Johannes Berkhof

Predicting optimal impact interventions in the post‐ HPV vaccination world

Abstract Prophylactic vaccination is a powerful tool that changes exposure to infections and associated morbidity of preventable diseases. We discuss the impact of pneumococci and human papillomavirus (HPV) vaccination on the population biology of the two micro‐organisms and related public health effects. Data on HPV type‐replacement in communities where vaccine‐covered HPVs are almost eliminated, and interactions of the remaining HPV types on the risk of cervical cancer are reviewed. Results of comprehensive models for European country‐specific conduction of cervical screening among HPV‐vaccinated and unvaccinated women, assuming different HPV‐vaccination coverage and strategies, are discussed in our policy‐oriented review. An acceptable balance of benefits and harms of cervical cancer screening in HPV vaccinated populations requires an understanding of cancer risks in differently vaccinated birth cohorts. Finally, the challenges are complex but can be met if strategies are applied that (i) as fast as possible achieve herd effect and (ii) use a risk‐based design of HPV screening.

Colposcopy referrals and CIN3 detection after triage by host cell DNA methylation and/or HPV genotyping in HPV positive women with low‐grade cytology from a population‐based Dutch primary HPV screening trial

AbstractHigh‐risk HPV (hrHPV)‐based screening has led to many unnecessary colposcopy referrals, mainly because of direct referral after low‐grade cytology (ASC‐US/LSIL). DNA methylation and genotyping tests on ASC‐US/LSIL samples have the potential to significantly improve the efficiency of screening. In this study, 12 triage strategies were constructed from FAM19A4/miR124‐2 or ASCL1/LHX8 methylation, HPV16/18 or HPV16/18/31/33/45 genotyping and 1‐year repeat cytology. The performance was evaluated on 215 hrHPV‐positive ASC‐US/LSIL samples from the IMPROVE trial (NTR5078). Performance was measured by colposcopy referral rate, positive predictive value (PPV) for detecting precancer (CIN3), and negative predictive value (NPV). To evaluate efficiency, strategies were ordered by the cumulative colposcopy referral rate after 1‐year cytology and compared by the marginal PPV to detect one additional CIN3 (mPPV). The most conservative strategy (referral when HPV16/18 and FAM19A4/miR124 methylation results are positive) had a direct referral rate of 5.2%, a cumulative referral rate after 1‐year cytology of 54.1%, and mPPV of 19.3%. Replacing HPV16/18 by HPV16/18/31/33/45 increased the cumulative 1‐year referral rate to 54.6%, and yielded an mPPV of 10.0%. Similar results were obtained for strategies with ASCL1/LHX8 methylation. Of all strategies, referral after an HPV16/18/31/33/45 positive, ASCL1/LHX8 methylation‐positive, and/or 1‐year cytology‐positive result yielded the highest direct and cumulative 1‐year colposcopy referral rates of 64.4% and 79.1%, respectively. The NPVs after 1‐year cytology varied between 98.1% and 99.4%, warranting a return to routine screening. Altogether, DNA methylation‐based triage strategies are recommended as they are discriminative for CIN3 and control the number of immediate colposcopy referrals.

Colposcopy Referral and CIN3+ Risk of Human Papillomavirus Genotyping Strategies in Cervical Cancer Screening

Abstract Background: High-risk human papillomavirus (hrHPV)-based cervical cancer screening in the Netherlands led to a substantial increase in number of colposcopy referrals and low-grade lesions detected. Genotyping strategies may be employed to lower the screening-related burden. Methods: We evaluated 14 triage strategies with genotyping (HPV16/18 or HPV16/18/31/33/45/52/58) for hrHPV-positive borderlineormilddyskaryosis (BMD)ornormal cytology,usingdata from a population-based hrHPV-based screening trial with 5-year interval (POBASCAM). We considered colposcopy referral at baseline, after 6-month repeat cytology and after 5-year hrHPV testing. Performance was evaluated by one-round positive and negative predictive value (PPVandNPV) for CIN3+ and by two-roundcolposcopy referral rate. To identify efficient strategies, they were ordered by the one-round colposcopy referral rate. Adjacent strategies were compared by the marginal PPV for detecting one additional CIN3+ (mPPV). Results: The most conservative strategy (repeat cytology after BMD and HPV16/18/31/33/45/52/58-positive normal cytology, next round otherwise) yielded an mPPV of 28%, NPV of 98.2%, and two-round colposcopy referral rate of 47.2%. Adding direct referral after BMD or genotype-positive BMD yielded an mPPV ≤ 8.2%, NPV ≥ 98.5% and an increase in colposcopy referral rate of 1.9% to 6.5%. Adding direct referral after HPV16/18-positive normal cytology yielded an mPPV ≤ 3.5%, NPV ≥ 99.5% and an increase in colposcopy referral rate of 13.9%. Conclusions: Direct colposcopy referral of women with BMD or normal cytology is unlikely to be efficient, but genotype-guided direct referral after BMD may be considered because the increase in colposcopies is limited. Impact: hrHPV screening programs can become very efficient when immediate colposcopy referral is limited to women at highest CIN3+ risk. See related In the Spotlight, p. 979

