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Editorial from Primary hrHPV DNA Testing in Cervical Cancer Screening: How to Manage Screen-Positive Women? A POBASCAM Trial Substudy

Recently, DNA methylation analysis of FAM19A4 in cervical scrapes has been shown to adequately detect high-grade cervical intraepithelial neoplasia and cervical cancer (≥ CIN3) in high-risk HPV (hrHPV)-positive women. Here, we compared the clinical performance of FAM19A4 methylation analysis to cytology and HPV16/18 genotyping, separately and in combination, for ≥ CIN3 detection in hrHPV-positive women participating in a prospective observational multi-center cohort study. The study population comprised hrHPV-positive women aged 18-66 years, visiting a gynecological outpatient clinic. From these women, cervical scrapes and colposcopy-directed biopsies (for histological confirmation) were obtained. Cervical scrapes were analyzed for FAM19A4 gene promoter methylation, cytology and HPV16/18 genotyping. Methylation analysis was performed by quantitative methylation-specific PCR (qMSP). Sensitivities and specificities for ≥ CIN3 were compared between tests. Stratified analyses were performed for variables that potentially influence marker performance. Of all 508 hrHPV-positive women, the sensitivities for ≥ CIN3 of cytology, FAM19A4 methylation analysis, and cytology combined with HPV16/18 genotyping were 85.6, 75.6 and 92.2%, respectively, with corresponding specificities of 49.8, 71.1 and 29.4%, respectively. Both sensitivity and specificity of FAM19A4 methylation analysis were associated with age (p ≤ 0.001 each). In women ≥ 30 years (n = 287), ≥ CIN3 sensitivity of FAM19A4 methylation analysis was 88.3% (95%CI: 80.2-96.5) which was noninferior to that of cytology [85.5% (95%CI: 76.0-94.0)], at a significantly higher specificity [62.1% (95%CI: 55.8-68.4) compared to 47.6% (95%CI: 41.1-54.1)]. In conclusion, among hrHPV-positive women from an outpatient population aged ≥ 30 years, methylation analysis of FAM19A4 is an attractive marker for the identification of women with ≥ CIN3.

Objective To study the source of human papillomavirus (HPV) in semen. Design Observational study (CCMO-NL3248800010). Setting Academic hospital-based laboratory. Patient(s) Healthy male volunteers (n = 213). Intervention(s) One penile scrape and three semen samples were obtained per participant for HPV-DNA testing by both GP5+/6+ polymerase chain reaction (PCR) and SPF10-PCR to detect moderate/high and low viral loads, respectively; flat penile lesions (FPL) were detected by penoscopy. Main Outcome Measure(s) HPV-DNA presence in semen and penile scrapes, and the presence of FPL. Result(s) HPV-DNA at moderate/high viral loads (i.e., GP5+/6+ PCR-positive) was detected in ≥1 semen sample(s) in 27% of participants. Most men with moderate/high viral loads in the penile scrape also had moderate/high viral loads in semen (85%). Men with a HPV-negative penile scrape were very unlikely to have moderate/high viral loads in semen (3%). The presence of HPV in semen was associated with the presence of HPV in the penile scrape also on a genotype-specific level. Having FPL was a risk factor for HPV presence in semen. Conclusion(s) HPV-DNA presence in semen of healthy men is common and associated with HPV infections of the penile epithelium. HPV-DNA presence in semen may result from desquamation of HPV-infected penile cells.

