Methylation markers FAM19A4 and miR124-2 as triage strategy for primary human papillomavirus screen positive women: A large European multicenter study.

In human papillomavirus (HPV) cervical cancer screening, cytology is used as triage to counter the low specificity of HPV testing. VALID-SCREEN is a EU-multicenter, retrospective study conducted to evaluate the clinical performance of the FAM19A4/miR124-2 methylation-based molecular triage test as a substitute or addition to cytology as reflex testing of HPV screen positive women. FAM19A4/miR124-2 methylation test (QIAsure Methylation Test) was evaluated in 2384 HPV-positive cervical screening samples, from women 29-76 years of age, derived from four EU countries. Specimens were collected in ThinPrep or SurePath media, HPV-status, concurrent cytology, and histology diagnosis were provided by the parent institutes. The control population consisted of women with no evidence of disease within 2 years of follow-up. A total of 899 histologies were retrieved; 527 showed no disease, 124 CIN2 (5.2%), 228 CIN3 (9.6%) and 20 cervical cancers (0.8%); 19 of 20 screen-detected cervical cancers were found methylation-positive (sensitivity 95%). Overall specificity of FAM19A4/miR124-2 methylation test was 78.3% (n = 2013; 95%CI: 76-80). The negative predictive value of hrHPV positive, methylation-negative outcomes were 99.9% for cervical cancer (N = 1694; 95%CI: 99.6-99.99), 96.9% for ≥CIN3 (95%CI: 96-98), and 93.0% for ≥CIN2 (95%CI: 92-94). Overall sensitivity for CIN3 using FAM19A4/miR124-2 methylation test was 77% (n = 228; 95%CI: 71-82). CIN3 sensitivity was uniform between centers independent of sample collection medias, DNA extraction methods and HPV screening tests. Being objectively reported compared to the subjectivity of cytology, equally performing across settings and screening methods, the FAM19A4/miR124-2 methylation constitute an alternative/supplement to cytology as triage method to be investigated in real-life pilot implementation.

p53 and p16 expression profiles in vulvar cancer: a translational analysis by the Arbeitsgemeinschaft Gynäkologische Onkologie Chemo and Radiotherapy in Epithelial Vulvar Cancer study group.

BACKGROUND: There are 2 known pathways for tumorigenesis of vulvar squamous cell carcinoma-a human papillomavirus-dependent pathway characterized by p16 overexpression and a human papillomavirus-independent pathway linked to lichen sclerosus, characterized by TP53 mutation. A correlation of human papillomavirus dependency with a favorable prognosis has been proposed. OBJECTIVE: The objective of the study was to further understand the role of human papillomavirus and p53 status in vulvar squamous cell carcinoma and characterize its clinical relevance. STUDY DESIGN: The Arbeitsgemeinschaft Gynaecological Oncology Chemo and Radiotherapy in Epithelial Vulvar Cancer-1 study is a retrospective cohort study of 1618 patients with primary vulvar squamous cell carcinoma Fédération Internationale de Gynécologie et d'Obstétrique stage ≥1B treated at 29 gynecologic cancer centers in Germany between 1998 and 2008. For this translational substudy, formalin-fixed paraffin-embedded tissue was collected. A tissue microarray was constructed (n=652 samples); p16 and p53 expression was determined by immunohistochemistry. Human papillomavirus status and subtype were analyzed by polymerase chain reaction. RESULTS: p16 immunohistochemistry was positive in 166 of 550 tumors (30.2%); p53 staining in 187 of 597 tumors (31.3%). Only tumors with available information regarding p16 and p53 immunohistochemistry and without p53 silent expression pattern were further analyzed (n=411); 3 groups were defined: p53+ (n=163), p16+/p53- (n=132), and p16-/p53- (n=116). Human papillomavirus DNA was detected in 85.6% of p16+/p53- tumors; human papillomavirus-16 was the most common subtype (86.3%). Patients with p16+ tumors were younger (64 vs 72 years for p53+, respectively, 69 years for p16-/p53- tumors; P<.0001) and showed lower rates of lymph-node involvement (28.0% vs 42.3% for p53+, respectively, 30.2% for p16-/p53- tumors; P=.050). Notably, 2-year-disease-free and overall survival rates were significantly different among the groups: disease-free survival, 47.1% (p53+), 60.2% (p16-/p53-), and 63.9% (p16+/p53-) (P<.001); overall survival, 70.4% (p53+), 75.4% (p16-/p53-), and 82.5% (p16+/p53-) (P=.002). In multivariate analysis, the p16+/p53- phenotype showed a consistently improved prognosis compared with the other groups (hazard ratio, 0.66; 95% confidence interval, 0.44-0.99; P=.042). CONCLUSION: p16 overexpression is associated with an improved prognosis whereas p53 positivity is linked to an adverse outcome. Our data support the hypothesis of a clinically relevant third subgroup of vulvar squamous cell carcinoma with a p53-/p16- phenotype showing an intermediate prognosis that needs to be further characterized.

