host. Assessment of HPV DNA methylation is hence a promising biomarker to triage HPV-positive cytology samples, but may have limited utility in young women. Future studies assessing the likely utility of HPV DNA methylation as a potential triage biomarker must take account of women’s age.

Background: Methylation of HPV16 DNA is a promising biomarker for triage of HPV positive cervical screening samples but the biological basis for the association between HPV-associated neoplasia and increased methylation is unclear. Objectives: To determine whether HPV16 DNA methylation was associated with viral integration, and investigate the relationships between viral DNA methylation, integration and gene expression. Study design: HPV16 DNA methylation, integration and gene expression were assessed using pyrosequencing, ligation-mediated PCR and QPCR, in biopsies from 25 patients attending a specialist vulval neoplasia clinic and in short-term clonal cell lines derived from vulval and vaginal neoplasia. Results: Increased methylation of the HPV16 L1/L2 and E2 regions was associated with integration of viral DNA into the host genome. This relationship was observed both in vivo and in vitro. Increased methylation of E2 binding sites did not appear to be associated with greater expression of viral early genes. Expression of HPV E6 and E7 did not correlate with either integration state or increased L1/L2 methylation. Conclusions: The data suggest that increased HPV DNA methylation may be partly attributable to viral integration, and provide a biological rationale for quantification of L1/L2 methylation in triage of HPV positive cervical screening samples.

Summary Background Vulval intraepithelial neoplasia is a skin disorder affecting the vulva that, if left untreated, can become cancerous. Currently, the standard treatment for patients with vulval intraepithelial neoplasia is surgery, but this approach does not guarantee cure and can be disfiguring, causing physical and psychological problems, particularly in women of reproductive age. We aimed to assess the activity, safety, and feasibility of two topical treatments—cidofovir and imiquimod—as an alternative to surgery in female patients with vulval intraepithelial neoplasia. Methods We recruited female patients (age 16 years or older) from 32 centres to an open-label, randomised, phase 2 trial. Eligibility criteria were biopsy-proven vulval intraepithelial neoplasia grade 3 and at least one lesion that could be measured accurately. We randomly allocated patients to topical treatment with either 1% cidofovir (supplied as a gel in a 10 g tube, to last 6 weeks) or 5% imiquimod (one 250 mg sachet for every application), to be self-applied three times a week for a maximum of 24 weeks. Randomisation (1:1) was done by stratified minimisation via a central computerised system, with stratification by hospital, disease focality, and presentation stage. The primary endpoint was a histologically confirmed complete response at the post-treatment assessment visit 6 weeks after the end of treatment (a maximum of 30 weeks after treatment started). Analysis of the primary endpoint was by intention to treat. Secondary outcomes were toxic effects (to assess safety) and adherence to treatment (to assess feasibility). We present results after all patients had reached the primary endpoint assessment point at 6 weeks; 2-year follow-up of complete responders continues. This trial is registered with Current Controlled Trials, ISRCTN 34420460. Findings Between Oct 21, 2009, and Jan 11, 2013, 180 participants were enrolled to the study; 89 patients were randomly allocated cidofovir and 91 were assigned imiquimod. At the post-treatment assessment visit, a complete response had been achieved by 41 (46%; 90% CI 37·0–55·3) patients allocated cidofovir and by 42 (46%; 37·2–55·3) patients assigned imiquimod. After 6 weeks of treatment, 156 (87%) patients (78 in each group) had adhered to the treatment regimen. Five patients in the cidofovir group and seven in the imiquimod group either withdrew or were lost to follow-up before the first 6-week safety assessment. Adverse events of grade 3 or higher were reported in 31 (37%) of 84 patients allocated cidofovir and 39 (46%) of 84 patients assigned imiquimod; the most frequent grade 3 and 4 events were pain in the vulva, pruritus, fatigue, and headache. Interpretation Cidofovir and imiquimod were active, safe, and feasible for treatment of vulval intraepithelial neoplasia and warrant further investigation in a phase 3 setting. Both drugs are effective alternatives to surgery for female patients with vulval intraepithelial neoplasia after exclusion of occult invasive disease. Funding Cancer Research UK.

