DNA methylation analysis of the genes FAM19A4 and miR124-2 has emerged as a promising triage strategy for high-risk (hr) human papillomavirus (HPV)-positive women in cervical screening. This study reports a prospective evaluation of the diagnostic accuracy of FAM19A4/miR124-2 methylation in a large population-based primary HPV-based screening cohort with 18 months of follow-up. In this prospective observational study, clinician-collected cervical samples from 3848 consecutive hrHPV-positive women who participated in the Dutch national primary HPV-based screening program were tested with the QIAsure Methylation Test, a quantitative methylation-specific PCR assessing FAM19A4 and miR124-2 methylation. Through linkage to the Dutch Nationwide Pathology Databank (Palga), 11 cases of cervical carcinoma, 293 cervical intraepithelial neoplasia grade 3 (CIN3), and 29 adenocarcinoma in situ (AIS) were identified during 18 months of follow-up. Clinical performance for CIN3, AIS, and cancer (CIN3+) detection was assessed based on (I) the threshold of the QIAsure Methylation Test, and (II) thresholds corresponding with clinical specificities of 70% and 80% in this hrHPV-positive study population. The sensitivity and specificity of FAM19A4/miR124-2 methylation for CIN3+ detection using the QIAsure Methylation Test threshold (I) were 57.4% (95% CI: 52.0-62.7) and 88.3% (95% CI: 87.2-89.5), respectively. At predefined specificities of 70% and 80%, FAM19A4/miR124-2 methylation (II) demonstrated a CIN3+ sensitivity of 75.4% (95% CI: 70.7-80.0) and 66.1% (95% CI: 61.0-71.2), respectively. In conclusion, FAM19A4/miR124-2 methylation analysis showed good triage performance in a primary HPV-based screening setting. These findings support the use of methylation-based testing as a triage strategy in primary HPV-based screening programs, with an appropriate follow-up policy for hrHPV-positive women who test methylation-negative.

DNA methylation of host-cell genes is an epigenetic process that regulates gene expression and is associated with cervical cancer development. Studies on the natural history of cervical intraepithelial neoplasia (CIN) and the molecular alterations associated with cervical carcinogenesis led to the identification of several host-cell DNA methylation markers. Over the past years, various studies on methylation markers have shown promising results in terms of diagnostic and prognostic value to improve cervical cancer screening and management of CIN. This review provides an overview of the clinical indications of host-cell DNA methylation markers in cervical screening and management of CIN, and outlines avenues for further applications.

Abstract Background A persisting high-risk human papillomavirus (HR-HPV) infection is causal for cervical cancer; however, there is limited population-based data on the prevalence of HPV infections in Germany. We assessed the age and type-specific HPV prevalence, and associated risk factors in HPV unvaccinated women aged 30 and above. Methods The MARZY prospective population-based cohort study was conducted between 2005 and 2012 in Mainz and Mainz-Bingen, Germany. Eligible women were randomly recruited from population registries and invited for cervical cancer screening (n = 5,275). A study swab (liquid-based cytology) was taken and HPV testing was performed with GP5+/6 + polymerase chain reaction (PCR) followed by genotyping. We assessed HPV types as HR-HPV, ‘moderate’ risk and low-risk (LR-HPV). Logistic regression was performed to identify factors associated with HPV infection, stratified by HPV types. Results 2,520 women were screened with a valid PCR result. Overall HPV prevalence was 10.6% (n = 266), with 6.5% HR-HPV positive (n = 165), 1.5% ’moderate’ risk type (n = 38) and 3.3% LR-HPV type (n = 84) positive. 8.9% had a single infection (n = 225) and 1.6% had multiple types (n = 41). The most common HR-HPV types were 16, 56, 52 and 31 and LR-HPV 90 and 42. Of 187 HR-HPV infections detected (among 165 women), 55.1% (n = 103) were with HPV types not covered by available bivalent or quadrivalent HPV vaccines. About 23% (n = 43) were of types not covered by the nonavalent vaccine (HPV 35, 39, 51, 56, 59). The HR and LR-HPV prevalence were highest in the age group 30–34 years (HR 9.8%, ‘moderate’ risk 3.0% and LR 5.6%), decreasing with increasing age. HR-HPV prevalence in women with normal cytology was 5.5%. In women with a high-grade squamous intraepithelial lesion (HSIL), prevalence was 66.7%. Women currently not living with a partner and current smokers had increased chances of an HR-HPV infection. Conclusion The overall population-based HPV prevalence was relatively high. An important share of prevalent HR-HPV infections constituted types not covered by current HPV vaccines. With the advent of HPV screening and younger vaccinated cohorts joining screening, HPV types should be monitored closely, also in older women who were not eligible for HPV vaccination.