Objectives: The interpretation of cervical biopsy specimens guides management of women with suspected cervical cancer precursors. However, morphologic evaluation is subjective and has low interobserver agreement. Addition of p16INK4a immunohistochemistry may improve interpretation. Methods: We performed a systematic review and meta-analysis of published data on interobserver agreement of p16INK4a positivity using p16INK4a immunohistochemistry and of cervical intraepithelial neoplasia grade 2 (CIN2+) and CIN grade 3 (CIN3+) classification using H&E morphology in conjunction with p16INK4a in comparison with H&E morphology alone. Results: The literature search revealed five eligible articles. The results show strong agreement of pathologists’ interpretation of cervical biopsy specimens as p16INK4a positive or negative (pooled κ = 0.90; 95% confidence interval [CI], 0.88–0.92) and significantly higher agreement for a CIN2+ diagnosis with H&E morphology in conjunction with p16INK4a (κ = 0.73; 95% CI, 0.67–0.79) compared with H&E morphology alone (κ = 0.41; 95% CI, 0.17–0.65). Also, a slightly higher agreement for CIN3+ can be observed (κ = 0.66; 95% CI, 0.39–0.94 for H&E morphology in conjunction with p16INK4a and κ = 0.61; 95% CI, 0.44–0.78 for H&E morphology alone), but this difference was not statistically significant. Conclusions: The published literature indicates improved interobserver agreement of the diagnosis of CIN2+ with the conjunctive use of H&E morphology with p16INK4a immunohistochemistry compared with H&E morphology alone.

Cytology-based nation-wide cervical screening has led to a substantial reduction of the incidence of cervical cancer in western countries. However, the sensitivity of cytology for the detection of high-grade precursor lesions or cervical cancer is limited; therefore, repeated testing is necessary to achieve program effectiveness. Additionally, adenocarcinomas and its precursors are often missed by cytology. Consequently, there is a need for a better screening test. The insight that infection with high-risk human papillomavirus (hrHPV) is the causal agent of cervical cancer and its precursors has led to the development of molecular tests for the detection of hrHPV. Strong evidence now supports the use of hrHPV testing in the prevention of cervical cancer. In this review, we will discuss the arguments in favor of, and concerns on aspects of implementation of hrHPV testing in primary cervical cancer screening, such as the age to start hrHPV-based screening, ways to increase screening attendance, requirements for candidate hrHPV tests to be used, and triage algorithms for screen-positive women.

OBJECTIVES To provide an early risk assessment of extending screening intervals beyond five years for a human papillomavirus (HPV) based cervical screening programme in the Netherlands. DESIGN 14 year follow-up of a population based randomised cohort from the POBASCAM randomised trial. SETTING Organised cervical screening in the Netherlands, based on a programme of three screening rounds (each round done every five years). PARTICIPANTS 43 339 women aged 29-61 years with a negative HPV and/or negative cytology test participating in the POBASCAM trial. INTERVENTIONS Women randomly assigned to HPV and cytology co-testing (intervention) or cytology testing only (control), and managed accordingly. MAIN OUTCOME MEASURES Cumulative incidence of cervical cancer and cervical intraepithelial neoplasia (CIN) grade 3 or worse (CIN3+). Associations with age were expressed as incidence rate ratios. In HPV positive women, reductions in CIN3+ incidence after negative cytology, HPV type 16/18 genotyping, and/or repeat cytology were estimated. RESULTS The cumulative incidence of cervical cancer (0.09%) and CIN3+ (0.56%) among HPV negative women in the intervention group after three rounds of screening were similar to the cumulative among women with negative cytology in the control group after two rounds (0.09% and 0.69%, respectively). Cervical cancer and CIN3+ risk ratios were 0.97 (95% confidence interval 0.41 to 2.31, P=0.95) and 0.82 (0.62 to 1.09, P=0.17), respectively. CIN3+ incidence was 72.2% (95% confidence interval 61.6% to 79.9%, Pfive years) should be implemented with risk stratification.

Background: High-risk human papillomavirus (hrHPV)-positive women require triage to identify those with cervical high-grade intraepithelial neoplasia and cancer (>= CIN3 (cervical intraepithelial neoplasia grade 3)). FAM19A4 methylation analysis, which detects advanced CIN and cancer, is applicable to different sample types. However, studies comparing the performance of FAM19A4 methylation analysis in hrHPV-positive self-samples and paired physician-taken scrapes are lacking. Methods: We compared the performance of FAM19A4 methylation analysis (and/or HPV16/18 genotyping) in self-samples and paired physician-taken scrapes for >= CIN3 detection in hrHPV-positive women (n = 450,18-66 years). Results: Overall FAM19A4 methylation levels between sample types were significantly correlated, with strongest correlation in women with >= CIN3 (Spearman's rho 0.697, P= 30 years, >= CIN3 sensitivity of FAM19A4 methylation analysis was 78.4% in self-samples and 88.2% in scrapes (ratio 0.89; CI: 0.75-1.05). In women = CIN3 sensitivities were 37.5% and 45.8%, respectively (ratio 0.82; CI: 0.55-1.21). In both groups, >= CIN3 specificity of FAM19A4 methylation analysis was significantly higher in self-samples compared with scrapes. Conclusions: FAM19A4 methylation analysis in hrHPV-positive self-samples had a slightly lower sensitivity and a higher specificity for >= CIN3 compared with paired physician-taken scrapes. With a similarly good clinical performance in both sample types, combined FAM19A4 methylation analysis and HPV16/18 genotyping provides a feasible triage strategy for hrHPV-positive women, with direct applicability on self-samples.

