Determination of viral load thresholds in cervical scrapings to rule out CIN 3 in HPV 16, 18, 31 and 33-positive women with normal cytology

An increased high-risk human papillomavirus (hrHPV) viral load in cervical scrapings has been proposed as a determinant for high-grade cervical intraepithelial neoplasia (CIN) and cervical cancer (> or =CIN 2), but data so far for HPV types different from HPV 16 are limited and inconsistent. In addition, a viral load threshold to distinguish hrHPV positive women without > or =CIN 2 still has not been defined. Here, we used baseline cervical scrapings of women with normal cytology participating in a large population-based cervical screening trial (i.e. POBASCAM) who were GP5+/6+-PCR positive for 4 common hrHPV types, i.e. HPV 16, 18, 31 or 33, as a reference to arbitrarily define various viral load thresholds (i.e. 25th, 33rd, 50th, 67th and 75th percentiles of the lowest viral load values) for distinguishing women having single infections with these types without high-grade CIN. Viral load assessment was performed by real time type-specific PCR. The viral load threshold values were subsequently validated on abnormal cervical scrapes of 162 women with underlying, histologically confirmed CIN lesions containing 1 of these 4 hrHPV types. All 59 women with CIN 3 had viral load levels that were higher than those of 33% of the women with normal cytology containing the respective hrHPV type detectable by GP5+/6+-PCR (i.e. higher than the 33rd percentile of viral load). By using this 33rd percentile viral load cut-off, sensitivity for CIN 3 of 100% (95% CI 93.9-100) was obtained. Hence, application of this viral load threshold would increase the specificity of HPV testing for HPV 16, 18, 31 and 33-associated prevalent CIN 3 without the cost of a marked reduction in sensitivity. In practice, on the basis of viral load analysis, a less aggressive management can be foreseen for 33% of the women with normal cytology participating in a population-based screening program who are GP5+/6+-PCR positive for HPV 16, 18, 31 or 33.     

Risk of high-grade cervical intra-epithelial neoplasia based on cytology and high-risk HPV testing at baseline and at 6-months

Adding a test for high-risk human papillomavirus (hrHPV) to cytological screening enhances the detection of high-grade cervical intraepithelial neoplasia (>or=CIN2), but data are required that enable long-term evaluation of screening. We investigated the >or=CIN2 risk for women participating in population-based screening as a function of hrHPV and cytology testing results at baseline and at 6 months. We included 2,193 women aged 30-60 years participating in a population-based screening trial who received colposcopy or a repeat testing advice at baseline. The main endpoint was histologically confirmed >or=CIN2 diagnosed within 36 months. hrHPV testing was more sensitive than cytology for >or=CIN2 (relative sensitivity 1.4, 95%CI: 1.3-1.5; absolute sensitivity 94.1 and 68.0%, respectively). The 18-month >or=CIN2 risks in women with a hrHPV-positive smear and in women with abnormal cytology were similar (relative risk 0.9, 95%CI: 0.8-1.1). Women with HPV16 and/or HPV18 had a higher >or=CIN2 risk than other hrHPV-positive women irrespective of the cytological grade. Repeat testing showed that both cytological regression and viral clearance were strongly associated with a decrease in >or=CIN2 risk. Notably, women who had a double negative repeat test at 6 months had a >or=CIN2 risk of only 0.2% (95%CI: 0.0-1.1) and hrHPV-negative women with baseline borderline or mild dyskaryosis and normal cytology at 6 months had a >or=CIN2 risk of 0% (95%CI: 0.0-0.8). Using hrHPV and/or cytology testing, risk of >or=CIN2 can be assessed more accurately by repeat testing than single visit testing. Hence, when hrHPV testing is implemented, patient management with repeat testing is a promising strategy to control the number of referrals for colposcopy.     