Background: Human papillomavirus (HPV) testing on self-samples is a valid tool for cervical cancer screening. HPV self-sample workflows need to be clinically validated to ensure safe use in screening. Objective: This study evaluated the fully automated NeuMoDx HPV Assay self-sample workflow that is compiled of the NeuMoDx HPV assay and the NeuMoDx 96/288 Molecular Systems, for clinical performance and reproducibility on Evalyn Brush-collected self-samples. Methods: The clinical performance of the NeuMoDx HPV Assay self-sample workflow for cervical intraepithelial neoplasia grade 2 or worse (CIN2+) and CIN3+ was evaluated on 987 self-samples obtained from women attending national organized HPV-based cervical cancer screening by a noninferiority analysis relative to reference workflows using either HPV-Risk Assay or high-risk HPV GP5+/6+-PCR. Intra- and inter-laboratory reproducibility of the NeuMoDx HPV Assay self-sample workflow using both NeuMoDx 96 and 288 Molecular Systems was assessed on 520 self-samples in three laboratories. Results: The clinical sensitivity and specificity of the NeuMoDx HPV Assay self-sample workflow for the detection of CIN2+ and CIN3+ were found to be non-inferior to the reference workflows using either HPV-Risk Assay or high-risk HPV GP5+/6+-PCR, with all p-values <0.034. The NeuMoDx HPV Assay self-sample workflow exhibited an intra-laboratory reproducibility of 94.4 % (95 %CI:92.5-96.1 %) with kappa value 0.86 (95 % CI:0.81-0.91). Inter-laboratory agreement was high (all >= 93.4 % and all kappa values >= 0.83). Conclusions: The NeuMoDx HPV Assay self-sample workflow demonstrated high clinical accuracy for CIN2+/3+ and high reproducibility. The NeuMoDx HPV Assay self-sample workflow can be considered suitable for cervical cancer screening purposes.

High-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.

Cervical cancer screening programs, including triage tests, need redesigning as human papillomavirus (HPV)-vaccinated women are entering the programs. Methylation markers offer a potential solution to reduce false-positive rates by identifying clinically relevant cervical lesions with progressive potential. In a nested case-control study, 9242 women who received the three-dose HPV16/18-vaccine at ages 12-15 or 18 in a community-randomized trial were included. Subsequently, they were re-randomized for either frequent or infrequent cervical cancer screening trials. Over a 15-year post-vaccination follow-up until 2022, 17 high-grade squamous intraepithelial lesion (HSIL) and 15 low-grade (LSIL) cases were identified at the 25-year screening round, alongside 371 age and community-matched HPV16/18-vaccinated controls. Methylation analyses were performed on cervical samples collected at age 25, preceding histologically confirmed LSIL or HSIL diagnoses. DNA methylation of viral (HPV16/18/31/33) and host-cell genes (EPB41L3, FAM19A4, and miR124-2) was measured, along with HPV-genotyping. No HPV16/18 HSIL cases were observed. The predominant HPV-genotypes were HPV52 (29.4%), HPV59/HPV51/HPV58 (each 23.5%), and HPV33 (17.7%). Methylation levels were generally low, with no significant differences in mean methylation levels of viral or host-cell genes between the LSIL/HSIL and controls. However, a significant difference in methylation levels was found between HSIL cases and controls when considering a combination of viral genes and EPB41L3 (p value = .0001). HPV-vaccinated women with HSIL had HPV infections with uncommon HPV types that very rarely cause cancer and displayed low methylation levels. Further investigation is warranted to understand the likely regressive nature of HSIL among HPV-vaccinated women and its implications for management.