Clinical validation of methylation biomarkers for optimal detection of high‐grade vulvar intraepithelial neoplasia

Abstract The precursor lesions of vulvar squamous cell carcinoma (VSCC) include human papillomavirus (HPV)‐associated and HPV‐independent squamous neoplasia with a varying cancer risk. Our study aimed to validate the accuracy of previously identified DNA methylation markers for detection of such high‐grade vulvar intraepithelial neoplasia (VIN). A large clinical series of 751 vulvar lesions, originally diagnosed as high‐grade VIN, were reassessed and categorized into HPV‐associated or HPV‐independent vulvar disease categories. Together with 113 healthy vulvar controls, all samples were tested for 12 methylation markers with quantitative multiplex methylation‐specific PCR (qMSP). Performance of individual markers and selection of an optimal marker panel for detection of high‐grade VIN was determined by logistic regression analysis. SST was the best‐performing individual marker (AUC 0.90), detecting 80% of high‐grade VIN cases, with excellent detection of HPV‐independent VIN (95%), known to have the highest cancer risk. Merely 2% of controls tested methylation positive for SST . Selection of a marker panel, including ZNF582 , SST and miR124‐2 , resulted in a comparably high accuracy for detection of high‐grade VIN (AUC 0.89). In conclusion, we clinically validated the accuracy of 12 DNA methylation markers for detection of high‐grade VIN. SST , as a sole marker or in a panel, provides an optimal diagnostic tool to distinguish high‐grade VIN in need of treatment, particularly HPV‐independent VIN, from low‐grade or reactive vulvar lesions. These findings warrant further prognostic validation of methylation biomarkers for cancer risk stratification of patients with VIN.

Performance of DNA methylation analysis of ASCL1, LHX8, ST6GALNAC5, GHSR, ZIC1 and SST for the triage of HPV‐positive women: Results from a Dutch primary HPV‐based screening cohort

AbstractMethylation of host‐cell deoxyribonucleic acid (DNA) has been proposed as a promising biomarker for triage of high‐risk (hr) human papillomavirus (HPV) positive women at screening. Our study aims to validate recently identified host‐cell DNA methylation markers for triage in an hrHPV‐positive cohort derived from primary HPV‐based cervical screening in The Netherlands. Methylation markers ASCL1, LHX8, ST6GALNAC5, GHSR, ZIC1 and SST were evaluated relative to the ACTB reference gene by multiplex quantitative methylation‐specific PCR (qMSP) in clinician‐collected cervical samples (n = 715) from hrHPV‐positive women (age 29‐60 years), who were enrolled in the Dutch IMPROVE screening trial (NTR5078). Primary clinical end‐point was cervical intraepithelial neoplasia grade 3 (CIN3) or cancer (CIN3+). The single‐marker and bi‐marker methylation classifiers developed for CIN3 detection in a previous series of hrHPV‐positive clinician‐collected cervical samples were applied. The diagnostic accuracy was visualised using receiver operating characteristic (ROC) curves and assessed through area under the ROC curve (AUC). The performance of the methylation markers to detect CIN3+ was determined using the predefined threshold calibrated at 70% clinical specificity. Individual methylation makers showed good performance for CIN3+ detection, with highest AUC for ASCL1 (0.844) and LHX8 (0.830). Combined as a bi‐marker panel with predefined threshold, ASCL1/LHX8 yielded a CIN3+ sensitivity of 76.9% (70/91; 95% CI 68.3‐85.6%) at a specificity of 74.5% (465/624; 95% CI 71.1‐77.9%). In conclusion, our study shows that the individual host‐cell DNA methylation classifiers and the bi‐marker panel ASCL1/LHX8 have clinical utility for the detection of CIN3+ in hrHPV‐positive women invited for routine screening.