Reliable identification of women with CIN3+ using hrHPV genotyping and methylation markers in a cytology-screened referral population

Cervical screening aims to identify women with high-grade squamous intraepithelial lesion/cervical intraepithelial neoplasia 2-3 (HSIL/CIN2-3) or invasive cervical cancer (ICC). Identification of women with severe premalignant lesions or ICC (CIN3+) could ensure their rapid treatment and prevent overtreatment. We investigated high-risk human papillomavirus (hrHPV) detection with genotyping and methylation of FAM19A4/miR124-2 for detection of CIN3+ in 538 women attending colposcopy for abnormal cytology. All women had an additional cytology with hrHPV testing (GP5+/6+-PCR-EIA+), genotyping (HPV16/18, HPV16/18/31/45), and methylation analysis (FAM19A4/miR124-2) and at least one biopsy. CIN3+ detection was studied overall and in women <30 (n = 171) and ≥30 years (n = 367). Positivity for both rather than just one methylation markers increased in CIN3, and all ICC was positive for both. Overall sensitivity and specificity for CIN3+ were, respectively, 90.3% (95%CI 81.3–95.2) and 31.8% (95%CI 27.7–36.1) for hrHPV, 77.8% (95%CI 66.9–85.8) and 69.3% (95%CI 65.0–73.3) for methylation biomarkers and 93.1% (95%CI 84.8–97.0) and 49.4% (95%CI 44.8–53.9) for combined HPV16/18 and/or methylation positivity. For CIN3, hrHPV was found in 90.9% (95%CI 81.6–95.8), methylation positivity in 75.8% (95%CI 64.2–84.5) and HPV16/18 and/or methylation positivity in 92.4% (95%CI 83.5–96.7). In women aged ≥30, the sensitivity of combined HPV16/18 and methylation was increased (98.2%, 95%CI 90.6–99.7) with a specificity of 46.3% (95%CI 40.8–51.9). Combination of HPV16/18 and methylation analysis was very sensitive and offered improved specificity for CIN3+, opening the possibility of rapid treatment for these women and follow-up for women with potentially regressive, less advanced, HSIL/CIN2 lesions.     

Age-dependent prevalence of 14 high-risk HPV types in the Netherlands: implications for prophylactic vaccination and screening

We determined the prevalence of type-specific hrHPV infections in the Netherlands on cervical scrapes of 45 362 women aged 18-65 years. The overall hrHPV prevalence peaked at the age of 22 with peak prevalence of 24%. Each of the 14 hrHPV types decreased significantly with age (P-values between 0.0009 and 0.03). The proportion of HPV16 in hrHPV-positive infections also decreased with age (OR=0.76 (10-year scale), 95% CI=0.67-0.85), and a similar trend was observed for HPV16 when selecting hrHPV-positive women with cervical intraepithelial neoplasia grade 2 or worse (CIN2+) (OR=0.76, 95% CI=0.56-1.01). In women eligible for routine screening (age 29-61 years) with confirmed CIN2+, 65% was infected with HPV16 and/or HPV18. When HPV16/18-positive infections in women eligible for routine screening were discarded, the positive predictive value of cytology for the detection of CIN2+ decreased from 27 to 15%, the positive predictive value of hrHPV testing decreased from 26 to 15%, and the predictive value of a double-positive test (positive HPV test and a positive cytology) decreased from 54 to 41%. In women vaccinated against HPV16/18, screening remains important to detect cervical lesions caused by non-HPV16/18 types. To maintain a high-positive predictive value, screening algorithms must be carefully re-evaluated with regard to the screening modalities and length of the screening interval.     

High-risk HPV type-specific clearance rates in cervical screening

We assessed clearance rates of 14 high-risk human papillomavirus (hrHPV) types in hrHPV-positive women with normal cytology and borderline/mild dyskaryosis (BMD) in a population-based cervical screening cohort of 44,102 women. The 6-month hrHPV type-specific clearance rates, that is, clearance of the same type as detected at baseline, in women with normal and BMD smears were 43% (95% confidence interval (CI) 39-47) and 29% (95% CI 24-34), respectively. Corresponding 18-month clearance rates were markedly higher, namely 65% (95% CI 60-69) and 41% (95% CI 36-47), respectively. The lowest clearance rates in women with normal cytology were observed for HPV16, HPV18, HPV31, and HPV33. Significantly reduced 18-month clearance rates at a significance level of 1% were observed for HPV16 (49%, 95% CI 41-59) and HPV31 (50%, 95% CI 39-63) in women with normal cytology, and for HPV16 (19%, 95% CI 12-29) in women with BMD. Among women who did not clear hrHPV, women with HPV16 persistence displayed an increased detection rate of >or=CIN3 (normal P<0.0001; BMD, P=0.005). The type-specific differences in clearance rates indicate the potential value of hrHPV genotyping in screening programs. Our data support close surveillance (i.e. referral directly, or within 6 months) of women with HPV16 and are inconclusive for surveillance of women with HPV18, HPV31, and HPV33. For the other hrHPV-positive women, it seems advisable to adopt a conservative management with a long waiting period, as hrHPV clearance is markedly higher after 18 months than after 6 months and the risk for >or=CIN3 is low.     