Despite first-void urine (FVU) being increasingly recognized as a credible specimen for human papillomavirus (HPV) detection, there is a lack of well-validated testing methods providing full quantitative genotyping required for vaccine impact monitoring from FVU samples. The Allplex HPV28 assay, capable of individually detecting 28 HPV genotypes, presents a promising method. We aimed to evaluate its genotype-specific performance on FVU samples, following optimization of FVU preanalytics. We selected 701 FVU samples collected using a Colli-Pee device (20 mL, with UCM), enriched for HPV-positivity (n = 630) based on previous testing with GP5+/6+-PCR-based reverse line blot (GP5+/6+ RLB) and E7-MPG after Amicon filtration (AF). We first evaluated the comparability and agreement of Allplex HPV28 genotype-specific positivity according to different preanalytics. Subsequently, we conducted the genotype-specific comparison of Allplex HPV28 with GP5+/6+ RLB AF and E7-MPG AF. No significant differences in HPV positivity by Allplex HPV28 testing were observed when comparing pre-centrifuged versus non-centrifuged DNA extraction, nor when comparing manual versus automated DNA extraction. Good genotype-specific agreement was observed between Allplex HPV28 and GP5+/6+ RLB AF, with Allplex HPV28 being slightly more sensitive for all 28 HPV genotypes (average Allplex HPV28:GP5+/6+ RLB AF ratio 1.729). Compared to E7-MPG AF, Allplex HPV28 exhibited lower sensitivity for all 21 overlapping HPV genotypes (average Allplex HPV28:E7-MPG AF ratio 0.588). The findings of this study, combined with practical considerations for real-world implementation, support the use of Allplex HPV28 testing after automated or manual DNA extraction without the requirement for pre-centrifugation, for HPV studies based on FVU samples, most notably those for vaccine impact monitoring on HPV prevalence.IMPORTANCEThis study provides the first analytical validation of the Allplex HPV28 genotyping assay for use in first-void urine samples, offering a reliable, non-invasive, and practical alternative to cervical samples for human papillomavirus (HPV) detection. It demonstrates a validated approach that supports the assay's potential application in real-world settings, including low- and middle-income countries, where non-invasive and widely acceptable sampling methods are crucial for maximizing population coverage and representativity. Given the urgent need for accurate and practical tools to monitor HPV vaccination impact, these findings offer a timely and impactful contribution to the field.

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.

As human papillomavirus (HPV) immunisation and HPV-based cervical cancer (CC) screening programmes expand across sub-Saharan Africa, we investigated the potential impact of human immunodeficiency virus (HIV) status on high-risk (HR)-HPV distribution among women with CC in Cote d'Ivoire. From July 2018 to June 2020, paraffin-embedded CC specimens diagnosed in Abidjan, Cote d'Ivoire were systematically collected and tested for HR-HPV DNA. Type-specific HR-HPV prevalence was compared according to HIV status. Of the 170 CC specimens analysed (median age 52 years, interquartile range: [43.0-60.0]), 43 (25.3%) were from women living with HIV (WLHIV) with a median CD4 count of 526 [373-833] cells/mm3 and 86% were on antiretroviral therapy (ART). The overall HR-HPV prevalence was 89.4% [95% CI: 84.7-94.1]. All were single HR-HPV infections with no differences according to HIV status (P = .8). Among HR-HPV-positive CC specimens, the most prevalent HR-HPV types were HPV16 (57.2%), HPV18 (19.7%), HPV45 (8.6%) and HPV35 (4.6%), with no significant differences according to HIV status. Altogether, infection with HPV16/18 accounted for 71.1% [95% CI: 55.9-86.2] of CC cases in WLHIV vs 78.9% [95% CI: 71.3-86.5] in women without HIV (P = .3). The study confirms the major role of HPV16/18 in CC in Cote d'Ivoire and should support a regional scale-up of HPV16/18 vaccination programmes regardless of HIV status. However, vaccines targeting additional HR-HPV types, including HPV45 and HPV35, could further decrease future CC incidence in Cote d'Ivoire, both for WLHIV and women without HIV.