Significant decline of HPV 6 infection and genital warts despite low HPV vaccination coverage in young women in Germany: a long-term prospective, cohort data analysis.

BACKGROUND: The introduction of human papillomavirus (HPV) vaccination has resulted in a remarkable decline of genital warts in women and men, but in Germany historical rates of vaccination are relatively low. We report long-term surveillance data on changes in HPV 6 and HPV 11 infection and the prevalence of genital warts in young women in the Wolfsburg HPV epidemiological study (WOLVES). METHODS: Women born in 1983/84, 1988/89, and 1993/94 participated in four cohorts between 2009/10 and 2014/15. Quadrivalent vaccination coverage and prevalence of HPV 6/11 infection and genital warts are reported for participants aged 19-22 years and 24-27 years at the time of sample collection. Statistical analyses were done to compare similarly aged participants using 2 × 2 contingency tables (Röhmel-Mansmann unconditional exact test; two-side alpha of 0.05). RESULTS: A total of 2456 women were recruited. Between 2010 and 2015, there was a statistically significant decrease in the prevalence of HPV 6 infection among women aged 24-27 years (2.1% versus 0.0%; P < 0.0001) and women aged 19-22 years (2.0% versus 0.0%; P = 0.0056). There was no significant decline in HPV 11 infection. In total, 52 of 2341 participants were diagnosed with genital warts. There was a statistically significant drop in the risk of developing genital warts in women aged 24-27 years between 2010 and 2015 (4.7% versus 1.7%, respectively; P = 0.0018). The overall risk of developing genital warts in women aged 19-27 years decreased from 3.1% in 2010 to 1.2% in 2015 (P = 0.0022). CONCLUSIONS: An increase in vaccination coverage was associated with a decreased prevalence of genital warts in young women. A protective effect greater than herd immunity alone was seen despite low vaccination rates. Quadrivalent vaccine had a protective effect on genital HPV 6 infection and an almost fully protective effect on the development of genital warts in the youngest population.

German evidence and consensus-based (S3) guideline: Vaccination recommendations for the prevention of HPV-associated lesions.

Anogenital and oropharyngeal infections with human papilloma viruses (HPV) are common. Clinically manifest disease may significantly impact quality of life; the treatment of HPV-associated lesions is associated with a high rate of recurrence and invasive neoplasms, such as cervical, anal, vulvar, penile, and oropharyngeal cancers, which are characterized by significant morbidity and mortality. Vaccination against HPV is an effective and safe measure for the primary prevention of HPV-associated lesions, but immunization rates are still low in Germany. The present publication is an abridged version of the German evidence and consensus-based guideline "Vaccination recommendations for the prevention of HPV-associated lesions", which is available on the website of the German Association of the Scientific Medical Societies (AWMF). On the basis of a systematic review with meta-analyses, a representative panel developed and agreed upon recommendations for the vaccination of different populations against HPV. In addition, consensus-based recommendations were developed for specific issues relevant to everyday practice. Based on current evidence and a representative expert consensus, these recommendations are intended to provide guidance in a field in which there is often uncertainty and in which both patients and health care providers are sometimes confronted with controversial and emotionally charged points of view.

Association of genomic variants at the human leukocyte antigen locus with cervical cancer risk, HPV status and gene expression levels.