Long‐term risk of cervical intraepithelial neoplasia grade 3 or worse according to high‐risk human papillomavirus genotype and semi‐quantitative viral load among 33,288 women with normal cervical cytology

In this prospective cohort study, we estimated the long‐term risk of cervical intraepithelial neoplasia grade 3 or cancer (CIN3+) by high‐risk human papillomavirus (hrHPV) genotype and semi‐quantitative viral load at baseline among 33,288 women aged 14–90 years with normal baseline cytology. During 2002–2005, residual liquid‐based cervical cytology samples were collected from women screened for cervical cancer in Copenhagen, Denmark. Samples were HPV‐tested with Hybrid Capture 2 (HC2) and genotyped with INNO‐LiPA. Semi‐quantitative viral load was measured by HC2 relative light units in women with single hrHPV infections. The cohort was followed in a nationwide pathology register for up to 11.5 years. In women aged ≥30 years at baseline, the 8‐year absolute risk for CIN3+ following baseline detection of HPV16 was 21.8% (95% confidence interval [CI]: 18.0–25.6%). The corresponding risks for HPV18, HPV31, HPV33, and other hrHPV types, respectively, were 12.8% (95% CI: 7.6–18.0%), 11.3% (95% CI: 7.7–14.9%), 12.9% (95% CI: 7.0–18.8%) and 3.9% (95% CI: 2.7–5.2%). Similar absolute risk estimates were observed in women aged <30 years. Higher HPV16‐viral load was associated with increased risk of CIN3+ (hazard ratio = 1.34, 95% CI: 1.10–1.64, per 10‐fold increase in viral load). A similar trend, although statistically nonsignificant, was found for viral load of HPV18. The 8‐year absolute risk of CIN3+ in women with HPV16‐viral load ≥100.0 pg/ml was 30.2% (95% CI: 21.9–38.6%). Our results support that hrHPV genotyping during cervical cancer screening may help identify women at highest risk of CIN3+.     

阴道镜结果正常或低度病变妇女发生宫颈癌前病变的前瞻性风险分层研究

  目的  探讨细胞学、高危型人乳头瘤病毒（high risk human papillomavirus,hrHPV）分型对于阴道镜结果正常或低级别鳞状上皮内病变（low-grade squamous intraepithelial lesion,LSIL）妇女的风险预测作用。  方法  基于1999年6月在山西省建立的宫颈癌筛查队列,以2005年随访时阴道镜结果为正常或低度病变的596例妇女为研究对象,于2010年和2014年进行随访。分析hrHPV阴性组、hrHPV阳性组、HPV16/18阳性组、细胞学LSIL以下组和细胞学LSIL及以上组发生宫颈上皮内瘤样病变2级及以上（cervical intraepithelial neoplasia grade 2 or worse,CIN2+）的瞬时、5年和9年累积风险和相对危险度。  结果  细胞学LSIL以下组发生CIN2+的瞬时、5年和9年累积风险分别为0.2%、2.8%和4.2%,细胞学LSIL及以上组相应的风险分别为14.7%（RR=73.8,95% CI为9.7~561.5）、40.0%（RR=16.0,95% CI为8.2~31.1）和51.4%（RR=15.0,95% CI为8.3~27.0）。hrHPV阴性组发生CIN2+的瞬时风险、5年和9年累积风险较低,分别为0.6%、2.7%和3.8%,hrHPV阳性和HPV16/18阳性组发生CIN2+的风险逐渐升高,其中HPV16/18阳性组的相应风险分别为13.2%（RR=23.4,95% CI为5.1~106.9）、36.9%（RR=15.4,95% CI为6.9~34.3）和42.6%（RR=14.1,95% CI为6.8~29.2）。  结论  阴道镜结果正常或LSIL妇女,若细胞学结果为LSIL及以上或HPV16/18阳性,未来进展为高度宫颈癌前病变的风险较高,细胞学和HPV16/18分型可用于该人群的临床分流管理。      

High‐risk human papillomavirus DNA load in a population‐based cervical screening cohort in relation to the detection of high‐grade cervical intraepithelial neoplasia and cervical cancer