a b s t r a c t Background: Studies have shown that self-sampling for hrHPV testing (HPV self-sampling) is highly acceptable to women, increases screening participation rate, and may therefore further reduce cervi- cal cancer incidence. However, it is important to clinically validate HPV self-sampling procedures for screening purposes. Objectives: Clinical validation of combined brush-based self-sampling with GP5+/6+-PCR EIA for primary cervical screening. In addition, HPV type-specific agreement between sample types and acceptability of brush-based self-sampling were evaluated. Study design: 135 women referred for colposcopy took a self-sample at home prior to vaginal- and cervical sampling by a gynaecologist. All women were biopsied for histology. HPV testing was done by GP5+/6+- PCR EIA, with genotyping by reverse line blotting (RLB). Acceptability of sampling methods was measured with a questionnaire. Results: In this outpatient population, hrHPV test results showed good concordance between self-samples and physician-taken cervical scrapes (86%, k = 0.70), with sensitivities and specificities for CIN2+ that did not differ significantly (93% and 51%, 91% and 51%, respectively (P = 1.0)). The clinical sensitivity of brush-based self-sampling combined with GP5+/6+-PCR EIA hrHPV testing for detection of CIN2+ was non-inferior to that of hrHPV testing on physician-taken cervical samples (P = 0.018). In addition, hrHPV genotyping results were highly concordant between sample types, with almost perfect agreement for HPV16 (k = 0.81) and HPV18 (k = 0.92). Finally, 91% of participants described brush-based self-sampling as easy-to-use. Conclusions: Brush-based self-sampling in combination with GP5+/6+-PCR EIA hrHPV testing is acceptable to women and valid for assessing the risk of CIN2+ in comparison to hrHPV testing on physician-taken scrapes. In addition, there was high concordance of HPV genotyping results. Therefore, this HPV self

Women who test positive for a high-risk type of the human papillomavirus (HPV) require triage testing to identify those women with cervical intraepithelial neoplasia grade 3 or cancer (>= CIN3). Although Pap cytology is considered an attractive triage test, its applicability is hampered by its subjective nature. This study prospectively compared the clinical performance of p16/Ki-67 dual-stained cytology to that of Pap cytology, with or without HPV16/18 genotyping, in high-risk HPV-positive women visiting gynecologic outpatient clinics (n=446 and age 18-66 years). From all women, cervical scrapes (for Pap cytology, HPV16/18 genotyping, and p16/Ki-67 dual-stained cytology) and colposcopy-directed biopsies were obtained. The sensitivity of p16/Ki-67 dual-stained cytology for >= CIN3 (93.8%) did neither differ significantly from that of Pap cytology (87.7%; ratio 1.07 and 95% confidence interval (CI): 0.97-1.18) nor from that of Pap cytology combined with HPV16/18 genotyping (95.1%; ratio 0.99 and 95% CI: 0.91-1.07). However, the specificity of p16/Ki-67 dual-stained cytology for >= CIN3 (51.2%) was significantly higher than that of Pap cytology (44.9%; ratio 1.14 and 95% CI: 1.01-1.29) and Pap cytology combined with HPV16/18 genotyping (25.8%; ratio 1.99 and 95% CI: 1.68-2.35). After exclusion of women who had been referred because of abnormal Pap cytology, the specificity of p16/Ki-67 dual-stained cytology for >= CIN3 (56.7%) remained the same, whereas that of Pap cytology (60.3%) increased substantially, resulting in a similar specificity of both assays (ratio 0.94 and 95% CI: 0.83-1.07) in this sub-cohort. In summary, p16/Ki-67 dual-stained cytology has a good clinical performance and is an interesting objective microscopy-based triage tool for high-risk HPV-positive women.