The human leukocyte antigen (HLA) locus on chromosome 6 has been reported to be associated with cervical cancer. We investigated two independent single-nucleotide polymorphisms in a large case-control series of cervical dysplasia and carcinoma that has been newly established by the German Cervigen Consortium, comprising a total of 2481 cases and 1556 healthy females. We find significant associations for both variants, rs9272117 at HLA-DQA1 and rs2844511 at MICA and HCP5, with cervical disease. Both variants showed evidence of association with invasive cervical cancer (rs9272117: OR 0.89, 95% CI 0.79-0.99, P = .036; rs2844511: OR 1.17, 95% CI 1.04-1.31, P = .008) and with high-grade dysplasia (rs9272117: OR 0.78, 95% CI 0.70-0.87, P = 7.1 × 10-6 ; rs2844511: OR 1.13, 95% CI 1.01-1.26, P = .035), as well as in a combined analysis of both groups (rs9272117: OR 0.83, 95% CI 0.75-0.91, P = 6.9 × 10-5 ; rs2844511: OR 1.14, 95% CI 1.04-1.26, P = .005). Variant rs2844511, but not rs9272117, also showed modest evidence of association with low-grade dysplasia (OR 1.26, 95% CI 1.04-1.54, P = .019). In case-only analyses, rs2844511 tended to predict HPV status (P = .044) and rs9272117 tended to associate with HPV16 (P = .022). RNA studies in cervical samples showed a significant correlation in the transcript levels of MICA, HCP5 and HLA-DQA1, suggesting extensive co-regulation. All three genes were upregulated in HPV16-positive samples. In stratified analyses, rs9272117 was associated with HLA-DQA1 levels, specifically in HPV-positive samples, while rs2844511 was associated with MICA and HCP5 levels. The risk allele of rs2844511 was required for correlations between MICA or HCP5 with HLA-DQA1. Altogether, our results support 6p21.32-33 as the first consistent cervical cancer susceptibility locus and provide evidence for a link between genetic risk variants, HPV16 status and transcript levels of HLA-DQA1, HCP5 and MICA, which may contribute to tumor immune evasion.

FAM19A4/miR124-2 methylation in invasive cervical cancer: A retrospective cross-sectional worldwide study.

Widespread adoption of primary human papillomavirus (HPV)-based screening has encouraged the search for a triage test which retains high sensitivity for the detection of cervical cancer and precancer, but increases specificity to avoid overtreatment. Methylation analysis of FAM19A4 and miR124-2 genes has shown promise for the triage of high-risk (hr) HPV-positive women. In our study, we assessed the consistency of FAM19A4/miR124-2 methylation analysis in the detection of cervical cancer in a series of 519 invasive cervical carcinomas (n = 314 cervical scrapes, n = 205 tissue specimens) from over 25 countries, using a quantitative methylation-specific PCR (qMSP)-based assay (QIAsure Methylation Test®). Positivity rates stratified per histotype, FIGO stage, hrHPV status, hrHPV genotype, sample type and geographical region were calculated. In total, 510 of the 519 cervical carcinomas (98.3%; 95% CI: 96.7-99.2) tested FAM19A4/miR124-2 methylation-positive. Test positivity was consistent across the different subgroups based on cervical cancer histotype, FIGO stage, hrHPV status, hrHPV genotype, sample type and geographical region. In conclusion, FAM19A4/miR124-2 methylation analysis detects nearly all cervical carcinomas, including rare histotypes and hrHPV-negative carcinomas. These results indicate that a negative FAM19A4/miR124-2 methylation assay result is likely to rule out the presence of cervical cancer.

Reduction of cervical cancer incidence within a primary HPV screening pilot project (WOLPHSCREEN) in Wolfsburg, Germany.

BACKGROUND: Randomised controlled trials showed human papillomavirus (HPV)-based screening leads to a significant reduction in cervical cancer incidence compared with cytology-based screening only. METHODS: Non-hysterectomised participants ≥30 years underwent co-testing with Papanicolaou (Pap) smear and HR-HPV testing (Hybrid Capture 2; HC2). Women with normal findings had their next screening round after 5 years, and HC2+ and Pap abnormal cases were immediately referred for colposcopy, while cases with discordant findings had repeat testing after 12 months with referral to colposcopy in cases with persistent positive findings. RESULTS: Twenty-six thousand six hundred and twenty-four women were recruited between February 2006 and December 2016. Two hundred and seventy-four CIN3+ cases were diagnosed (270 HPV+, 4 HPV-), including 31 invasive cervical cancers (29 HPV+, 2 HPV-). No CIN3+ was detected in HPV- women with abnormal cytology. We observed a significant decline in the 5-year incidence of CIN3+ (from 0.96% [95% CI 0.85-1.09%] to 0.16% [95% CI 0.10-0.25%]; p < 0.0001) and cervical cancer (from 0.10% [95% CI 0.07%-0.15%] to 0.025% [95% CI 0.01-0.08%]; p = 0.01) between the first and subsequent rounds. Approximately 90% (246/274) of CIN3+ cases were diagnosed at first colposcopy. CONCLUSIONS: The decline in disease rates with 5-yearly co-testing seems mainly attributable to HPV testing since no CIN3+ occurred in HPV-/Pap+ women.

