Human papillomavirus genotypes and risk of persistence and progression in women undergoing active surveillance for cervical intraepithelial neoplasia grade 2.

BACKGROUND: In recent years, active surveillance has been introduced as an alternative to excisional treatment in younger women with cervical intraepithelial neoplasia grade 2 because regression rates are high and excisional treatment is associated with increased risk of preterm birth. However, early identification of women at increased risk of persistence/progression is important to ensure timely treatment. Evidence is limited on biomarkers that may be used to identify women at increased risk of persistence/progression. OBJECTIVE: This study aimed to describe human papillomavirus HPV type-specific persistence/progression in women undergoing active surveillance for cervical intraepithelial neoplasia grade 2. STUDY DESIGN: We conducted a historical cohort study of women aged 23 to 40 years diagnosed with cervical intraepithelial neoplasia grade 2 at Aarhus University Hospital from 2000 to 2010. Women were identified through the Danish Pathology Data Bank (DPDB) and were considered as undergoing active surveillance if they had a first record of a cervical biopsy within 2 years after index diagnosis and no loop electrosurgical excision procedure before this. Human papillomavirus genotyping was performed on archived tissue samples using the HPV SPF10-DEIA-LiPA25 system (DNA ELISA [enzyme-linked immunosorbent assay] HPV SPF10 kit and RHA HPV SPF10-LiPA25 kit). Persistence/progression was defined as having a record of cervical intraepithelial neoplasia grade ≥2 in the DPDB determined on the last and worst diagnosis on a biopsy or loop electrosurgical excision procedure specimen during follow-up. We estimated the relative risk (95% confidence interval) of persistence/progression using a modified Poisson model. RESULTS: A total of 455 women were included. Two-thirds were aged ≤30 years (73.8%) at index diagnosis, and nearly half had a high-grade index cytology (48.8%). Overall, 52.2% of all women had cervical intraepithelial neoplasia grade ≥2 during follow-up; 70.5% were human papillomavirus-16-positive and 29.5% were positive for other human papillomavirus types. Human papillomavirus-16 was associated with a significantly higher risk of persistence/progression (relative risk, 1.64; 95% confidence interval, 1.37-1.95) compared with non-human papillomavirus-16. The risk of persistence/progression was highest in human papillomavirus-16-positive women with a high-grade index cytology compared with human papillomavirus-16-positive women with a low-grade cytology (relative risk, 1.29; 95% confidence interval, 1.03-1.61), whereas no differences were observed across age groups. CONCLUSION: The highest risk of persistence/progression was observed among human papillomavirus-16-positive women, particularly those with associated high-grade cytology. These findings suggest that early excisional treatment should be considered in this group of women.

FAM19A4/miR124-2 Methylation Testing and Human Papillomavirus (HPV) 16/18 Genotyping in HPV-Positive Women Under the Age of 30 Years.

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.

Evaluation of DNA methylation biomarkers ASCL1 and LHX8 on HPV-positive self-collected samples from primary HPV-based screening.

BACKGROUND: Host-cell DNA methylation analysis can be used to triage women with high-risk human papillomavirus (HPV)-positive self-collected cervicovaginal samples, but current data are restricted to under-/never-screened women and referral populations. This study evaluated triage performance in women who were offered primary HPV self-sampling for cervical cancer screening. METHODS: Self-collected samples from 593 HPV-positive women who participated in a primary HPV self-sampling trial (IMPROVE study; NTR5078), were tested for the DNA methylation markers ASCL1 and LHX8 using quantitative multiplex methylation-specific PCR (qMSP). The diagnostic performance for CIN3 and cervical cancer (CIN3 + ) was evaluated and compared with that of paired HPV-positive clinician-collected cervical samples. RESULTS: Significantly higher methylation levels were found in HPV-positive self-collected samples of women with CIN3 + than control women with no evidence of disease (P values <0.0001). The marker panel ASCL1/LHX8 yielded a sensitivity for CIN3 + detection of 73.3% (63/86; 95% CI 63.9-82.6%), with a corresponding specificity of 61.1% (310/507; 95% CI 56.9-65.4%). The relative sensitivity for detecting CIN3+ was 0.95 (95% CI 0.82-1.10) for self-collection versus clinician-collection, and the relative specificity was 0.82 (95% CI 0.75-0.90). CONCLUSIONS: The ASCL1/LHX8 methylation marker panel constitutes a feasible direct triage method for the detection of CIN3 + in HPV-positive women participating in routine screening by self-sampling.