Vulval intraepithelial neoplasia is a precursor of vulval cancer and is commonly caused by infection with Human Papillomavirus (HPV). Development of topical treatments for vulval intraepithelial neoplasia requires appropriate in vitro models. This study evaluated the feasibility of primary culture of vulval intraepithelial neoplasia biopsy tissue to produce cell lines for use as in vitro models. A potentially immortal cell line was produced which gave rise to three monoclonal lines. These lines were characterized for HPV genomic integration and for viral gene expression using ligation-mediated PCR and quantitative PCR. Distinct patterns of viral integration and gene expression were observed among the three lines. Integration and expression data were validated using deep sequencing of mRNA. Gene ontology analyses of these data also demonstrated that expression of the HPV16 E4 and E5 proteins resulted in substantial changes in the composition of the cell membrane and extracellular space, associated with alterations in cell adhesion and differentiation. These data illustrate the diverse patterns of HPV gene expression potentially present within a single lesion. The derived cell lines provide useful models to investigate the biology of vulval intraepithelial neoplasia and the interactions between different HPV gene products and potential therapeutic agents.

Background: In 2008 a human papillomavirus (HPV) vaccination programme for cervical cancer prevention was implemented in the UK. Surveillance of vaccine uptake, impact on prevalence of HPV infection and cervical cancer incidence were identified as key measures to evaluate the intervention. Objective: To determine baseline HPV prevalence in unvaccinated women and predict impact of HPV vaccination on high-grade cervical disease (CIN2+). Study design: A pseudo-anonymous prospective cohort was sampled on entry to the routine cervical screening programme between March 2009 and November 2010. In total, 13,306 eligible females were identified and high-risk (hrHPV) type specific status determined. Potential impact of prophylactic vaccination on CIN2+ was calculated by applying HPV vaccine clinical trial data to the baseline HPV type-specific data. Results: Of 13,306 samples tested, 3545 (26.6%) were confirmed positive for at least one hrHPV type and 1325 (10%) were positive for low risk HPV. HPV16 was the predominant type detected in cases positive with either single or multiple hrHPV infection(s) (5.2% and 4.7%, respectively). Based on hrHPV type-specific data, Gardasil would have prevented 33.2% HPV16/18 unrelated CIN2+ compared to 47.1% for Cervarix. This difference was not statistically significant. Conclusion: Prior to the introduction of the HPV vaccine, approximately one-quarter of young women were positive for hrHPV and one-tenth positive for HPV16. Post-vaccination, we anticipate a substantial absolute risk reduction in high-grade cervical disease associated with both targeted and non-targeted hrHPV types. There is no significant difference between the two commercially available vaccines in terms of clinical impact.

Key content Human papillomavirus (HPV) is associated with neoplastic disease at sites other than the cervix. Cancer caused by HPV may behave differently to other cancers at the same site. The presence of HPV alone is not sufficient to demonstrate a causal association. HPV vaccination has the potential to impact on disease burden beyond cervical cancer. Learning objectives To understand the spectrum of disease caused by HPV. To review the management of HPV-associated disease in pregnancy. To be aware that future research into HPV-related disease may alter management. Ethical issues What advice should women be given regarding their sexual partners? Should boys be offered HPV vaccination?

Background: Human papillomavirus (HPV) testing is more sensitive than cytology for detecting high-grade cervical intraepithelial neoplasia (CIN). We evaluated the performance of high-risk HPV (hrHPV) testing in routine screening. Methods: In all, 25 871 women (29–61) enrolled in our population-based cohort study were offered both cytology and hrHPV testing. High-risk HPV-positive women with normal cytology and an age-matched subcohort of hrHPV-negative women with normal cytology were invited for repeat testing after 1 and/or 2 years and were referred for colposcopy if they presented with abnormal cytology and/or a positive hrHPV test. The hrHPV-positive women with borderline or mild dyskaryosis (BMD) and all women with moderate dyskaryosis or worse (>BMD) were directly referred for colposcopy. Women with BMD and an hrHPV-negative test were advised to repeat cytology at 6 and 18 months and were referred for colposcopy if the repeat cytology test was abnormal. The main outcome measure was CIN grade 3 or worse (CIN3+). Results were adjusted for non-attendance at repeat testing. Results: The hrHPV-positive women with abnormal cytology had a CIN3+ risk of 42.2% (95% confidence interval (CI): 36.4–48.2), whereas the hrHPV-positive women with normal cytology had a much lower risk of 5.22% (95% CI: 3.72–7.91). In hrHPV-positive women with normal cytology, an additional cytology step after 1 year reduced the CIN3+ risk to only 1.6% (95% CI: 0.6–4.9) if the repeat test was normal. The CIN3+ risk in women with hrHPV-positive normal cytology was higher among women invited for the first time (29–33 years of age) (9.1% 95% CI: 5.6–14.3) than among older women (3.0% 95% CI: 1.5–5.5). Conclusion: Primary hrHPV screening with cytology triage in women aged ⩾30 years is an effective way to stratify women on CIN3+ risk and seems a feasible alternative to cytological screening. Repeat cytology after 1 year for hrHPV-positive women with normal cytology is however necessary before returning women to routine screening.