Intra- and inter-laboratory agreement of the FAM19A4/mir124-2 methylation test: Results from an international study.

BACKGROUND: HPV-based cervical screening detects women at an increased risk of cervical cancer and precancer. To differentiate among HPV-positive women those with (pre)cancer, triage testing is necessary. The detection of cancer-associated host-cell DNA methylation (FAM19A4 and hsa-mir124-2) in cervical samples has shown valuable as triage test. This multicenter study from 6 collaborating European laboratories and one reference laboratory was set out to determine the intra- and inter-laboratory agreement of FAM19A4/mir124-2 DNA methylation analysis utilizing the QIAsure Methylation Test. METHODS: Agreement analysis for the QIAsure Methylation Test was assessed on high-risk HPV-positive cervical specimens (n = 1680) both at the level of the assay and at the full workflow, including bisulfite conversion. RESULTS: Intra- and inter-laboratory assay agreement were 91.4% (534/584; 95% CI 88.9-93.5; κ = 0.82) and 92.5% (369/399; 95% CI 90.0-94.7; κ = 0.83), respectively. The inter-laboratory workflow (bisulfite conversion and assay combined) agreement was 90.0% (627/697; 95% CI 87.5%-92.0%; κ = 0.76). CONCLUSION: These data show that the QIAsure Methylation Test performs robust and reproducible in different laboratory contexts. These results support the use of the QIAsure Methylation Test for full molecular screening for cervical cancer, including primary HPV testing and triage testing by methylation analysis.

Sustained Immunogenicity of 2-dose Human Papillomavirus 16/18 AS04-adjuvanted Vaccine Schedules in Girls Aged 9-14 Years: A Randomized Trial.

Background: We previously reported the noninferiority 1 month after the last dose of 2-dose human papillomavirus 16/18 AS04-adjuvanted (AS04-HPV-16/18) vaccine schedules at months 0 and 6 (2D_M0,6) and months 0 and 12 (2D_M0,12) in girls aged 9-14 years compared with a 3-dose schedule at months 0, 1, and 6 (3D_M0,1,6) in women aged 15-25 years. Here, we report the results at study end (month 36 [M36]). Methods: Girls were randomized 1:1 and received 2 vaccine doses either 6 months (2D_M0,6) or 12 months apart (2D_M0,12); women received 3 doses at months 0, 1, and 6 (3D_M0,1,6). Endpoints included noninferiority of HPV-16/18 antibodies for 2D_M0,6 versus 3D_M0,1,6; 2D_M0,12 versus 3D_M0,1,6; and 2D_M0,12 versus 2D_M0,6; and assessment of neutralizing antibodies, T cells, B cells, and safety. Results: At M36, the 2D_M0,6 and 2D_M0,12 schedules remained noninferior to the 3D_M0,1,6 schedule in terms of seroconversion rates and 3D/2D geometric mean titers for anti-HPV-16 and anti-HPV-18. All schedules elicited sustained immune responses up to M36. Conclusions: Both 2-dose schedules in young girls remained noninferior to the 3-dose schedule in women up to study conclusion at M36. The AS04-HPV-16/18 vaccine administered as a 2-dose schedule was immunogenic and well tolerated in young girls.

Incomplete excision of cervical precancer as a predictor of treatment failure: a systematic review and meta-analysis.