Exposure to cyan or red light inhibits the axial growth of zebrafish eyes.

Myopia, or nearsightedness, is the most common type of refractive error and is characterized by a mismatch between the optical power and ocular axial length. Light, and more specifically the spectral composition of light, has been known to influence myopic axial growth. In this pilot study, we exposed zebrafish to illuminations that vary in spectral composition and screened for changes in axial length. The illumination spectra included narrow band ultra-violet A (UVA) (peak wavelength 369 nm), violet (425 nm), cyan (483 nm), green/yellow (557 nm), and red (633 nm) light, as well as broad band white light (2700 K and 6500 K), dim white light and broad spectrum (day) light. We found that rearing zebrafish in cyan or red light leads to a reduction of the ocular axial length. The results of this pilot study may contribute to new perspectives on the role of light and lighting as an intervention strategy for myopia control.

High prevalence of HPV16 and high-grade cytology in women undergoing active surveillance for cervical intraepithelial neoplasia grade 2.

INTRODUCTION: Many countries have adopted active surveillance in women with cervical intraepithelial neoplasia grade 2 (CIN2), leaving the lesion untreated. However, there is a lack of consensus on the eligibility criteria for active surveillance across countries, with some abstaining from active surveillance in women with human papilloma virus 16 (HPV16) or a high-grade cytology. Here, we aimed to describe the distribution of HPV genotypes, age, and cytology in women undergoing active surveillance for CIN2. MATERIAL AND METHODS: We conducted a single-center cross-sectional study on women aged 23-40 undergoing active surveillance for CIN2 during 2000-2010. Women were identified through the Danish Pathology Data Bank (DPDB) at Aarhus University Hospital, Denmark. We collected information on basic characteristics and results of histopathological examinations via DPDB. Women were deemed eligible for inclusion if they had a subsequent biopsy after index CIN2, and had no prior record of CIN2+, hysterectomy, or cone biopsy. Archived biopsies underwent HPV genotyping using the HPV SPF10 - DEIA-LiPA25 system, and the diagnosis was re-evaluated by three expert pathologists. We used the Chi squared-test (p-value) for comparison across groups. RESULTS: We identified 3623 women with CIN2 of whom 455 (12.6%) were included. Most women were 30 years or younger (73.8%), and half (48.8%) had a high-grade index cytology. The prevalence of any high-risk HPV was 87.0%, with HPV16 being the most prevalent genotype (35.6%). The prevalence of HPV16 was significantly higher in women aged 30 or younger (39.3%) compared to women older than 30 years (25.2%) (p = 0.006). Upon expert review, 261 (57.4%) had CIN2 confirmed, whereas 56 (12.3%) were upgraded to CIN3 and 121 (26.6%) were downgraded to CIN1/normal. While the HPV16 prevalence was similar between community and expert confirmed CIN2, the prevalence of HPV16 was significantly higher in women with expert CIN3 compared to women with expert CIN1/normal (64.3% vs. 19.0%, p = 0.001). CONCLUSIONS: The high prevalence of HPV16 and high-grade cytology imply that these women may be perceived as a high-risk population and non-eligible for active surveillance in countries outside Denmark. Future studies should investigate the importance of HPV, age, cytology, and expert review on risk of progression to help refine criteria for active surveillance.

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.

Methylation 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.