Risk-stratification of HPV-positive women with low-grade cytology by FAM19A4/miR124-2 methylation and HPV genotyping

Abstract Background The introduction of primary HPV screening has doubled the number of colposcopy referrals because of the direct referral of HPV-positive women with a borderline or mild dyskaryosis (BMD) cytology (ASC-US/LSIL) triage test. Further risk-stratification is warranted to improve the efficiency of HPV-based screening. Methods This study evaluated the discriminative power of FAM19A4/miR124-2 methylation, HPV16/18 genotyping and HPV16/18/31/33/45 genotyping in HPV-positive women with BMD (n = 294) in two Dutch screening trials. Absolute CIN3+ risks and colposcopy referrals within one screening round were calculated. Results Methylation analysis discriminated well, yielding a CIN3+ risk of 33.1% after a positive result and a CIN3+ risk of 9.8% after a negative result. HPV16/18 and HPV16/18/31/33/45 genotyping resulted in a 27.6% and 24.6% CIN3+ risk after a positive result, and a 13.2% and 9.1% CIN3+ risk after a negative result. Colposcopy referral percentages were 41.2%, 43.2%, and 66.3% for FAM19A4/miR124-2 methylation, HPV16/18 and HPV16/18/31/33/45 genotyping, respectively. The CIN3+ risk after a negative result could be lowered to 2.8% by combining methylation and extended genotyping, at the expense of a higher referral percentage of 75.5%. Conclusion The use of FAM19A4/miR124-2 methylation and/or HPV genotyping in HPV-positive women with BMD can lead to a substantial reduction in the number of direct colposcopy referrals.

Pricing of HPV tests in Italian tender-based settings

Human papillomavirus (HPV) testing has been recommended by the WHO as the first choice method in cervical cancer screening. So far, only a limited number of countries have implemented primary HPV testing, partly because of the assumed high costs of HPV testing. We assessed tender-based prices of HPV testing in Italy, where programmatic HPV-based screening has been implemented at the regional level. Procurement notices and awards, published between 2014 and December 2021, were retrieved from the European online platform for public procurement. The unit price per HPV test was calculated as the ratio of the contract award price and contract volume. The association between the unit price and contract volume, calendar year, number of offers, region's per capita gross domestic product and population density was assessed by linear regression. Fractional polynomials were used to describe the association between the unit price and contract volume. We retrieved data from 29 procurement procedures. The median unit price per HPV test was €10.75, ranging from €4.30 to €204.80. The unit price was not higher than €5 for 6 out of 11 contract awards with a volume of at least 100,000 tests. After discarding two low-volume contracts with very high contract prices (€182.40 and €204.80), volume explained 86.5% of the variation in unit price. The unit price was not associated with other variables. The Italian experience showed that the tender-based unit price of an HPV test is very low when procured at high volume, indicating that there is no reason for countries to further delay the implementation of HPV-based screening because of prohibitively high HPV testing costs.

Clinical Regression of High-Grade Cervical Intraepithelial Neoplasia Is Associated With Absence of FAM19A4/miR124-2 DNA Methylation (CONCERVE Study)

PURPOSE Cervical screening can prevent cancer by detection and treatment of cervical intraepithelial neoplasia grade 2 or 3 (CIN2/3). Screening also results in considerable overtreatment because many CIN2/3 lesions show spontaneous regression when left untreated. In this multicenter longitudinal cohort study of women with untreated CIN2/3, the prognostic value of FAM19A4/miR124-2 methylation was evaluated for clinical regression. PATIENTS AND METHODS Women with CIN2/3 were prospectively followed for 24 months. Surgical excision was replaced by a wait-and-see policy. FAM19A4/miR124-2 methylation was evaluated on all clinician-collected samples and self-collected samples collected at baseline. Every 6 months, human papillomavirus (HPV) testing and cytology were conducted on a clinician-collected sample, and a colposcopic examination was performed by a gynecologist to exclude progression. At the final study visit, two biopsies were taken. Clinical regression was defined as histologically confirmed absence of CIN2+ or an HPV-negative clinician-collected sample with normal cytology. Regression incidences were estimated using the Kaplan-Meier method. RESULTS One hundred fourteen women (median age, 30 years; range, 20-53 years) were included, 80 of whom were diagnosed with CIN2 and 34 with CIN3. During the study, 65.8% of women (75/114) did not receive surgical treatment. Women with a negative FAM19A4/miR124-2 result on the baseline clinician-collected sample showed more clinical regression (74.7%) than women with a positive methylation result (51.4%, P = .013). Regression in women with a negative FAM19A4/miR124-2 methylation test was highest when cytology was atypical squamous cells of undetermined significance/low-grade squamous intraepithelial lesion (88.4%) or HPV16 was negative (85.1%). CONCLUSION Most women with untreated CIN2/3 and a negative baseline FAM19A4/miR124-2 methylation test showed clinical regression. Methylation, in combination with cytology or HPV genotyping, can be used to support a wait-and-see policy in women with CIN2/3.