In a population‐based cervical screening cohort, we determined the value of type‐specific viral load assessment for the detection of high‐grade cervical intraepithelial neoplasia and cervical cancer (≥CIN2). Viral load was determined by type‐specific real‐time PCR in women with single HPV16,‐18,‐31 and ‐33 infections, as determined by GP5+/6+‐PCR. Study endpoints were the detection of cumulative ≥CIN2 or ≥CIN3 within 18 months of follow‐up. High viral loads of HPV16,‐31, and ‐33 were predictive for ≥CIN2 (relative risk of 1.6 (95% CI: 1.3–1.9), 1.7 (95% CI: 1.1–2.7) and 1.9 (95% CI: 1.1–3.1) per 10‐fold change in viral load, respectively). For HPV18, the relative risk was of similar magnitude (1.5, 95% CI: 0.7–3.1), though not significant (p = 0.3). Subsequently, we determined the sensitivities of viral load for ≥CIN2 and ≥CIN3 in HPV DNA‐positive women using viral load thresholds previously defined in a cross‐sectional study. These thresholds were based on the 25th, 33rd and 50th percentiles of type‐specific HPV16,‐18,‐31 or ‐33 viral load values found in women with normal cytology. For all types, combined sensitivities for ≥CIN2 were 93.5%, 88.8% and 77.7% for the 25th, 33rd and 50th percentile thresholds, respectively. Response‐operator‐characteristics (ROC) curve analysis showed that viral load testing on HPV DNA‐positive women in addition to or instead of cytology may result in an increased sensitivity for ≥CIN2, but at the cost of a marked decrease in specificity in relation to cytology. Similar results were obtained when using ≥CIN3 as endpoint. In conclusion, in a cervical screening setting viral load assessment of HPV16, 18, 31 and 33 has no additive value to stratify high‐risk HPV GP5+/6+‐PCR‐positive women for risk of ≥CIN2 or ≥CIN3. © 2008 Wiley‐Liss, Inc.     

Human papillomavirus type-specific 18-month risk of high-grade cervical intraepithelial neoplasia in women with a normal or borderline/mildly dyskaryotic smear.

INTRODUCTION: High-risk human papillomavirus (hrHPV) DNA testing is an increasingly used instrument in cervical cancer prevention along cervical cytology. The inclusion of hrHPV testing in cervical screening requires efficient management as many hrHPV infections are transient. We investigated the potential value of hrHPV genotyping in normal and borderline/mildly dyskaryotic (BMD) smears. MATERIALS AND METHODS: From a screening population of 44,102 women in the Netherlands, we included hrHPV-positive women with a normal or BMD smear. We assessed the type-specific 18-month risk of high-grade cervical intraepithelial neoplasia (CIN). RESULTS: In hrHPV-positive women, 18-month risk of CIN grade 3 or invasive cancer (> or =CIN3) was 6% [95% confidence interval (95% CI), 4-9] after normal cytology and 20% (95% CI, 16-25) after BMD. If positive for HPV16, > or =CIN3 risks were 14% (95% CI, 9-21) and 37% (95% CI, 28-48), respectively. In the subset of hrHPV-positive women without HPV16, HPV18 was associated with an increased risk of high-grade CIN after normal cytology and HPV31 and HPV33 were associated with an increased risk, particularly after BMD. HPV16 and HPV18 were also associated with an increased risk of high-grade CIN in women with an hrHPV-positive normal baseline smear and a repeat normal smear at 6 months. DISCUSSION: HrHPV-positive women without type 16, 18, 31, or 33 had a relatively low risk of high-grade CIN. Among women with baseline normal cytology and among women with a baseline and repeat normal smear, HPV16/18-positive women showed an increased risk of high-grade CIN. This warrants more aggressive management of HPV16/18-positive women compared with other hrHPV-positive women.     