Further detailed description of methods

ABSTRACT Endometrial cancer is the sixth most common cancer in women globally and has a rising incidence, accounting for 417,000 new diagnoses and more than 97,000 deaths in 2020. Early detection is critical because of a poor advanced-stage prognosis and high relapse risk. Postmenopausal bleeding precedes endometrial cancer in 90% of cases, yet only 5% to 10% of patients with this symptom have an underlying malignancy. Recent cytology research has demonstrated endometrial cancer cells in vaginal samples, suggesting urine and cervicovaginal self-samples may allow convenient and minimally invasive screening. In addition, screening samples for DNA methylation in tumor suppressor genes can increase testing efficacy of triaging patients with postmenopausal bleeding as it does not require intact tumor cells. This study aimed to evaluate the diagnostic performance of endometrial cancer detection using DNA methylation analysis in paired urine, cervicovaginal self-samples, and clinician-taken cervical scrapes. Paired samples from participants of the SOLUTION1 study with endometrial cancer were collected between October 2016 and August 2020. A complete urine void and cervicovaginal self-sample was collected at home, whereas the cervical scrape was taken by a clinician in the operating room before surgery. Each patient had a paired urine, cervicovaginal self-sample, and clinician-taken cervical scrape available for methylation analysis. Controls were collected from the Urine Controls (URIC) biobank and Dutch national cervical cancer screening program. Promoter hypermethylation of the ADCYAP1, BHLHE22, CDH13, CDO1, GALR1, GHSR, HAND2, SST, and ZIC1 genes was tested using quantitative methylation-specific polymerase chain reaction. Optimal 3-marker combinations were determined for each sample type using multivariable logistic regression analysis, and diagnostic performances of each marker and 3-marker panels were assessed by leave-one-out cross validation. A total of 103 endometrial cancer patients were included in this study, with 317 unpaired samples of control women. Methylation levels of all markers were significantly higher in all 3 sample types of endometrial cancer patients compared with healthy control women. In urine, the non–cross-validated area under the curve of the DNA methylation markers ranged between 0.61 and 0.93, in cervicovaginal self-samples between 0.62 and 0.91, and in clinician-taken cervical scrapes between 0.61 and 0.95. Most markers (7/9) showed the highest performance in urine, with the remaining 2 showing best performance in clinician-taken cervical scrapes. The optimal 3-marker combination panels showed area under the curve values of 0.95 (05% confidence interval [CI], 0.92–0.98) for urine, 0.94 (95% CI, 0.90–0.97) for cervicovaginal self-samples, and 0.97 (95% CI, 0.96–0.99) for clinician-taken cervical scrapes. The sensitivity and specificity of the optimal urine testing panel were both 90%, were 89% sensitivity and 92% specificity for cervicovaginal self-samples, and were 93% sensitivity and 90% specificity for clinician-taken cervical scrapes. The leave-one-out cross-validation analysis revealed virtually equal performances in marker panels for each of the 3 testing modalities. The results of this study demonstrate high diagnostic potential of DNA methylation testing in minimally and noninvasive samples for endometrial cancer detection, emphasizing the potential for patient-friendly home-based sample collection.