Background Human Papillomavirus (HPV) testing has been evaluated as a test of cure in patients following treatment of high-grade cervical intraepithelial neoplasia (CIN2+). Studies show that women who are HPV and cytology negative post treatment can be safely returned to routine recall. The management strategy for HPV positive women requires confirmation. Objective To evaluate the clinical utility of the PapilloCheck® genotyping assay for predicting disease recurrence in a test of cure setting. Study design Ninety-eight women (19–52 years) treated for CIN2+ by large loop excision of the transformation zone (LLETZ) were evaluated with samples taken before and 6 months after treatment for HPV testing. Cytology and histology were available from recruitment until 24 months post treatment. Results Recurrent disease was evident in 4% of patients with 2 cases low-grade and 2 cases of high-grade disease. In women with no disease recurrence, 40% (95% CI 30.42–51.05%) were high risk (HR) HPV negative post LLETZ. Both cases with high-grade disease had persistent HPV16 infection. Genotyping before and after treatment revealed 83% (95% CI 75.74–88.78%) of total viral infections were cleared and 17% (95% CI 11.22–24.26) viral infections persisted. Post treatment, combined cytology and HPV test results predicted CIN2+ with 100% sensitivity, 91.7% specificity, 100% NPV and 20% PPV and measuring viral persistence marginally increased specificity and PPV. Conclusion Post treatment, cytology combined with a single HR HPV test has high sensitivity and specificity for predicting disease recurrence. HPV genotyping before and after LLETZ identifies persistent viral infections and could help refine patient management.

Background Cervical screening detects precancerous cells and routine screening could be improved by testing for Human Papillomavirus (HPV), the virus that causes cervical cancer. HPV infection is common and the benefit of HPV testing would be identification of women who are HPV negative and at low risk of developing cancer. Study design The aim of this study was to evaluate the Greiner Bio-one PapilloCheck ® micro-array assay (PapilloCheck) for detection of HPV in comparison with Hybrid Capture II (hc2) and PCR-enzyme immunoassay (PCR-EIA) using the GP5/6+ primers. Results Samples from a cytologically defined population ( n = 878) were analysed and 187 samples also had histology information. Overall, 674 out of 878 samples gave a consistent result (76.8%; 95% CI 73.83–79.52%) on all three platforms. The genotype results obtained by PapilloCheck and PCR-EIA were compared and 94% were consistent (95% CI 92.1–96.4%). The main difference was the poor Kappa agreement for detection of high risk (HR) type 35 (Kappa = 0.190) with all inconsistent results being HR positive by PCR-EIA assay but negative on the PapilloCheck platform. There was no statistically significant difference between the performance of each assay when HR HPV positive samples were linked with clinical result (cytology and histology grade). PapilloCheck detected the highest number of HR HPV infections in samples with histology confirmed as CIN1, CIN2 and CIN 3 (76.6%, 85% and 91.7%, respectively). Conclusions Overall, PapilloCheck proved to be a sensitive, reproducible, robust molecular assay for HPV genotyping with the potential for high throughput of specimens in a clinical setting.

In this cross-sectional population-based study we determine human papillomavirus (HPV) prevalence in South Wales to provide comprehensive baseline data for future assessment of the impact of prophylactic HPV vaccination and to help inform future screening strategies. Liquid-based cytology samples from women attending routine cervical screening were collected (n=10 000: mean age 38 years, 93% cytology negative, and 64.8% from the 50% least deprived LSOA according to social deprivation score (SDS)). High-Risk (HR) and Low-Risk HPV screening was performed using HPV PCR-EIA with genotyping of HR positives and data correlated with age, SDS and cytology. Overall HPV prevalence was 13.5% (9.3% age standardised) and the most frequent HR types were HPV 16, 31, 18 and 58. In HR HPV-positive cases 42.4% had a single HR type and they were predominant in women with severe cytological abnormalities. Here, 66% of all HR HPV cases were in women aged 30 years of age or less and SDS had no significant effect on HPV status. HPV prevalence increased significantly with degree of dyskarosis from 7% in cytology negative samples to 80% in samples with severe cytological abnormalities (P-value