Combined use of cytology,p16 immunostaining and genotyping for triage of women positive for high-risk human papillomavirus at primary screening

Human papillomavirus (HPV) testing is very sensitive for primary cervical screening but has low specificity. Triage tests that improve specificity but maintain high sensitivity are needed. Women enrolled in the experimental arm of Phase 2 of the New Technologies for Cervical Cancer randomized controlled cervical screening trial were tested for high-risk HPV (hrHPV) and referred to colposcopy if positive. hrHPV-positive women also had HPV genotyping (by polymerase chain reaction with GP5+/GP6+ primers and reverse line blotting), immunostaining for p16 overexpression and cytology. We computed sensitivity, specificity and positive predictive value (PPV) for different combinations of tests and determined potential hierarchical ordering of triage tests. A number of 1,091 HPV-positive women had valid tests for cytology, p16 and genotyping. Ninety-two of them had cervical intraepithelial neoplasia grade 2+ (CIN2+) histology and 40 of them had CIN grade 3+ (CIN3+) histology. The PPV for CIN2+ was >10% in hrHPV-positive women with positive high-grade squamous intraepithelial lesion (61.3%), positive low-grade squamous intraepithelial lesion (LSIL+) (18.3%) and positive atypical squamous cells of undetermined significance (14.8%) cytology, p16 positive (16.7%) and, hierarchically, for infections by HPV33, 16, 35, 59, 31 and 52 (in decreasing order). Referral of women positive for either p16 or LSIL+ cytology had 97.8% sensitivity for CIN2+ and women negative for both of these had a 3-year CIN3+ risk of 0.2%. Similar results were seen for women being either p16 or HPV16/33 positive. hrHPV-positive women who were negative for p16 and cytology (LSIL threshold) had a very low CIN3+ rate in the following 3 years. Recalling them after that interval and referring those positive for either test to immediate colposcopy seem to be an efficient triage strategy. The same applies to p16 and HPV16.     

Identification of HPV genotypes causing cervical precancer using tissue-based genotyping

Identification of high-risk human papillomavirus genotypes causing cervical precancer is crucial for informing HPV vaccine development and efficacy studies, and for determining which types to include in next-generation genotyping assays. Co-occurrence of hrHPV infections is common and complicates carcinogenicity assessment; accurate attribution requires tissue-based genotyping of precancers. We included all women with cervical intraepithelial neoplasia Grade 2 or worse (CIN2+) from the Biopsy Study, an observational study of 690 women enrolled between 2009 and 2012 at the University of Oklahoma. Tissue-based genotyping, including whole tissue sections (WTS) and laser-capture microdissection (LCM), was performed on all precancers with multiple hrHPV infections detected in cytology, totaling over 1,800 HPV genotyping assays. Genotype attribution was compared to hierarchical and proportional hrHPV-type attribution models. Of 276 women with CIN2+, 122 (44.2%) had multiple hrHPV genotypes in cytology. Of 114 women with genotyping data, 94 had one or more hrHPV detected in tissue. Seventy-one women (75.5%) had a single causal hrHPV genotype, while 23 women had multiple hrHPV genotypes causing CIN2+. Ten women had multiple causal infections in a single biopsy, contrary to the previous notion that each lesion is caused by a single type only. While HPV16 was the predominant causal hrHPV genotype using all approaches, the hierarchical model overattributed HPV16, whereas other causal hrHPV genotypes, particularly HPV18 and HPV35, were underattributed. Understanding true causal genotypes is important for the evaluation of vaccine efficacy, to estimate the extent of unmasking, and for type-specific risk assessment in screening and management.     

Primary cervical cancer screening with HPV testing compared with liquid-based cytology: results of round 1 of a randomised controlled trial �C the HPV FOCAL Study

Background:

Round 1 data of human papillomavirus (HPV) FOCAL, a three-arm, randomised trial, which aims to establish the efficacy of HPV DNA testing as a primary screen for cervical cancer, are presented.

Methods:

The three arms are: Control arm – liquid based cytology with atypical squamous cells of unknown significance (ASC-US) triage with hrHPV testing; Intervention Arm – hrHPV at entry with liquid-based cytology (LBC) triage of hrHPV positives, with exit screen at 4 years; Safety check arm – hrHPV at entry with LBC triage of hrHPV positives with exit screen at 2 years.

Results:

A total of 6154 women were randomised to the control arm and 12 494 to the HPV arms (intervention and safety check). In the HPV arm, the baseline cervical intraepithelial neoplasia (CIN)2+ and CIN3+ rate was 9.2/1000 (95%CI; 7.4, 10.9) and 4.8/1000 (95%CI; 3.6, 6.1), which increased to 16.1/1000 (95%CI 13.2, 18.9) for CIN2+ and to 8.0/1000 (95%CI; 5.9, 10.0) for CIN3+ after subsequent screening of HPV-DNA-positive/cytology-negative women. Detection rate in the control arm remained unchanged after subsequent screening of ASC-US-positive/hrHPV DNA-negative women at 11.0/1000 for CIN2+ and 5.0/1000 for CIN3+.

