Combined HPV 16 E2 and L1 methylation predict response to treatment with cidofovir and imiquimod in patients with vulval intraepithelial neoplasia.

BACKGROUND: Topical cidofovir and imiquimod can effectively treat approximately 55% of patients with vulval intraepithelial neoplasia (VIN), thus avoiding the need for surgery. Human papillomavirus (HPV) E⁢2 gene methylation predicts response to treatment but a methylation measurement is only obtainable in approximately 50% of patients. OBJECTIVE: This work aimed to determine if the applicability and predictive power of the E⁢2 methylation assay could be improved by combining it with the components of a host and viral DNA methylation panel (S5) that has been found to predict disease progression in patients with cervical intraepithelial neoplasia. METHODS: HPV E2 methylation and S5 classifier score were measured in fresh tissue samples collected pre-treatment from 132 patients with biopsy-proven VIN grade 3 who participated in a multicentre clinical trial and were randomised to treatment with cidofovir or imiquimod. RESULTS: Combining HPV16 E⁢2 and HPV16 L⁢1 methylation provides a biomarker that is both predictive of response to topical treatment and that can produce a clinically applicable result for all patients. Patients with HPV 16 L⁢1^high and HPV 16 E⁢2^high (36/132 (27.3%)) were more likely to respond to treatment with cidofovir (12/15 (80.0%)) than imiquimod (9/21 (42.9%)) (p= 0.026). Patients with HPV 16 L⁢1^low or HPV 16 E⁢2^low (including those with no HPV/unassessable methylation) were more likely to respond to imiquimod: 23/50 (46.0%) vs 31/46 (67.4%) (p= 0.035). CONCLUSIONS: Combined HPV E⁢2 and L⁢1 methylation is a potential predictive marker in treatment for all patients with VIN. These findings justify validation in a prospective trial.

Predicting regression of cervical intraepithelial neoplasia grade 2 in women under 25 years.

BACKGROUND: A number of retrospective and prospective studies have documented substantial rates of regression in cervical intraepithelial neoplasia grade 2 lesions in young women. Initial observational management of cervical intraepithelial neoplasia grade 2 is increasingly accepted as appropriate for women under 25 years of age with screen-detected abnormalities and is included in a number of clinical guidelines. However, there has been a paucity of large prospective studies on observational management with strict inclusion criteria. A number of important questions remain, specifically regarding the clinical variables that are associated with the risk of progression or persistence of disease. To investigate these factors and to ensure that young women with cervical intraepithelial neoplasia grade 2 undergoing observational management were being managed in a well-monitored and an appropriately informed fashion, we conducted a large, multicenter prospective study on observational management of cervical intraepithelial neoplasia grade 2 in women under 25 years. OBJECTIVE: This study aimed to determine the regression rates and clinical, cytologic, and pathologic predictors of regression of cervical intraepithelial neoplasia grade 2 in women under 25 years undergoing observational management over 24 months. STUDY DESIGN: This study was a multicenter prospective study on observational management of cervical intraepithelial neoplasia grade 2 (ie, repeat colposcopy, cytology, and cervical biopsy every 6 months) for up to 24 months. A total of 615 consenting women under 25 years with newly-diagnosed, biopsy-proven cervical intraepithelial neoplasia grade 2 were recruited (from 2010 to 2016) through 16 hospital-based colposcopy units in New Zealand and Australia. RESULTS: At completion, 326 women had confirmed regression, 156 had persistent high-grade cervical intraepithelial neoplasia grade 2 or 3 or adenocarcinoma in situ, and 24 had unconfirmed regression (ie, first regression at the 24-month follow-up). A total of 109 women did not complete the protocol (41 because of delayed follow-up, 41 lost to follow-up, 22 elected treatment, 4 refused a biopsy, and 1 died of an unrelated cause). Confirmed regression was observed in 53% (326 of 615) of all women enrolled in the study and, when missing data were imputed, it was estimated that 64% of women (95% confidence interval, 60%-68%) would have experienced regression. Similarly, lesions regressed in 64% (326 of 506) of women who completed the observational protocol. Based on a multivariable analysis, detection of human papillomavirus 16 in a liquid-based cytology sample at the time of initial colposcopy decreased the chance of regression by 31% (risk ratio, 0.69; 95% confidence interval, 0.56-0.86; P<.001). In addition, at initial colposcopy, low-grade or normal colposcopic impression, later year of diagnosis, low-grade or normal cytology, and being a nonsmoker were all independently associated with an increased chance of regression. CONCLUSION: More than half of women under 25 years with cervical intraepithelial neoplasia grade 2 will regress to cervical intraepithelial neoplasia grade 1 or normal within 24 months without destructive treatment. The absence of human papillomavirus 16 is the most important predictor of regression.