Human papillomavirus (HPV) is responsible for 99.7% of cervical cancer. There are over 200 types of HPV, and much is now known about the virus, including its life cycle, the function of its proteins and the mechanism by which the neoplastic transformation of keratinocytes occurs. The causal relationship between HPV and anogenital neoplasia has been confirmed by epidemiological evidence. Several methods of HPV detection have been proposed, and studies are ongoing to examine whether these tests might be incorporated into a cervical screening programme. The observation that HPV-related disease is more common in immunosuppressed patients has led to research into the host response to HPV and the development of vaccinations. Although consistent clinical responses have not been seen with therapeutic vaccination, trials of prophylactic vaccination have shown encouraging results. Public knowledge regarding the causal link between HPV and cervical neoplasia is poor and will provide a challenge for vaccination programmes.

The objective of this study was to investigate variation in human papillomavirus (HPV) type-16 load within histologically defined grades of cervical intraepithelial neoplasia. Two hundred and thirty-seven liquid based cytology samples were collected from women attending colposcopy clinics, DNA was extracted, and presence of virus determined by PCR-enzyme immunoassay. Quantitative real-time PCR was used to determine viral load for 70 HPV-16 positive single infections. Viral load was expressed as the ratio of copies of the viral L1 gene to copies of the human beta-globin gene. Measurements varied from 0.019 to 4,194 HPV genomes per cell. Our data demonstrate that in cervical neoplasia, HPV load tends to correlate with disease severity, but that the number of viral genomes/cell varies considerably within histological grades. This variation within disease grades currently limits the clinical utility of viral load measurement.

The objective of this study was to describe human papillomavirus (HPV) prevalence in South Wales in relation to age, cytology and social deprivation. This was an unlinked, prospective, anonymous, population-based study. DNA was purified from 1911 liquid-based cytology samples (mean age 37.7 years, cytology 93.2% negative, social deprivation average score 17.9) using quality assured techniques and the presence of virus determined by PCR-Enzyme Immuno Assay (PCR-EIA). 209 (10.9%) samples contained high-risk (HR) HPV infection of which 36.4% had multiple HR-HPV types. The most frequent HR types were HPV 16 (19.6%), HPV 35 (9.5%), HPV 66 (9.2%), HPV 59 (8.5%) and HPV 56 (7.6%). There was a strong association between HPV infection and cytological abnormality. Significantly more HR-HPV infections were detected in women under the age of 30 years (68.9% of all HR-HPV infections Fisher's exact test P=0.0001) compared to 30 years and above. There was no difference in HPV prevalence between different socioeconomic groups. The data presented suggest a different HPV type distribution in South Wales in comparison to that reported for other populations.

Purpose: The purpose is to study the immunogenicity of heterologous prime-boost human papillomavirus (HPV) oncogene vaccination in patients with anogenital intraepithelial neoplasia (AGIN). Experimental Design: Twenty-nine women with high-grade AGIN received three i.m. doses of TA-CIN (HPV-16 L2/E6/E7 protein) at four weekly intervals followed by a single dermal scarification of vaccinia HPV-16/18 E6/E7 and were followed up for 12 weeks. Immunity to HPV-16 was assessed by lymphoproliferation, IFN-γ enzyme-linked immunospot (ELISPOT), and ELISA. Results: The patient group significantly responded to TA-CIN and not to the control antigen HPV-6 L2/E7 at all postvaccination time points when compared with baseline responses (P ≤ 0.05). Ten of the patients showed at least a 3-fold increase in TA-CIN-specific proliferation at one or more time points after vaccination. Comparison of stimulation with HPV-16 E6- or E7-GST fusion proteins showed that proliferative responses were biased to HPV-16 E6. This bias was also seen by IFN-γ ELISPOT using overlapping peptides, with HPV-16 E6- or E7-specific T cells being detected in 9 and 2 patients, respectively. In addition, vaccination resulted in the induction of antibodies against the HPV-16 oncoproteins. Of the 6 clinical responders, 2 patients showed both a proliferative TA-CIN-specific response and an E6-specific IFN-γ response, whereas 3 other patients displayed E6-specific reactivity only. Stable disease was recorded in 19 patients, 8 of whom showed a concomitant TA-CIN-specific proliferative and/or E6-specific T-cell response. Of the 4 progressors, 2 failed to make a T-cell response and 2 responded by either proliferation or E6 ELISPOT alone. Conclusions: The prime-boost regimen is immunogenic in AGIN patients (humoral and cellular immunity), but there is no simple relationship between induction of systemic HPV-16-specific immunity and clinical outcome. Other factors that may play a role in the eradication of long-term established AGIN lesions need to be determined to identify the patient group that would benefit from immunotherapy with the vaccines used in this study.