Conclusion:

After subsequent screening of women who were either hrHPV positive/cytology negative or ASC-US positive/HPV negative, women randomised to the HPV arms had increased CIN2+ detection compared with women randomised to the cytology arm.     

Validation of a DNA methylation HPV triage classifier in a screening sample

High‐risk human papillomavirus (hrHPV) DNA tests have excellent sensitivity for detection of cervical intraepithelial neoplasia 2 or higher (CIN2+). A drawback of hrHPV screening, however, is modest specificity. Therefore, hrHPV‐positive women might need triage to reduce adverse events and costs associated with unnecessary colposcopy. We compared the performance of HPV16/18 genotyping with a predefined DNA methylation triage test (S5) based on target regions of the human gene EPB41L3, and viral late gene regions of HPV16, HPV18, HPV31 and HPV33. Assays were run using exfoliated cervical specimens from 710 women attending routine screening, of whom 38 were diagnosed with CIN2+ within a year after triage to colposcopy based on cytology and 341 were hrHPV positive. Sensitivity and specificity of the investigated triage methods were compared by McNemar's test. At the predefined cutoff, S5 showed better sensitivity than HPV16/18 genotyping (74% vs 54%, P = 0.04) in identifying CIN2+ in hrHPV‐positive women, and similar specificity (65% vs 71%, P = 0.07). When the S5 cutoff was altered to allow equal sensitivity to that of genotyping, a significantly higher specificity of 91% was reached (P < 0.0001). Thus, a DNA methylation test for the triage of hrHPV‐positive women on original screening specimens might be a valid approach with better performance than genotyping.     

Evaluation of 14 triage strategies for HPV DNA-positive women in population-based cervical screening

High-risk human papillomavirus (hrHPV) testing has a higher sensitivity but lower specificity than cytology for detection of high-grade intraepithelial neoplasia (CIN). To avoid over-referral to colposcopy and overtreatment, hrHPV-positive women require triage testing and/or followup. A total of 25,658 women (30-60 years) enrolled in a population-based cohort study had an adequate baseline Pap smear and hrHPV test. The end-point was cumulative two-year risk of CIN grade 3 or worse (CIN3+). In a post-hoc analysis, fourteen triage/followup strategies for hrHPV-positive women (n = 1,303) were evaluated for colposcopy referral rate, positive (PPV) and negative predictive value (NPV). Five strategies involved triage testing without a repeat test and nine strategies involved triage testing followed by one repeat testing. The tests were cytology, hrHPV, HPV16/18 genotyping and HPV16/18/31/33/45 genotyping. Results were adjusted for women in the cohort study who did not attend repeat testing. Of the strategies without repeat testing, combined cytology and HPV16/18/31/33/45 genotyping gave the highest NPV of 98.9% (95%CI 97.6-99.5%). The corresponding colposcopy referral rate was 58.1% (95%CI 55.4-60.8%). Eight of the nine strategies with retesting had an estimated NPV of at least 98%. Of those, cytology triage followed by cytology at 12 months had a markedly lower colposcopy referral rate of 33.4% (95%CI 30.2-36.7%) than the other strategies. The NPV of the latter strategy was 99.3% (95%CI 98.1-99.8%). Triage hrHPV-positive women with cytology, followed by repeat cytology testing yielded a high NPV and modest colposcopy referral rate and appear to be the most feasible management strategy.     

Comparing the performance of FAM19A4 methylation analysis,cytology and HPV16/18 genotyping for the detection of cervical (pre)cancer in high‐risk HPV‐positive women of a gynecologic outpatient population (COMETH study)

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.     