BACKGROUND: Incomplete excision of cervical precancer is associated with therapeutic failure and is therefore considered as a quality indicator of clinical practice. Conversely, the risk of preterm birth is reported to correlate with size of cervical excision and therefore balancing the risk of adequate treatment with iatrogenic harm is challenging. We reviewed the literature with an aim to reveal whether incomplete excision, reflected by presence of precancerous tissue at the section margins, or post-treatment HPV testing are accurate predictors of treatment failure. METHODS: We did a systematic review and meta-analysis to assess the risk of therapeutic failure associated with the histological status of the margins of the tissue excised to treat cervical precancer. We estimated the accuracy of the margin status to predict occurrence of residual or recurrent high-grade cervical intraepithelial neoplasia of grade two or worse (CIN2+) and compared it with post-treatment high-risk human papillomavirus (HPV) testing. We searched for published systematic reviews and new references from PubMed-MEDLINE, Embase, and CENTRAL and did also a new search spanning the period Jan 1, 1975, until Feb 1, 2016. Studies were eligible if women underwent treatment by excision of a histologically confirmed CIN2+ lesion, with verification of presence or absence of CIN at the resection margins; were tested by cytology or HPV assay between 3 months and 9 months after treatment; and had subsequent follow-up of at least 18 months post-treatment including histological confirmation of the occurrence of CIN2+. Primary endpoints were the proportion of positive section margins and the occurrence of treatment failure associated with the marginal status, in which treatment failure was defined as occurrence of residual or recurrent CIN2+. Information about positive resection margins and subsequent treatment failure was pooled using procedures for meta-analysis of binomial data and analysed using random-effects models. FINDINGS: 97 studies were eligible for inclusion in the meta-analysis and included 44 446 women treated for cervical precancer. The proportion of positive margins was 23·1% (95% CI 20·4-25·9) overall and varied by treatment procedure (ranging from 17·8% [12·9-23·2] for laser conisation to 25·9% [22·3-29·6] for large loop excision of the transformation zone) and increased by the severity of the treated lesion. The overall risk of residual or recurrent CIN2+ was 6·6% (95% CI 4·9-8·4) and was increased with positive compared with negative resection margins (relative risk 4·8, 95% CI 3·2-7·2). The pooled sensitivity and specificity to predict residual or recurrent CIN2+ was 55·8% (95% CI 45·8-65·5) and 84·4% (79·5-88·4), respectively, for the margin status, and 91·0% (82·3-95·5) and 83·8% (77·7-88·7), respectively, for high-risk HPV testing. A negative high-risk HPV test post treatment was associated with a risk of CIN2+ of 0·8%, whereas this risk was 3·7% when margins were free. INTERPRETATION: The risk of residual or recurrent CIN2+ is significantly greater with involved margins on excisional treatment; however, high-risk HPV post-treatment predicts treatment failure more accurately than margin status. FUNDING: European Federation for Colposcopy and Institut national du Cancer (INCA).

Evaluating HPV-negative CIN2+ in the ATHENA trial.

A post hoc analysis of the ATHENA study was performed to determine whether true HPV-negative cervical lesions occur and whether they have clinical relevance. The ATHENA database was searched for all CIN2 or worse (CIN2+) cases with cobas HPV-negative results and comparison was made with Linear Array (LA) and Amplicor to detect true false-negative HPV results. Immunostaining with p16 was performed on these cases to identify false-positive histology results. H&E slides were re-reviewed by the study pathologists with knowledge of patient age, HPV test results and p16 immunostaining. Those with positive p16 immunostaining and/or a positive histopathology review underwent whole tissue section HPV PCR by the SPF10/LiPA/RHA system. Among 46,887 eligible women, 497 cases of CIN2+ were detected, 55 of which tested negative by the cobas(®) HPV Test (32 CIN2, 23 CIN3/ACIS). By LA and/or Amplicor, 32 CIN2+ (20 CIN2, 12 CIN3/ACIS) were HPV positive and categorized as false-negatives by cobas HPV; nine of 12 false-negative CIN3/ACIS cases were p16+. There were 23 cases (12 CIN2, 11 CIN3/ACIS) negative by all HPV tests; seven of 11 CIN3/ACIS cases were p16+. H&E slides were available for six cases for re-review and all were confirmed as CIN3/ACIS. Tissue PCR was performed on the six confirmed CIN3/ACIS cases (and one without confirmation): four were positive for HPV types not considered oncogenic, two were positive for oncogenic genotypes and one was indeterminate. In summary, subanalysis of a large cervical cancer screening study did not identify any true CIN3/ACIS not attributable to HPV.

A randomized study of hexaminolevulinate photodynamic therapy in patients with cervical intraepithelial neoplasia 1/2.