Background: Data on the psychosocial burden of human papillomavirus (HPV)-related diseases other than cervical cancer are scarce. The objectives of this study were to measure and compare the psychosocial burden and the impact on health-related quality of life (HRQoL) of HPV-related lower genital tract diseases and genital warts (GW) using several generic and disease-specific instruments. Methods. Overall, 842 individuals with normal cervical cytology (n = 241), borderline nuclear abnormalities and/or mild dyskaryosis (n = 23), cervical intraepithelial neoplasia (CIN)1 (n = 84), CIN2/3 (n = 203), vulval intraepithelial neoplasia (VIN)2/3 (n = 43), GW (n = 186) and a history of GW (non-current) (n = 62) were included. The generic European Quality of Life Index Version 5D (EQ-5D) questionnaire was completed by patients with GW and VIN2/3. Sexual functioning was evaluated using the Change in Sexual Functioning Questionnaire (CSFQ). Psychosocial impact was measured in women using the HPV Impact Profile (HIP) questionnaire. HRQoL was assessed using a GW-specific questionnaire, the Cuestionario Especifico en Condilomas Acuminados (CECA) (completed by patients with GW and history of GW). For each instrument, scores were compared between groups using the Student's t-test. In addition, utility loss due to GW and VIN2/3 was evaluated by comparing mean EQ-5D scores weighted by age and sex with the UK general population normal values. Results: A significant psychosocial impact was found in women diagnosed with HPV-related genital diseases, particularly in those with GW. The health state of younger adults with GW was significantly impaired compared with UK normal values (mean EQ-5D index score 0.86 vs 0.94, p < 0.001 for 18-24-year-olds; 0.87 vs 0.93, p = 0.030 for 25-34-year-olds). VIN2/3 was found to have a significant negative impact on sexual functioning, and women with VIN2/3 had a highly impaired health state compared with women in the UK general population (weighted mean EQ-5D index score 0.72 vs 0.89, p < 0.001; weighted mean Visual Analogue Scale score 62 vs 85, p < 0.001). Conclusions: HPV-related lower genital tract lesions and GW significantly impair psychosocial wellbeing and HRQoL. The psychosocial aspects of HPV-related diseases need to be considered when evaluating the potential benefit of HPV vaccination. © 2013 Dominiak-Felden et al.; licensee BioMed Central Ltd.

The synthesis and in vitro biological evaluation of novel phosphonamidate and phosphonodiamidate prodrugs of adefovir and tenofovir are reported. The selected synthetic approach from free phosphonic acid via bis-trimethylsilyl ester intermediates affords (l)-alanine ester derivatives in 10–70% yields. When assessed for their anti-HIV activity, all the prodrugs showed submicromolar activity. Noteworthy, the most potent derivative in the adefovir series contained a 5,6,7,8-tetrahydronaphtyl group, herein reported for the first time as an aryl moiety in a ProTide. A pronounced cytostatic activity of the above prodrugs is also reported. Selected compounds were tested for their antiproliferative activity against HPV-transformed cells and they were found significantly more active in comparison to their parent compounds. In this study a slightly improved activity of the adefovir derivatives over those of tenofovir was also noticed. However, no specificity for naturally HPV-transformed cell lines was observed.