Women treated for CIN2/3 remain at increased risk of recurrent CIN and cervical cancer, and therefore posttreatment surveillance is recommended. This post hoc analysis evaluates the potential of methylation markers ASCL1/LHX8 and FAM19A4/miR124‐2 for posttreatment detection of recurrent CIN2/3. Cervical scrapes taken at 6 and 12 months posttreatment of 364 women treated for CIN2/3 were tested for methylation of ASCL1/LHX8 and FAM19A4/miR124‐2 using quantitative multiplex methylation‐specific PCR. Performance of the methylation tests were calculated and compared with the performance of HPV and/or cytology. Methylation levels of recurrent CIN were compared between women with a persistent HPV infection, and women with an incident HPV infection or without HPV infection. Recurrent CIN2/3 was detected in 42 women (11.5%), including 28 women with CIN2 and 14 with CIN3. ASCL1/LHX8 tested positive in 13/14 (92.9%) of recurrent CIN3 and 13/27 (48.1%) of recurrent CIN2. FAM19A4/miR124‐2 tested positive in 14/14 (100%) of recurrent CIN3 and 10/27 (37.0%) of recurrent CIN2. Combined HPV and/or methylation testing showed similar positivity rates as HPV and/or cytology. The CIN2/3 risk at 12 months posttreatment was 30.8% after a positive ASCL1/LHX8 result at 6 months posttreatment. Methylation levels of CIN2/3 in women with a persistent HPV infection were significantly higher compared with women with an incident or no HPV infection. In conclusion, posttreatment monitoring by methylation analysis of ASCL1/LHX8 and FAM19A4/miR124‐2 showed a good performance for the detection of recurrent CIN. DNA methylation testing can help to identify women with recurrent CIN that require re‐treatment.

AIMS:Adequate diagnosis of human papillomavirus (HPV)-associated high-grade squamous intraepithelial lesion (HSIL) and HPV-independent vulvar intraepithelial neoplasia (VIN) is essential but can be challenging. We comprehensively characterized a large population-based series of vulvar lesions, originally reported as high-grade VIN, and assessed the cancer risk.METHODS AND RESULTS:Baseline high-grade VIN of 751 patients were categorized by histopathological reassessment, integrating the results of immunohistochemistry (p16INK4a , p53, Ki-67) and HPV DNA testing. Integrated analyses resulted in 88.4% HPV-associated lesions (77.0% HSIL, 10.9% low-grade SIL [LSIL], and 0.4% vulvar squamous cell carcinoma [VSCC]), 10.9% HPV-independent lesions (6.1% HPV-independent VIN, 4.7% nondysplastic lesions, and 0.1% VSCC) and 1.1% inconclusive lesions. HSIL demonstrated p16INK4a block-positivity in 99.0%, increased Ki-67 in ≥2/3rd of the epithelium in 93.6%, and HPV positivity in 99.6%. In HSIL, a p53 wildtype mid-epithelial staining pattern was common (51.6%) while this was not observed in HPV-independent lesions. HPV-independent VIN harboured mutant p53 patterns in 65.2% and showed a wide morphological spectrum, ranging from differentiated to nondifferentiated ('HPV-associated-like', in 41.3%). Kaplan-Meier analyses showed a 10-year cancer risk of 8.0% in HPV-associated HSIL, 67.4% in HPV-independent VIN/p53mutant, and 27.8% in HPV-independent VIN/p53wildtype. Strikingly, the 10-year cancer risk was 73.3% in HPV-independent VIN with nondifferentiated ('HPV-associated-like') morphology.CONCLUSION:Immunohistochemistry by p16INK4a and p53 is highly recommended for optimal categorization into HPV-associated and HPV-independent VIN, which is of utmost importance given the different cancer risk. The high cancer risk of HPV-independent VIN underscores the need for surgical treatment and close follow-up, especially in case of a p53 mutant pattern and/or nondifferentiated morphology.