OBJECTIVE: The objective of the study was to investigate the efficacy and safety of hexaminolevulinate (HAL) photodynamic therapy (PDT), a novel therapy for women with cervical intraepithelial neoplasia (CIN)1/2, to define the appropriate population and endpoints for a phase 3 program. STUDY DESIGN: This was a double-blind, randomized, placebo-controlled, dose-finding study that included a total of 262 women with biopsy-confirmed CIN 1/2 based on local pathology. Patients received 1 or 2 topical treatments of HAL hydrochloride 0.2%, 1%, 5%, and placebo ointment and were evaluated for response after 3-6 months based on biopsy, Papanicolaou test, and oncogenic human papillomavirus (HPV) test. All efficacy analyses were performed on blinded central histology review to avoid interreader variability. Adverse events, blood biochemistry, and vital signs were assessed after 3 months. RESULTS: There were no statistically significant differences between placebo and either the CIN 1 or combined CIN 1/2 populations. A clear dose effect with a statistically significant response in the HAL 5% group of 95% (18/19 patients) compared to 57% (12/21 patients) in the placebo group (P < .001) was observed at 3 months in women with CIN 2, including an encouraging 83% (5/6 patients) clearance of HPV 16/18 compared to 33% (2/6 patients) in the placebo group at 6 months. The treatment was easy to use and well accepted by patients and gynecologists. Only local self-limiting adverse reactions including discharge, discomfort, and spotting were reported. CONCLUSION: HAL PDT is a novel therapy that shows promise in the treatment of CIN 2 including clearance of oncogenic HPV, but not of CIN 1. The positive risk/benefit balance makes HAL PDT a tissue-preserving alternative in women of childbearing age who wish to preserve the cervix. Confirmatory studies are planned.

Utility and Reproducibility of the International Federation for Cervical Pathology and Colposcopy Classification of Transformation Zones in Daily Practice: A Multicenter Study of the German Colposcopy Network.

OBJECTIVE: To compare the distribution of International Federation for Cervical Pathology and Colposcopy (IFCPC) transformation zone (TZ) types among women in different age groups referred to 8 colposcopy clinics. MATERIALS AND METHODS: Between February 2012 and February 2013, we prospectively collected individual patient data from 8 clinics within the German Colposcopy Network (G-CONE). Data were analyzed using ODSdysplasie, software designed to allow continuous quality assessment in colposcopy clinics. The distribution of IFCPC-classified TZ was compared between different centers for the following age groups: younger than 30 years, between 30 and 50 years, and older than 50 years. RESULTS: Of 3,761 patients included in the analysis, 2,153 (57%) were classified as having type 2 TZ, 906 (24%) as type 1 TZ, and 702 (19%) as type 3 TZ. Type 3 TZ was the most commonly reported type in women older than 50 years (70%). We found that the relative distribution of type 3 TZ between age groups was similar in the participating colposcopy clinics. However, there was evidence of heterogeneous distribution of types 1 and 2 TZ between age groups in different clinics, ranging from 7.8% to 66.4% for type 1 TZ in women younger than 30 years and 28.9% to 78.1% for type 2 TZ in women 30 to 50 years old. CONCLUSIONS: Although IFCPC type 3 TZ seems to be a reproducible finding, the distribution of types 1 and 2 TZ showed significant heterogeneity. A more precise anatomic distinction between types 1 and 2 TZ in the IFCPC terminology could improve reporting of colposcopy findings.

Early detection of CIN3 and cervical cancer during long-term follow-up using HPV/Pap smear co-testing and risk-adapted follow-up in a locally organised screening programme.