Recently, DNA methylation analysis of FAM19A4 in cervical scrapes has been shown to adequately detect high-grade cervical intraepithelial neoplasia and cervical cancer (≥ CIN3) in high-risk HPV (hrHPV)-positive women. Here, we compared the clinical performance of FAM19A4 methylation analysis to cytology and HPV16/18 genotyping, separately and in combination, for ≥ CIN3 detection in hrHPV-positive women participating in a prospective observational multi-center cohort study. The study population comprised hrHPV-positive women aged 18-66 years, visiting a gynecological outpatient clinic. From these women, cervical scrapes and colposcopy-directed biopsies (for histological confirmation) were obtained. Cervical scrapes were analyzed for FAM19A4 gene promoter methylation, cytology and HPV16/18 genotyping. Methylation analysis was performed by quantitative methylation-specific PCR (qMSP). Sensitivities and specificities for ≥ CIN3 were compared between tests. Stratified analyses were performed for variables that potentially influence marker performance. Of all 508 hrHPV-positive women, the sensitivities for ≥ CIN3 of cytology, FAM19A4 methylation analysis, and cytology combined with HPV16/18 genotyping were 85.6, 75.6 and 92.2%, respectively, with corresponding specificities of 49.8, 71.1 and 29.4%, respectively. Both sensitivity and specificity of FAM19A4 methylation analysis were associated with age (p ≤ 0.001 each). In women ≥ 30 years (n = 287), ≥ CIN3 sensitivity of FAM19A4 methylation analysis was 88.3% (95%CI: 80.2-96.5) which was noninferior to that of cytology [85.5% (95%CI: 76.0-94.0)], at a significantly higher specificity [62.1% (95%CI: 55.8-68.4) compared to 47.6% (95%CI: 41.1-54.1)]. In conclusion, among hrHPV-positive women from an outpatient population aged ≥ 30 years, methylation analysis of FAM19A4 is an attractive marker for the identification of women with ≥ CIN3.

Background: High-risk human papillomavirus (hrHPV) DNA positive women require triage testing to identify those with high-grade cervical intraepithelial neoplasia or cancer (>= CIN2). Objective: Comparing three triage algorithms (1) E7 mRNA testing following HPV16/18/31/33/45/52/58 genotyping (E7 mRNA test), (2) HPV16/18 DNA genotyping and (3) cytology, for >CIN2 detection in hrHPV DNA-positive women. Study design: hrHPV DNA-positive women aged 18-63 years visiting gynecology outpatient clinics were included in a prospective observational cohort study. From these women a cervical scrape and colposcopy-directed biopsies were obtained. Cervical scrapes were evaluated by cytology, HPV DNA genotyping by bead-based multiplex genotyping of GP5+6+-PCR-products, and presence of HPV16/18/31/33/45/52/58 E7 mRNA using nucleic acid sequence-based amplification (NASBA) in DNA positive women for respective HPV types. Sensitivities and specificities for >= CIN2 were compared between E7 mRNA test and HPV16/18 DNA genotyping in the total group (n = 348), and E7 mRNA test and cytology in a subgroup of women referred for non-cervix-related gynecological complaints (n = 133). Results: Sensitivity for >= CIN2 of the E7 mRNA test was slightly higher than that of HPV16/18 DNA genotyping (66.9% versus 60.9%; ratio 1.10, 95% CI: 1.0002-1.21), at similar specificity (54.8% versus 52.3%; ratio 1.05, 95% CI: 0.93-1.18). Neither sensitivity nor specificity of the E7 mRNA test differed significantly from that of cytology (sensitivity: 68.8% versus 75.0%; ratio 0.92, 95% CI: 0.72-1.17; specificity: 59.4% versus 65.3%; ratio 0.91, 95% CI: 0.75-1.10). Conclusion: For detection of >= CIN2 in hrHPV DNA-positive women, an algorithm including E7 mRNA testing following HPV16/18/31/33/45/52/58 DNA genotyping performs similar to HPV16/18 DNA genotyping or cytology. (C) 2015 Elsevier B.V. All rights reserved.