Complete pathological response following levonorgestrel intrauterine device in clinically stage 1 endometrial adenocarcinoma: Results of a randomized clinical trial.

PURPOSE: Intrauterine levonorgestrel (LNG-IUD) is used to treat patients with endometrial adenocarcinoma (EAC) and endometrial hyperplasia with atypia (EHA) but limited evidence is available on its effectiveness. The study determined the extent to which LNG-IUD with or without metformin (M) or weight loss (WL) achieves a pathological complete response (pCR) in patients with EAC or EHA. PATIENTS AND METHODS: This phase II randomized controlled clinical trial enrolled patients with histologically confirmed, clinically stage 1 FIGO grade 1 EAC or EHA; a body mass index > 30 kg/m2; a depth of myometrial invasion of less than 50% on MRI; a serum CA125 ≤ 30 U/mL. All patients received LNG-IUD and were randomized to observation (OBS), M (500 mg orally twice daily), or WL (pooled analysis). The primary outcome measure was the proportion of patients developing a pCR (defined as absence of any evidence of EAC or EHA) after 6 months. RESULTS: From December 2012 to October 2019, 165 patients were enrolled and 154 completed the 6-months follow up. Women had a mean age of 53 years, and a mean BMI of 48 kg/m2. Ninety-six patients were diagnosed with EAC (58%) and 69 patients with EHA (42%). Thirty-five participants were randomized to OBS, 36 to WL and 47 to M (10 patients were withdrawn). After 6 months the rate of pCR was 61% (95% CI 42% to 77%) for OBS, 67% (95% CI 48% to 82%) for WL and 57% (95% CI 41% to 72%) for M. Across the three treatment groups, the pCR was 82% and 43% for EHA and EAC, respectively. CONCLUSION: Complete response rates at 6 months were encouraging for patients with EAC and EHA across the three groups. TRIAL REGISTRATION: U.S. National Library of Medicine, NCT01686126.

Cytopathological review of cervical pathology: Impact for women and follow-up results.

BACKGROUND: Cervical screening programs have had an important effect on the reduction of cervical cancer rates. Comprehensive programs require access to pathological review to improve the sensitivity of screening cytology and the specificity of diagnostic histology. AIMS: To determine the number of cases where cervical cytology or histology was amended at cytopathological review; whether amendments were 'upgrades' or 'downgrades', and how amendments aligned with follow-up results for these patients. MATERIALS AND METHODS: A retrospective cohort study was performed of all patients reviewed from January 2016 to December 2017 (n = 287 cases, from 254 patients) at colposcopy multidisciplinary meetings at Wellington Hospital, a tertiary referral hospital. Where amendments to cytology or histology were made, follow-up results were retrieved where available (85.7% and 84.2% respectively). RESULTS: Cytology or histology was amended in 24.7% of cases. Smear cytology was amended in 16.7%. Where cytology was upgraded (n = 9), 44% had subsequent results of equal or higher grade including one case of adenocarcinoma. Where cytology was downgraded (n = 19), 93.8% (81.9-100%) had follow-up studies showing equal or lower results. Cervical biopsy histology was amended in 12.2% of cases (upgraded n = 19, downgraded n = 6). Large loop excision of the transformation zone or cone biopsy histology was amended in three cases (7.9%). CONCLUSIONS: Cytopathological review appears to improve the specificity of the comprehensive cervical screening program, leading to a reduction in unnecessary treatment. Additionally, a small number of cases of malignant or premalignant disease were detected.

Pathological process has a crucial role in sentinel node biopsy for vulvar cancer.

OBJECTIVES: To report the interim findings of an audit of the outcomes of sentinel node (SN) biopsy performed as a replacement for groin node dissection in women with early stage vulvar cancer in routine clinical practice in Australia and New Zealand. METHODS: A prospective multi-center study in 8 participating centers. Eligible patients had squamous cell carcinomas clinically restricted to the vulva <4 cm in diameter. SN procedures and pathological assessment were to be performed in accordance with the methods published by the GROINSS-V collaboration [1]. RESULTS: 130 women with apparent early stage vulvar cancer were enrolled. Seventeen women subsequently did not meet the eligibility criteria and were excluded. SNs were identified in 111/113 of the remaining women. Twenty-two women had positive nodes. Sixteen of these women had at least 12 months follow up and 7 (44%) had recurrent disease. Eighty-nine women had only negative nodes. Seventy-four of these women had at least 12 months follow up and 6 (8%) had recurrent disease (including 2 [2.7%] with recurrence in the groin). On subsequent review of the two women with negative SNs who had groin recurrences, it was found that the recommended pathology protocol had not been followed. In both cases, SN metastases were identified following serial sectioning of the nodes. CONCLUSIONS: SN biopsy is feasible in routine clinical practice. However, undetected metastases in a removed SN may be associated with groin recurrence. To ensure patient safety, strict adherence to the pathology protocol is an essential component in the utilization of the sentinel lymph node technique in vulvar cancer.