DNA methylation changes in human papillomavirus type 16 (HPV16) DNA are common and might be important for identifying women at increased risk of cervical cancer. Using recently published data from Costa Rica we developed a classification score to differentiate women with cervical intraepithelial neoplasia grade 2 or 3 (CIN2/3) from those with no evident high-grade lesions. Here, we aim to investigate the performance of the score using data from the UK. Exfoliated cervical cells at baseline and 6-months follow-up were analyzed in 84 women selected from a randomized clinical trial of women undergoing surveillance for low-grade cytology. Selection of women for the methylation study was based on detectable HPV16 in the baseline sample. Purified DNA was bisulfite converted, amplified and pyrosequenced at selected CpG sites in the viral genome (URR, E6, L1 and L2), with blinding of laboratory personnel to the clinical data. The primary measure was a predefined score combining the mean methylation in L1 and any methylation in L2. At the second follow-up visit, 73/84 (87%) women were HPV16 positive and of these 25 had a histopathological diagnosis of CIN2/3. The score was significantly associated with CIN2/3 (area under curve = 0.74, p = 0.002). For a cutoff with 92% sensitivity, colposcopy could have been avoided in 40% (95% CI 27–54%) of HPV16 positive women without CIN2/3; positive predictive value was 44% (32–58%) and negative predictive value was 90% (71–97%). We conclude that quantitative DNA methylation assays could help to improve triage among HPV16 positive women.

Background: Persistent infection with Human Papillomavirus (HPV) type 16 causes the majority of cervical cancers. Genital HPV infection is very common, but neoplastic progression is uncommon. There is an urgent need to identify biomarkers associated with cervical neoplasia that can be used to triage women who test positive for HPV. Objectives: To assess the ability of quantitative measurement of HPV16 DNA methylation to separate samples of different cytology grades and cervical cancers, and determine which of the assessed regions of the HPV genome and individual CpGs are most informative. Study design: DNA methylation was quantified by pyrosequencing of bisulphite converted DNA from liquid based cytology samples from 17 women with normal cytology and 20 women with severe dyskaryosis, and from fixed tissue from 24 women with cervical cancer. Methylation was assessed in the HPV Long Control Region (LCR), E2 and L1/L2 regions. Results: In cervical cancers, increased HPV DNA methylation was present in all regions. Increased methylation was also observed in severely dyskaryotic relative to normal samples, but only in the E2 and L1/L2 regions. The ability of methylation based classifiers to separate the three classes of material was assessed by ROC curve analyses. The best separation between normal and dyskaryotic samples was achieved by assessment of the L1/L2 CpGs at nucleotide positions 5600 and 5609 (AUC = 0.900, 95% CI: 0.793–1). Conclusions: This study demonstrates the potential of quantification of HPV DNA methylation as a biomarker of cervical neoplasia. An algorithm considering methylation at specific L1/L2 CpGs appeared the most promising model.

Cervical screening identifies many women with low-grade abnormalities. In vitro and in vivo studies have shown that diindolylmethane (DIM) could potentially halt (cervical) carcinogenesis. We report on a randomised controlled trial of the effect of DIM in women with low-grade cervical cytological abnormalities. Methods: We conducted a pragmatic double-blind, randomised controlled trial of 150 mg DIM (from BioResponse DIM) or placebo daily for 6 months in women with newly diagnosed, low-grade cytological abnormalities. Randomisation was in the ratio 2 (DIM) to 1 (placebo). All women were invited for colposcopy at 6 months with biopsy of any abnormality. Results: Of the 551 randomised women available for analysis, 9% on DIM and 12% on placebo had cervical intraepithelial neoplasia-2 (CIN2) or worse after 6-month supplementation (risk ratio (RR) 0.7 (95% confidence interval (CI): 0.4–1.2)), whereas 4.6% and 5.1%, respectively, had CIN3 or worse (RR 0.9 (95% CI: 0.4–2.0)). A total of 27.3% of women on DIM and 34.3% on placebo had no sign of disease (negative cytology, colposcopy and human papilloma virus (HPV) tests) at 6 months (RR 0.8 (95% CI: 0.6–1.0)). Of those HPV-positive at baseline, 69% (114 out of 166) of the DIM group were positive at 6 months compared with 61% (43 out of 71) of the placebo group: RR 1.1 (95% CI: 0.9–1.4). Diindolylmethane supplementation was well tolerated. Conclusion: The results suggest that short-term DIM supplementation (150 mg day−1) is well tolerated, but is unlikely to have an effect on cytology or HPV infection. Uncertainty remains regarding its effect on CIN2+.