PDF file 180K, Supplementary experimental procedures Supplementary table and figure legends Supplementary references

AbstractBackground“REACH‐Bhutan” aimed to evaluate the feasibility and clinical performance of a community‐based screening program for cervical cancer in rural Bhutan using self‐collected samples for high‐risk human papillomavirus (HR‐HPV) testing.MethodsIn April/May 2016, 2590 women aged 30–60 years were screened across rural Bhutan by providing a self‐collected sample for careHPV testing. All careHPV‐positive women, plus a random sample of careHPV‐negative women, were recalled for colposcopy and biopsy. Self‐samples also underwent GP5+/6+ polymerase chain reaction (PCR)‐based HR‐HPV DNA detection and genotyping. Cross‐sectional screening indices were estimated against histological high‐grade squamous intraepithelial lesions or worse (hHSIL+), including imputation of hHSIL+ in women without colposcopy.ResultsHR‐HPV positivity was 10.2% by careHPV and 14.8% by GP5+/6+ PCR. Twenty‐two cases of hHSIL+ were histologically diagnosed, including one invasive cancer; an additional 7 hHSIL+ were imputed in women without colposcopy. HR‐HPV testing by GP5+/6+ showed higher sensitivity for hHSIL+ (89.7%, 95% CI 72.6–97.8) than careHPV (75.9%, 95% CI 56.5–89.7). Negative predictive value was also slightly higher for GP5+/6+ (99.9%, 95% CI 99.6–100) than careHPV (99.7%, 95% CI 99.4–99.9). Specificity, however, was lower for GP5+/6+ (86.1%, 95% CI 84.6–87.4) than careHPV (90.6%, 95% CI 89.4–91.7), as was positive predictive value (6.9%, 95% CI 4.5–9.9 vs. 8.5%, 95% CI 5.4–12.6). Of 377 HR‐HPV‐positive women by GP5+/6+, 173 (45.9%) were careHPV‐positive, including 54.7% HPV16‐positive and 30.2% HPV18‐positive women.ConclusionsThe final REACH‐Bhutan results show that screening for cervical cancer with self‐collection of samples and HR‐HPV testing, in addition to our previous report of achieving high participation, can also perform well to detect women with hHSIL+.

Supplementary Figure S1: Overview of study populations. Supplementary Figure S2: Correlation heatmap of all 72 hrHPV-positive self-samples analyzed by Infinium Methylation 450K Array. Supplementary Figure S3: The effect of informative co-data. Supplementary Figure S4: Approach to build a 3-gene methylation classifier applicable to both lavage and brush self-samples. Supplementary Figure S5: DNA methylation levels of all 28 methylation targets in hrHPV-positive lavage self-samples. Supplementary Figure S6: DNA methylation-positivity determined by the 3-gene methylation classifier. Supplementary Figure S7: Classification tree model for lavage self-samples. Supplementary Figure S8: Classification tree model for brush self-samples. Supplementary Figure S9: DNA methylation-positivity in hrHPV-positive self-samples from women with glandular lesions and cancer. Supplementary Figure S10: Predicted probabilities in self-samples from women with and without CIN3. Supplementary Figure S11: DNA methylation levels of ASCL1, LHX8 and ST6GALNAC5 in self-samples from women with HPV16 or with other hrHPV types (non-HPV16). Supplementary Table S1: Primers and probes used in multiplex qMSP. Supplementary Table S2: Logistic regression analysis results of the 3-gene methylation classifier for CIN3 detection in hrHPV-positive self-samples. Supplementary Table S3: The comparison of the performance of logistic regression and CART models.