The interpretation of cervical biopsy specimens guides management of women with suspected cervical cancer precursors. However, morphologic evaluation is subjective and has low interobserver agreement. Addition of p16(INK4a) immunohistochemistry may improve interpretation.We performed a systematic review and meta-analysis of published data on interobserver agreement of p16(INK4a) positivity using p16(INK4a) immunohistochemistry and of cervical intraepithelial neoplasia grade 2 (CIN2+) and CIN grade 3 (CIN3+) classification using HE morphology in conjunction with p16(INK4a) in comparison with HE morphology alone.The literature search revealed five eligible articles. The results show strong agreement of pathologists' interpretation of cervical biopsy specimens as p16(INK4a) positive or negative (pooled κ = 0.90; 95% confidence interval [CI], 0.88-0.92) and significantly higher agreement for a CIN2+ diagnosis with HE morphology in conjunction with p16(INK4a) (κ = 0.73; 95% CI, 0.67-0.79) compared with HE morphology alone (κ = 0.41; 95% CI, 0.17-0.65). Also, a slightly higher agreement for CIN3+ can be observed (κ = 0.66; 95% CI, 0.39-0.94 for HE morphology in conjunction with p16(INK4a) and κ = 0.61; 95% CI, 0.44-0.78 for HE morphology alone), but this difference was not statistically significant.The published literature indicates improved interobserver agreement of the diagnosis of CIN2+ with the conjunctive use of HE morphology with p16(INK4a) immunohistochemistry compared with HE morphology alone.

Background: High-risk human papillomavirus (hrHPV) testing has higher sensitivity but lower specificity than cytology for cervical (pre)-cancerous lesions. Therefore, triage of hrHPV-positive women is needed in cervical cancer screening. Methods: A cohort of 1,100 hrHPV-positive women, from a population-based screening trial (POBASCAM: n = 44,938; 29–61 years), was used to evaluate 10 triage strategies, involving testing at baseline and six months with combinations of cytology, HPV16/18 genotyping, and/or repeat hrHPV testing. Clinical endpoint was cervical intraepithelial neoplasia grade 3 or worse (CIN3+) detected within four years; results were adjusted for women not attending repeat testing. A triage strategy was considered acceptable, when the probability of no CIN3+ after negative triage (negative predictive value, NPV) was at least 98%, and the CIN3+ risk after positive triage (positive predictive value, PPV) was at least 20%. Results: Triage at baseline with cytology only yielded an NPV of 94.3% [95% confidence interval (CI), 92.0–96.0] and a PPV of 39.7% (95% CI, 34.0–45.6). An increase in NPV, against a modest decrease in PPV, was obtained by triaging women with negative baseline cytology by repeat cytology (NPV 98.5% and PPV 34.0%) or by baseline HPV16/18 genotyping (NPV 98.8% and PPV 28.5%). The inclusion of both HPV16/18 genotyping at baseline and repeat cytology testing provided a high NPV (99.6%) and a moderately high PPV (25.6%). Conclusions: Triaging hrHPV-positive women by cytology at baseline and after 6 to 12 months, possibly in combination with baseline HPV16/18 genotyping, seems acceptable for cervical cancer screening. Impact: Implementable triage strategies are provided for primary hrHPV screening in an organized setting. Cancer Epidemiol Biomarkers Prev; 23(1); 55–63. ©2013 AACR.