International audience; Vulval intraepithelial neoplasia is a precursor of vulval carcinoma, and is frequently associated with Human Papillomavirus (HPV) infection. Estimates of HPV prevalence in vulval intraepithelial neoplasia vary widely in the UK. The objective of this study was to assess HPV infection in a sample of women with vulval intraepithelial neoplasia, confirmed histologically, and determine the proportion of disease associated with HPV types targeted by prophylactic HPV vaccines. HPV infection was assessed in biopsies from 59 patients using the Greiner Bio-One PapilloCheck® DNA chip assay. Valid results were obtained for 54 cases. HPV infection was present in 43 of the 54 cases (79.6%: 95% CI 67.1-88.2%). The most common HPV types were HPV 16 (33/54: 61.1%), HPV 33 (8/54: 14.8%), HPV 6 (5/54: 9.3 %) and HPV 42 (3/54: 5.6%). The mean age of HPV positive women was significantly less than the mean age of HPV negative women. This is the largest UK series of vulval intraepithelial neoplasia in which HPV type has been investigated, and 34/54 (63.0 %, 95% CI: 49.6-78.6 %) cases were associated with HPV 16/18, which are targeted by current prophylactic HPV vaccines.

OBJECTIVES: To quantify the effect of demographic, psychological and disease-related factors on Quality of life (QoL) outcomes in women with high-grade vulval intraepithelial neoplasia (VIN2-3). To obtain qualitative data on the effect of disease and treatment in these women and their partners. To assess the participants' perception of their risk of developing of vulval cancer and its relation to QoL outcomes. METHODS: A questionnaire was constructed using existing instruments to measure the effect of demographic, psychological and disease-related factors on QoL outcomes. Free text space was provided for qualitative data. The questionnaire was mailed to women attending two specialist VIN clinics. RESULTS: One hundred and fifty women were invited for the study. Eighty-two responded (54.6%) of which forty-four (53.6%) were sexually active. Demographic factors (age and or living situation) had a significant effect on emotional health and body image. Psychological factors (anxiety and depression) had a significant effect on all aspects of QoL. Disease-related factors did not have a measurable effect on QoL outcomes, although the qualitative data revealed that various aspects of VIN had affected the lives of these women and their partners. There was a significant positive association between a perceived risk of developing vulval cancer with worsening general and emotional health. CONCLUSION: Psychological co-morbidity and various demographic factors should be considered while managing women with VIN. Accurate information regarding the development of vulval cancer should be given. The findings of this preliminary study will assist the construction of VIN-specific QoL instruments in the future.

Background: In 2003 the NHS Cervical Screening Programme in England changed the age at which women are first invited for cervical screening from 20 to 25 years. The aim of this review was to assess the evidence for benefit and harm of undertaking cervical screening in Wales for women aged 20–24 years. Method: A literature review looking for evidence of the effectiveness and potential harmful effects of cervical screening was undertaken. Welsh data for the number of cases of high-grade cervical intraepithelial neoplasia (CIN3) were examined and rates of invasive cervical cancer (1981–2003) for young women calculated. The medical notes of women less than 25 years old diagnosed with cervical cancer were reviewed and a cost analysis was performed. Results: The literature review failed to identify any randomized controlled studies of the effectiveness of cervical screening in young women, but demonstrated that organized screening programmes result in a decrease in the incidence of cervical carcinoma. Following the introduction of the Welsh organized call/recall cervical screening programme in 1988, cervical cancer has been reduced by 58% in women aged 20–24 years and 45% in women aged 25–29 years (mean age-specific rate per 100,000 women aged 20–24 years: 4.2 in 1981–88 compared with 2.2 in 1989–2003). If these changes can be attributed to the screening of women aged 20–24, then the costs of at least £82,500 are estimated to prevent one cervical cancer in this age group. Conclusions: The incidence of cervical cancer in young women has halved since the introduction of the Welsh-organized call–recall cervical screening programme. In Wales we recommend that women continue to be invited for cervical screening from 20 years of age. This will provide the information required to compare the incidence and stage at diagnosis of cervical cancer in young women invited for first time cervical screening at different ages across the UK.

This chapter discusses the importance of providing women with information about human papillomavirus and its role as the causative agent in the development of cervical cancer. Studies assessing current levels of knowledge among the general public are reviewed. What women need to know will be covered, followed by possible strategies for implementation. There are no randomised controlled trials to review regarding information for women about human papillomavirus but a number of well-conducted qualitative studies explore the issues that are faced both by the women undergoing cervical screening and their healthcare providers.