Background Rwanda was the first African country to implement national human papillomavirus (HPV) vaccination (against types HPV6, 11, 16, and 18). In 2011, a school-based catch-up programme was initiated to vaccinate girls aged younger than 15 years but it also reached older girls in schools. We aimed to estimate the population-level effect of HPV vaccination on HPV prevalence. Methods Cross-sectional surveys were done between July, 2013, and April, 2014 (baseline), and between March, 2019, and December, 2020 (repeat), in sexually active women aged 17-29 years at health centres in the Nyarugenge District of Kigali, Rwanda. HPV prevalence was assessed in cervical cell samples collected by a health-care worker in PreservCyt solution (Cytyc, Boxbourough, MA, USA) and tested using a general primer (GP5+ or GP6+)-mediated PCR. Adjusted overall, total, and indirect (herd immunity) vaccine effectiveness was computed as the percentage of HPV detection among all women and among unvaccinated women. Findings 1501 participants completed the baseline survey and 1639 completed the repeat survey. HPV vaccine-type prevalence in participants aged 17-29 years decreased from 12% (173 of 1501) in the baseline survey to 5% (89 of 1639) in the repeat survey, with an adjusted overall vaccine effectiveness of 47% (95% CI 31 to 60) and an adjusted indirect vaccine effectiveness of 32% (9 to 49). Among participants aged 17-23 years, who were eligible for catch-up vaccination, the adjusted overall vaccine effectiveness was 52% (35 to 65) and the adjusted indirect vaccine effectiveness was 36% (8 to 55), with important heterogeneity according to education (overall vaccine effectiveness was 68% [51 to 79] in participants with & GE;6 years of school completed and 16% [-34 to 47] in those with <6 years) and HIV status (overall vaccine effectiveness was 55% [36 to 69] for HIV-negative participants and 24% [-62 to 64] for HIV-positive participants). Interpretation In Rwanda, the prevalence of vaccine-targeted HPV types has been significantly decreased by the HPV vaccine programme, most notably in women who were attending school during the catch-up programme in 2011. HPV vaccine coverage and population-level impact is expected to increase in future cohorts who are eligible for routine HPV vaccination at age 12 years.

Figure S1: Overview of MSP results in Verification II. Figure S2: Representative MSP results in cervical tissue specimens. Figure S3: Validation of GHSR, SST and ZIC1 by qMSP in hrHPV-positive cervical scrapes. Figure S4: No association of FAM19A4, CADM1, MAL and miR124-2 methylation levels with 3q gain. Table S1: Number of samples used per study item.

PDF file - 46K, Metabolite analysis. Mean fractions of parent 11Cerlotinib, nonpolar metabolites and polar metabolites for groups 1 (filled symbols) and 2 (open symbols). Vertical bars indicate standard deviations.

Abstract Background High-grade squamous intraepithelial lesions (HSIL) or cervical intraepithelial neoplasia (CIN) grade 2/3 lesions in human papillomavirus (HPV)–positive women <30 years of age have high spontaneous regression rates. To reduce overtreatment, biomarkers are needed to delineate advanced CIN lesions that require treatment. We analyzed the FAM19A4/miR124-2 methylation test and HPV16/18 genotyping in HPV-positive women aged <30 years, aiming to identify CIN2/3 lesions in need of treatment. Methods A European multicenter retrospective study was designed evaluating the FAM19A4/miR124-2 methylation test and HPV16/18 genotyping in cervical scrapes of 1061 HPV-positive women aged 15–29 years (690 ≤CIN1, 166 CIN2, and 205 CIN3+). A subset of 62 CIN2 and 103 CIN3 were immunohistochemically characterized by HPV E4 expression, a marker for a productive HPV infection, and p16ink4a and Ki-67, markers indicative for a transforming infection. CIN2/3 lesions with low HPV E4 expression and high p16ink4a/Ki-67 expression were considered as nonproductive, transforming CIN, compatible with advanced CIN2/3 lesions in need of treatment. Results FAM19A4/miR124-2 methylation positivity increased significantly with CIN grade and age groups (<25, 25–29, and ≥30 years), while HPV16/18 positivity was comparable across age groups. FAM19A4/miR124-2 methylation positivity was HPV type independent. Methylation-positive CIN2/3 lesions had higher p16ink4a/Ki-67-immunoscores (P = .003) and expressed less HPV E4 (P = .033) compared with methylation-negative CIN2/3 lesions. These differences in HPV E4 and p16ink4a/Ki-67 expression were not found between HPV16/18–positive and non-16/18 HPV–positive lesions. Conclusions Compared with HPV16/18 genotyping, the FAM19A4/miR124-2 methylation test detects nonproductive, transforming CIN2/3 lesions with high specificity in women aged <30 years, providing clinicians supportive information about the need for treatment of CIN2/3 in young HPV-positive women.