We evaluated compliance with human papillomavirus (HPV) testing and risk-adapted patient pathways and monitored changes in high-grade cervical disease during long-term follow-up. Women aged >30 years attending routine screening for cervical cancer were managed according to results from first-round screening tests (cytology and high-risk HPV; Hybrid Capture 2). Between February 2006 and January 2011, 19,795 of 19,947 women agreed to participate, of whom 4,067 proceeded to a second screening round 5 years after recruitment. Predefined endpoints were compliance, grade 3 cervical intraepithelial neoplasia or cancer (CIN3+), new HPV infection, HPV persistence and abnormal smears in round 2. A total of 765 of 19,795 women (3.9%) in round 1 and 41 of 4,067 (1.0%) in round 2 were referred for colposcopy. Compliance rates with colposcopy were 93.1 and 92.7%, respectively, while histological assessment was performed in 680 of 712 (95.5%) and 36 of 38 (94.7%), respectively. CIN3+ rates were 172 of 19,795 (0.87%; 95% confidence intervals: 0.7-1.0) in round 1 and 2 of 4,064 (0.05%; 95% confidence intervals: 0.006-0.2) in round 2; the difference was statistically significant (Fisher's exact test, p<0.001). After 5 years, the incidence of new HPV infection was 124 of 3,906 (3.2%) and HPV persistence was observed in 22 of 161 (13.7%). Locally organised HPV/cytology co-testing is feasible and acceptable to women. Risk-adapted management rapidly detected a high rate of prevalent CIN3+, while the subsequent long-term risk of new high-grade cervical disease was surprisingly low. It remains unclear if this phenomenon is explained by CIN3 mostly occurring early in life or by modifying the natural course of HPV infection with colposcopy and histological assessment.

Laser-skinning colpectomy for extended vaginal intraepithelial neoplasia and microinvasive cancer.

OBJECTIVE: The aim of this study is to analyze the efficacy of colposcopic-guided laser-skinning colpectomy to treat extended high-grade vaginal intraepithelial neoplasia (VaIN). METHODS: Retrospective review of 33 heavily pretreated patients with high-grade VaIN extending over 20-100% of the vaginal surface treated between 2003 and 2013 with colposcopic-guided laser-skinning colpectomy. The vaginal epithelium including all VaIN lesions was excised in one piece with a depth of 2-3mm. RESULTS: Vaginal cancer was diagnosed in 10 patients (nine microinvasive squamous cell carcinoma and one vaginal carcinoma). No serious adverse events related to laser-skinning colpectomy were observed. Of 33 patients, 23 were followed up with cytology and colposcopy for at least 12months at our institution (median follow 26.5months; range 12-104months), while five had a shorter follow-up, four an external follow-up and one patient was lost. Of 23 patients with follow-up ≥12months, 20 were disease free after a single laser-skinning colpectomy (overall cure rate 87.0%). Moderate shortening of the vagina was observed in two patients and another two required reconstruction of vaginal strictures during long-term follow-up. CONCLUSION: Laser-skinning colpectomy appears to be a feasible treatment for extended high-risk VaIN3. The procedure avoids the mutilation associated with colpectomy and allows early diagnosis and staging of invasive disease.

Treatment of extramammary Paget disease of the vulva with imiquimod: a retrospective, multicenter study by the German Colposcopy Network.

BACKGROUND: Extramammary Paget disease (EMPD) is a very rare genital neoplasia associated with a high frequency of local recurrences. Surgical excision is the standard treatment, but results in mutilating procedures in patients with advanced or recurrent disease. Case reports have shown clinical responses to imiquimod in patients with EMPD, but this therapy has not been evaluated systematically. OBJECTIVE: The aim of this study was to evaluate imiquimod as local treatment of first-time and recurrent EMPD. METHODS: All cases of biopsy-proven EMPD of the vulva treated within the German Colposcopy Network or other institutions specializing in vulvar diseases in Germany were included in this retrospective analysis. RESULTS: A total of 21 women with EMPD treated with imiquimod were identified: 11 (52.4%) achieved complete response, 6 (28.6%) achieved partial response, and there were no cases of progressive disease. The dose and duration of imiquimod differed between patients. The mean duration of treatment exceeded 16 weeks in women achieving complete response. LIMITATIONS: EMPD is rare and this retrospective study is limited by the small number of patients identified. CONCLUSION: When associated cancers and invasive growth are excluded, imiquimod appears to be a useful treatment option for recurrent EMPD and may avoid extensive mutilating surgical treatment.

Efficacy and safety of hexaminolevulinate photodynamic therapy in patients with low-grade cervical intraepithelial neoplasia.