Persistent human papillomavirus (HPV) type 16 and 18 infection can lead to pre-malignant and malignant diseases of the lower genital tract. Several lines of evidence suggest that T cell responses can control HPV infection. However, relative to other human viruses, strong effector memory T cell responses against HPV have been difficult to detect. We used an in vitro stimulation step prior to enzyme-linked immunospot assays to identify IFN-γ-secreting T cells specific for HPV16 and 18 E6/E7 peptides. This allowed the detection of HPV-specific CD4+ T cells that were not evident in direct ex vivo assays. T cell responses against HPV16 or 18 peptides were detected in healthy volunteers (7/9) and patients with lower genital tract neoplasia (10/20). Importantly, this assay allowed tracking of vaccine-induced T cell responses in nine patients, following inoculation with a live recombinant vaccinia virus (HPV16 and 18 E6/E7, TA-HPV). Novel vaccine-induced T cell responses were demonstrated in five patients, but no clinical responses (lesion regressions) were seen. For one vaccinated patient, the T cell response was mapped to a single dominant HPV18 E7 epitope and this response was sustained for >3 years. Our data suggest that systemic memory T cells against HPV16 and 18, induced naturally or by TA-HPV vaccination, are relatively rare. Nevertheless, the assay system developed allowed estimation of magnitude, epitope specificity, and longevity of vaccine-induced CD4+ T cell responses. This will be useful for vaccine design and measurement of immunological endpoints in clinical trials.

The link between human papillomavirus (HPV) and cervical cancer raises the question, can the burden of cervical cancer be reduced using HPV vaccination strategies? The clinical trials to date will be reviewed, along with the challenges and potential for future development.

It has been proposed that women who have a negative colposcopic examination or who have no cervical intraepithelial neoplasia (CIN) on colposcopic biopsy can be safely returned to routine screening with the next visit being three or five years later. We present data regarding 551 women who had colposcopy in Wales for a low-grade cytological abnormality and who were followed through Cervical Screening Wales for subsequent CIN. Of 436 women declared CIN free initially, 26 (6.0%) had high-grade CIN diagnosed on follow-up. We suggest that additional screening at an interval of less than three years should be offered to women with a negative colposcopy or a biopsy without CIN.

The objective of this study was to determine the clinical effectiveness of a prime-boost human papillomavirus (HPV) vaccine regimen. A nonrandomized phase II prime-boost vaccine trial was conducted. Women with biopsy-proven anogenital intraepithelial neoplasia (AGIN) 3 were vaccinated with three doses of a recombinant fusion protein comprising HPV 16, E6/E7/L2 (TA-CIN) followed by one dose of a recombinant vaccinia virus encoding HPV 16 and 18 E6/E7 (TA-HPV). Clinical responses were evaluated by serial photographs, symptomatology, and biopsies before and after vaccination. Twenty-nine women were vaccinated; 27 with vulval intraepithelial neoplasia 3 and 2 with vaginal intraepithelial neoplasia grade 3. Clinical responses were seen in five women (17%), with one complete and five partial responses. Fifteen women (62%) had symptomatic improvement. No serious adverse effects were recorded. This is the first trial of a prime-boost vaccination regimen using heterologous HPV vaccines (TA-CIN followed by TA-HPV) in the management of AGIN. Since the prime-boost approach in this cohort offered no significant advantages over single TA-HPV vaccination, there are no further studies planned using this protocol. Future studies are warranted to define responders to immunotherapy.

Objectives. Cidofovir is an acyclic nucleoside phosphonate, with broad spectrum anti-viral activity. It has previously been used in a number of clinical settings, including high grade intraepithelial disease in the cervix and low grade vulval disease. This pilot study was set up to assess whether Cidofovir might be useful in treating high grade vulval intraepithelial neoplasia. Methods. Women with high grade non-cervical anogenital intraepithelial neoplasia were treated with a topical formulation of 1% Cidofovir in Unguentum Merck. Safety, side effects, clinical response and viral status were assessed. Clinical response was measured using symptoms, clinical (and photographic) appearance, viral status and histology. Results. 12 women with high grade vulval, vaginal or perianal intraepithelial neoplasia were recruited, 10 of whom completed follow up. Diseased tissue underwent ulceration in the majority of cases, with no effect seen on neighbouring normal skin. Four women had complete regression of disease, with resolution of long standing symptoms as well as histological and viral clearance. Three women had a partial response. Two women did not respond, and one woman made a complete symptomatic response but was found to have invasive disease at final biopsy. Conclusion. These complete responses, in women with long standing disease, together with preservation of normal tissue, suggest that topical treatment with Cidofovir may have a place in the therapeutic armamentarium of high grade vulval intraepithelial neoplasia.