Specimen self‐collection and HPV DNA screening in a pilot study of 100,242 women

Since cervical cancer remains common in Mexico despite an established cytology screening program, the Ministry of Health recently introduced pilot front‐line HPV testing into the Mexican cervical cancer screening program (CCSP). Here, we present the key field performance metrics of this population‐based study. High‐risk HPV DNA (hrHPV) testing was conducted on self‐collected vaginal specimens from 100,242 women aged 25–75 years residing in Morelos State. All hrHPV positive women and a random sample of 3.2% (n = 2,864) of hrHPV negative participants were referred for colposcopic examination. The main disease endpoint of interest was cervical intraepithelial neoplasia grade 2 or higher (CIN2+). We calculated relative risk, positive predictive value and negative predictive value adjusted for screening test verification bias. The overall prevalence of hrHPV was 10.8% (95%CI 10.6–11.0). Women positive for hrHPV had a relative risk of 15.7 for histologically detectable CIN2+. The adjusted positive predictive value of the hrHPV test was 2.4% (95%CI 2.1–2.7); whereas the adjusted negative predictive value was 99.8% (95%CI 99.8–99.9). These findings suggest that large‐scale vaginal hrHPV testing in a middle‐income country can identify women at greater risk of advanced cervical abnormalities in a programmatically meaningful way but care is warranted to ensure that disease not detectable at colposcopy is kept to a minimum. PASS shows areas that need improvement and sets the stage for wider use of hrHPV screening of self‐collected vaginal specimens in Mexico.     

Guidelines for human papillomavirus DNA test requirements for primary cervical cancer screening in women 30 years and older

Given the strong etiologic link between high-risk HPV infection and cervical cancer high-risk HPV testing is now being considered as an alternative for cytology-based cervical cancer screening. Many test systems have been developed that can detect the broad spectrum of hrHPV types in one assay. However, for screening purposes the detection of high-risk HPV is not inherently useful unless it is informative for the presence of high-grade cervical intraepithelial neoplasia (CIN 2/3) or cancer. Candidate high-risk HPV tests to be used for screening should reach an optimal balance between clinical sensitivity and specificity for detection of high-grade CIN and cervical cancer to minimize redundant or excessive follow-up procedures for high-risk HPV positive women without cervical lesions. Data from various large screening studies have shown that high-risk HPV testing by hybrid capture 2 and GP5+/6+-PCR yields considerably better results in the detection of CIN 2/3 than cytology. The data from these studies can be used to guide the translation of high-risk HPV testing into clinical practice by setting standards of test performance and characteristics. On the basis of these data we have developed guidelines for high-risk HPV test requirements for primary cervical screening and validation guidelines for candidate HPV assays.     

POBASCAM, a population-based randomized controlled trial for implementation of high-risk HPV testing in cervical screening: design, methods and baseline data of 44,102 women

Cytological cervical screening is rather inefficient because of relatively high proportions of false negative and false positive smears. To evaluate the efficiency of high-risk human papillomavirus (hrHPV) testing, by GP5+/6+ PCR-enzyme immunoassay (EIA), in conjunction with cytology (Intervention Group) to that of the classical cytology (Control Group), we initiated the Population Based Screening Study Amsterdam (POBASCAM). POBASCAM is a population-based randomized controlled trial for implementation of hrHPV testing in cervical screening. The outcome measure is the proportion of histologically confirmed > or =CIN3 lesions in each study arm up to and including the next screening round after 5 years. We present the design, methods and baseline data of POBASCAM. When, in the next 5 years, the follow-up will be completed, the data obtained will be used in model studies, including a cost-effectiveness study, to advise the Dutch Ministry of Public Health in deciding whether cervical screening should be based on combined hrHPV and cytology testing instead of cytology alone. Between January 1999 and September 2002, 44,102 women (mean age = 42.8 years; range = 29-61) that participated in the regular Dutch screening program were included in our study. In the Intervention Group the distribution of cytology and hrHPV by cytology class was as follows: normal cytology 96.6% (3.6% hrHPV positive); borderline and mild dyskaryosis (BMD) 2.5% (34.6% hrHPV positive); and moderate dyskaryosis or worse (>BMD) 0.8% (88.3% hrHPV positive), i.e., 0.4% moderate dyskaryosis (82.9% hrHPV positive), 0.3% severe dyskaryosis (92.5% hrHPV positive), 0.1% carcinoma in situ (95.2% hrHPV positive), <0.1% suspected for invasive cancer (hrHPV positive 100.0%). In the Control Group 96.5% of the women had normal cytology, 2.4% BMD and 0.8% >BMD, i.e., 0.4% moderate dyskaryosis, 0.3% severe dyskaryosis, 0.1% carcinoma in situ, <0.1% suspected for invasive cancer. The presence of hrHPV was age-dependent, decreasing from 12.0% at 29-33 years to 2.4% at 59-61 years. Among women with a positive hrHPV test, the prevalence of BMD was age-dependent ranging from 20.2% at 29-33 years to 7.8% at 54-58 years. In contrast, the risk of >BMD of 13.7% among women with a positive hrHPV test was not age-dependent. Our study indicates that large-scale hrHPV testing by GP5+/6+ PCR-EIA in the setting of population-based cervical screening is practically feasible, is accepted by both participating women and general practitioners and yields highly reproducible results.     