OBJECTIVE: Non-surgical therapies are needed to reduce the rate of progression of low-grade cervical intraepithelial neoplasia (CIN 1) to high grade CIN (CIN 2/3). The aim of this study was to assess the efficacy and safety of hexaminolevulinate (HAL) photodynamic therapy (PDT) in the treatment of patients with CIN 1. STUDY DESIGN: This phase IIa prospective double-blind study randomized patients with CIN 1 into three groups: HAL vaginal suppository, placebo vaginal suppository or follow-up only. Patients in the first two groups received HAL or placebo suppositories 5 hours before illumination with 50 J/cm(2) red coherent light (633 nm) using a special light catheter. All patients had a follow up including colposcopy, cytology and human papilloma virus (HPV) testing 3 and 6 months and additional biopsy 6 months after PDT. The main outcome measure was efficacy, defined as complete histologic remission 6 months after PDT. Secondary outcomes were histologic remission 3 months and HPV eradication 6 months after first PDT. RESULTS: Seventy patients were randomized: 47 to HAL, 12 to placebo, 11 to follow up only. After 6 months CIN lesions had cleared in 57% of patients in the HAL-PDT group compared to 25% in the combined control group (per protocol population, P = 0.04). Twenty-six patients (37%) reported 44 adverse events (AEs), of which 40 were mild or moderate. Nineteen treatment-related AEs were reported by 15 patients (32%) in the HAL PDT group, one in the placebo PDT group (8%), and none in the follow-up group. The most common adverse events were local discomfort including mild pain/cramping (11) and leucorrhoea (2). CONCLUSION: HAL PDT shows a favorable efficacy and safety profile and represents a promising alternative to observation and surgical procedures in patients with CIN 1.

Annual Papanicolaou screening for 5 years among human papillomavirus-negative women.

BACKGROUND: Primary human papilloma virus (HPV) screening is more effective than cytology in reducing the risk of cervical cancer, but screening intervals should be extended in HPV-negative women. However, some Markov models predicted that long intervals are associated with an excess risk of cervical cancer. The aim of this analysis was to estimate the real-life risks and benefits of annual Papanicolaou (Pap) screening in HPV-negative women with normal cytology. METHODS: Women with negative Hybrid Capture 2 (HC2) results and normal cytology at the time of inclusion in the Hannover HPV screening trial underwent annual Pap smears for 5 years. A subgroup was randomly selected for retesting with cytology, HC2, and colposcopy 60-68 months after recruitment. RESULTS: Of 4236 women included, 3406 had at least one Pap smear, but only 1185 attended all five annual screening visits. The proportion of women with at least one abnormal smear was 14.4% in 60 months. The probability of abnormal smears increased continuously over time. No case of ≥ CIN2+ was observed during 5 years. Of 605 women selected for subgroup analysis, 292 agreed to be retested (48.3%). The rate of high-risk HPV at 60-68 months was 3.0% (9/296). CONCLUSIONS: The long-term risk of high-grade neoplasia after an initial negative HC2 test and normal cytology result was low, while the rate of false-positive abnormal Pap smears was significant and increased constantly over time. Pap smear screening of HPV-negative women more frequently than every 5 years could be potentially harmful and seems to be of little clinical value.

Accuracy of colposcopy management to detect CIN3 and invasive cancer in women with abnormal screening tests: results from a primary HPV screening project from 2006 to 2011 in Wolfsburg, Germany.

OBJECTIVE: Combining HPV and Pap screening achieves very good risk stratification and sensitive detection of CIN3 and cancer (CIN3 +), but poorer specificity, and may result in an increased risk of glandular and new lesions during follow-up. We examined if this phenomenon may compromise the accuracy of colposcopy. METHODS: As part of a primary HPV screening pilot project comprising 19,624 participants aged over 30 years, the failure rate to detect CIN3 at first visit was measured over a five-year period to assess the quality of colposcopy as an overall management concept. Management relied on excisional biopsies in all HSIL cytology or major findings on colposcopy, endocervical assessment in type 3 transformation zones (TZ) and guided biopsies in type 1 or 2 TZ. RESULTS: Of 667 women referred for colposcopy because of atypical Pap smears and/or HPV persistency, 171 were diagnosed with CIN3+. All 18 cancers and 140/153 CIN3 cases were diagnosed at the first visit. Of 13 CIN3 observed during follow-up, five were classified as new cases, five as definite and three as probably colposcopy failures, giving a failure rate of 4.7% (8/171). Only three failures were related to false-negative punch biopsies while five occurred because of false-negative endocervical assessment in type 3 TZ. CONCLUSIONS: Colposcopy management following defined pathways was safe in this HPV screening program with an acceptable failure rate. Further improvements may depend on developing better methods for endocervical assessment rather than for ectocervical biopsies.