Contains fulltext : 118480.pdf (Publisher’s version ) (Open Access) BACKGROUND: Attendance rates of cervical screening programs can be increased by offering HPV self-sampling to non-attendees. Acceptability, DNA yield, lavage volumes and choice of hrHPV test can influence effectiveness of the self-sampling procedures and could therefore play a role in recruiting non-attendees. To increase user-friendliness, a frequently used lavage sampler was modified. In this study, we compared this second generation lavage device with the first generation device within similar birth cohorts. METHODS: Within a large self-sampling cohort-study among non-responders of the Dutch cervical screening program, a subset of 2,644 women received a second generation self-sampling lavage device, while 11,977 women, matched for age and ZIP-code, received the first generation model. The second generation device was different in shape, color, lavage volume, and packaging, in comparison to its first generation model. The Cochran's test was used to compare both devices for hrHPV positivity rate and response rate. To correct for possible heterogeneity between age and ZIP codes in both groups the Breslow-Day test of homogeneity was used. A T-test was utilized to compare DNA yields of the obtained material in both groups. RESULTS: Median DNA yields were 90.4 mug/ml (95% CI 83.2-97.5) and 91.1 mug/ml (95% CI 77.8-104.4, p= 0.726) and hrHPV positivity rates were 8.2% and 6.9% (p= 0.419) per sample self-collected by the second - and the first generation of the device (p= 0.726), respectively. In addition, response rates were comparable for the two models (35.4% versus 34.4%, p= 0.654). CONCLUSIONS: Replacing the first generation self-sampling device by an ergonomically improved, second generation device resulted in equal DNA yields, comparable hrHPV positivity rates and similar response rates. Therefore, it can be concluded that the clinical performance of the first and second generation models are similar. Moreover, participation of non-attendees in cervical cancer screening is probably not predominantly determined by the type of self-collection device.

Study question Is the presence of human papillomavirus (HPV) in semen associated with impairment of semen quality? Summary answer In a large cohort of males seeking fertility evaluation, no associations were observed between seminal HPV presence and semen parameters. What is known already HPV is commonly detected in semen samples. Whether the presence of HPV is related to impairment of semen quality, remains unclear. Study design, size, duration This cross-sectional study included a cohort of 430 males. Participants/materials, setting, methods Male partners in couples seeking fertility evaluation provided one semen sample per person. Semen samples were tested for HPV-DNA using GP5+/6+-PCR. Sperm concentration was counted and motility was assessed in a Makler counting chamber at a magnification of ×200. The presence of antisperm antibodies was assessed by a mixed agglutination reaction (MAR)-test. Main results and the role of chance Overall HPV was detected in 14.9% (64/430) of semen samples, including 2.1% (9/430) that contained both high-risk (hr) HPV and low-risk (lr) HPV types, 8.8% (38/430) with exclusively hrHPV types and 4.0% (17/430) with exclusively lrHPV types. The presence of HPV in semen was not associated with the age of the participants, seminal pH, semen volume, total sperm count, sperm concentration, progressive motility or the presence of antisperm antibodies. Limitations, reasons for caution This study did not observe an association between HPV presence in semen and impairment of semen quality. However, we cannot exclude an effect of seminal HPV on early embryo development and clinical reproductive outcomes. Wider implications of the findings As HPV is frequently present in semen, screening of donor semen for HPV should be considered to prevent iatrogenic cervical HPV infections in the recipient. However our findings do not support standardized HPV testing of semen in the diagnostic work-up of subfertile couples. Study funding/competing interests This study was sponsored by an unrestricted grant of Stichting Researchfonds Pathology Amsterdam, the Netherlands. P.J.F.S. has been on the speakers bureau of Roche, Gen-Probe, Abbott, Qiagen and Seegene and has been a consultant for Crucell B.V. J.B. has been on the speakers bureau of Qiagen and has been a consultant for Roche, DDL Diagnostic Laboratory, GlaxoSmithKline and Merck. D.A.M.H. has been member of the scientific advisory boards of Amgen and Pfizer, and has been on the speakers bureau of Hologic/Gen-Probe. C.J.L.M.M. has been on the speakers bureau of GlaxoSmithKline, Qiagen, Merck, Roche, Menarini and Seegene, has served occasionally on the scientific advisory board of GlaxoSmithKline, Qiagen, Merck, Roche and Genticel, and has occasionally been a consultant for Qiagen. Formerly, C.J.L.M.M. was a minority shareholder of Delphi Biosciences, which bankrupted in 2014. C.J.L.M.M. is a minority shareholder of Diassay B.V. P.J.F.S., D.A.M.H. and C.J.L.M.M. have minority stake in Self-Screen B.V., a spin-off company of VU University Medical Center. R.L., M.G.D., P.G.A.H., D.T.M.P., and I.H. do not have any conflicts of interest to disclose. Trial registration number Not applicable.