Human papillomavirus E7 protein detection as a method of triage to colposcopy of HPV positive women,in comparison to genotyping and cytology. Final results of the PIPAVIR study

The objective of the presented cross‐sectional‐evaluation‐screening study is the clinical evaluation of high‐risk(hr)HPVE7‐protein detection as a triage method to colposcopy for hrHPV‐positive women, using a newly developed sandwich‐ELISA‐assay. Between 2013‐2015, 2424 women, 30‐60 years old, were recruited at the Hippokratio Hospital, Thessaloniki/Greece and the Im Mare Klinikum, Kiel/Germany, and provided a cervical sample used for Liquid Based Cytology, HPV DNA genotyping, and E7 detection using five different E7‐assays: “recomWell HPV16/18/45KJhigh”, “recomWell HPV16/18/45KJlow”, “recomWell HPV39/51/56/59”, “recomWell HPV16/31/33/35/52/58” and “recomWell HPVHRscreen” (for 16,18,31,33,35,39,45,51,52,56,58,59 E7), corresponding to different combinations of hrHPVE7‐proteins. Among 1473 women with eligible samples, those positive for cytology (ASCUS+ 7.2%), and/or hrHPV DNA (19.1%) were referred for colposcopy. Cervical Intraepithelial Neoplasia grade 2 or worse (CIN2+) was detected in 27 women (1.8%). For HPV16/18‐positive women with no triage, sensitivity, positive predictive value (PPV) and the number of colposcopies needed to detect one case of CIN2+ were 100.0%, 11.11% and 9.0 respectively. The respective values for E7‐testing as a triage method to colposcopy ranged from 75.0‐100.0%, 16.86‐26.08% and 3.83‐5.93. Sensitivity and PPV for cytology as triage for hrHPV(non16/18)‐positive women were 45.45% and 27.77%; for E7 test the respective values ranged from 72.72‐100.0% and 16.32‐25.0%. Triage of HPV 16/18‐positive women to colposcopy with the E7 test presents better performance than no triage, decreasing the number of colposcopies needed to detect one CIN2+. In addition, triage of hrHPV(non16/18)‐positive women with E7 test presents better sensitivity and slightly worse PPV than cytology, a fact that advocates for a full molecular screening approach.     

The clinical value of HPV genotyping in triage of women with high‐risk‐HPV‐positive self‐samples

Cytology alone, or combined with HPV16/18 genotyping, might be an acceptable method for triage in hrHPV‐cervical cancer screening. Previously studied HPV‐genotype based triage algorithms are based on cytology performed without knowledge of hrHPV status. The aim of this study was to explore the value of hrHPV genotyping combined with cytology as triage tool for hrHPV‐positive women. 520 hrHPV‐positive women were included from a randomised controlled self‐sampling trial on screening non‐attendees (PROHTECT‐3B). Eighteen baseline triage strategies were evaluated for cytology and hrHPV genotyping (Roche Cobas 4800) on physician‐sampled triage material. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), referral rate, and number of referrals needed to diagnose (NRND) were calculated for CIN2+ and CIN3+. A triage strategy was considered acceptable if the NPV for CIN3+ was ≥98%, combined with maintenance or improvement of sensitivity and an increase in specificity in reference to the comparator, being cytology with a threshold of atypical cells of undetermined significance (ASC‐US). Three triage strategies met the criteria: HPV16+ and/or ≥LSIL; HPV16+ and/or ≥HSIL; (HPV16+ and/or HPV18+) and/or ≥HSIL. Combining HPV16+ and/or ≥HSIL yielded the highest specificity (74.9%, 95% CI 70.5–78.9), with a sensitivity (94.4%, 95% CI 89.0–97.7) similar to the comparator (93.5%, 95% CI 87.7–97.1), and a decrease in referral rate from 52.2% to 39.5%. In case of prior knowledge of hrHPV presence, triage by cytology testing can be improved by adjusting its threshold, and combining it with HPV16/18 genotyping. These strategies improve the referral rate and specificity for detecting CIN3+ lesions, while maintaining adequate sensitivity.