Patterns of referral and uptake of BReast CAncer (BRCA) gene testing of eligible women with ovarian cancer in New Zealand.

AIMS: To determine the proportion of eligible patients with high-grade serous carcinoma of the ovary, fallopian tube or peritoneum discussed at gynaecological oncology multidisciplinary meetings (MDMs) in New Zealand and subsequently referred for genetic counselling and BRCA pathogenic variant testing. METHODS: Eligible cases were identified from Auckland, Wellington, Christchurch and Dunedin gynaecologic oncology MDM databases between 1 January 2015 to 31 December 2016. Patients who met the eligibility criteria for genetics referral were identified, and cross-referenced against genetic services databases to ascertain the rates of referrals received, the numbers attending appointments, genetic testing offered and range of results. RESULTS: During the two-year period, 205 patients were eligible for referral. Of these, 143 (70%) patients were referred for genetic counselling with 128 (90%) of this group recommended for BRCA pathogenic variant testing. Of the 126 who undertook the test, results were available for 120 (95%). Nineteen patients (16%) tested positive for a germline BRCA pathogenic variant. CONCLUSIONS: The New Zealand rate of referral to genetic counselling for women with high-grade serous cancer, (HGSC), of the ovary, fallopian tube or peritoneum diagnosed between 2015-2016 is encouraging when compared with others internationally. The rate of BRCA positive pathogenic variants is comparable to international data.

Human Papillomavirus DNA Methylation Predicts Response to Treatment Using Cidofovir and Imiquimod in Vulval Intraepithelial Neoplasia 3.

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 <4% predicted response with 70.6% sensitivity and 62.5% specificity.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.

Human papillomavirus type 16 L1/L2 DNA methylation shows weak association with cervical disease grade in young 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 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.

Activity, safety, and feasibility of cidofovir and imiquimod for treatment of vulval intraepithelial neoplasia (RT³VIN): a multicentre, open-label, randomised, phase 2 trial.

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.

Increased methylation of Human Papillomavirus type 16 DNA correlates with viral integration in Vulval Intraepithelial Neoplasia.

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.

mRNA sequencing of novel cell lines from human papillomavirus type-16 related vulval intraepithelial neoplasia: consequences of expression of HPV16 E4 and E5.

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.

PMPA and PMEA prodrugs for the treatment of HIV infections and human papillomavirus (HPV) associated neoplasia and cancer.

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.

UK population based study to predict impact of HPV vaccination.

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.

Quantitative measurement of Human Papillomavirus type 16 L1/L2 DNA methylation correlates with cervical disease grade.

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.

Impact of human papillomavirus-related genital diseases on quality of life and psychosocial wellbeing: results of an observational, health-related quality of life study in the UK.

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.

HPV16 L1 and L2 DNA methylation predicts high-grade cervical intraepithelial neoplasia in women with mildly abnormal cervical cytology.

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.

Should human papillomavirus DNA testing be offered in combination with cytology or as a sole primary screening test in cervical cancer prevention?

Evaluation of: Rijkaart DC, Berkhof J, van Kemenade FJ et al. HPV DNA testing in population-based cervical screening (VUSA-Screen study): results and implications. Br. J. Cancer 106(5), 975-981 (2012). Previous studies have shown that the combination of high-risk human papillomavirus (HPV) testing and cytology increases sensitivity for the detection of cervical intraepithelial neoplasia 3, a surrogate end point for the reduction of cervical cancer. This paper addressed three questions regarding the implementation of high-risk HPV testing within a cervical screening program. The main question addressed was whether high-risk HPV testing should be provided as a standalone primary screen or in combination with cytology. Management of HPV-positive women and the optimum age for HPV testing were also examined. Results identified limited benefit from co-testing compared with HPV testing alone and emphasized the importance of repeat testing for HPV-positive women with negative cytology triage at baseline, in all women from 30 years of age onwards.

Single negative colposcopy: is it enough to rule out high-grade disease?

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.

Human Papillomavirus genotype testing combined with cytology as a 'test of cure' post treatment: the importance of a persistent viral infection.

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.