Analysis of cervical HPV infections among unvaccinated young adult women to inform vaccine strategies in this age group: the Costa Rica HPV Vaccine Trial.

INTRODUCTION: Human papillomavirus (HPV) vaccines protect against incident HPV infections, which cause cervical cancer. OBJECTIVES: We estimated the prevalence and incidence of HPV infections in young adult women to understand the impact of an HPV vaccination programme in this population. METHODS: We collected cervical specimens from 6322 unvaccinated women, aged 18-37 years, who participated in the Costa Rica Vaccine Trial and its long-term follow-up. Women were followed for (median) 4.8 years and had (median) 4.0 study visits. Cervical specimens were tested for the presence/absence of 25 HPV genotypes. For each age band, we estimated the percentage of women with 1+ prevalent or 1+ incident HPV infections using generalised estimating equations. We also estimated the prevalence and incidence of HPV as a function of time since first sexual intercourse (FSI). RESULTS: The model estimated HPV incident infections peaked at 28.0% (95% CI 25.3% to 30.9%) at age 20 years then steadily declined to 11.8% (95% CI 7.6% to 17.8%) at age 37 years. Incident oncogenic HPV infections (HPV16/18/31/33/35/39/45/51/52/56/58/59) peaked and then declined from 20.3% (95% CI 17.9% to 22.9%) to 7.7% (95% CI 4.4% to 13.1%); HPV16/18 declined from 6.4% (95% CI 5.1% to 8.1%) to 1.1% (95% CI 0.33% to 3.6%) and HPV31/33/45/52/58 declined from 11.0% (95% CI 9.3% to 13.1%) to 4.5% (95% CI 2.2% to 8.9%) over the same ages. The percentage of women with 1+ incident HPV of any, oncogenic, non-oncogenic and vaccine-preventable (HPV16/18, HPV31/33/45, HPV31/33/45/52/58, and HPV6/11) types peaked <1 year after FSI and steadily declined with increasing time since FSI (p for trends <0.001). We observed similar patterns for model estimated HPV prevalences. CONCLUSION: Young adult women may benefit from HPV vaccination if newly acquired vaccine-preventable oncogenic infections lead to cervical precancer and cancer. HPV vaccination targeting this population may provide additional opportunities for primary prevention. TRIAL REGISTRATION NUMBER: NCT00128661.

Evidence of latent HPV infection in older Danish women with a previous history of cervical dysplasia.

INTRODUCTION: Understanding whether human papillomavirus (HPV) may establish latency in the uterine cervix is important. A better understanding of HPV natural history is useful for clinical counseling of women attending screening and to accurately inform health prevention strategies such as screening and HPV vaccination. We evaluated the extent of latent HPV infections in older women with a history of abnormal cytology. MATERIAL AND METHODS: We conducted a cross-sectional study in Aarhus, Denmark, from March 2013 through April 2015. Women were enrolled if they underwent cervical amputation or total hysterectomy because of benign disease. Prior to surgery, women completed a questionnaire and a cervical smear was collected for HPV testing and morphological assessment. For evaluation of latency (i.e., no evidence of active HPV infection, but HPV detected in the tissue), we selected women with a history of abnormal cervical cytology or histology, as these women were considered at increased risk of harboring a latent infection. Cervical tissue underwent extensive HPV testing using the SPF10-DEIA-LipA25 assay. RESULTS: Of 103 women enrolled, 26 were included in this analysis. Median age was 55 years (interquartile range [IQR] 52-65), and most women were postmenopausal and parous. The median number of sexual partners over the lifetime was six (IQR 3-10), and 85% reported no recent new sexual partner. Five women (19.2%) had evidence of active infection at the time of surgery, and 19 underwent latency evaluation. Of these, a latent infection was detected in 11 (57.9%), with HPV16 being the most prevalent type (50%). Nearly 80% (n = 14) of the 18 women with a history of previous low-grade or high-grade cytology with no treatment had an active or latent HPV infection, with latent infections predominating. HPV was detected in two of the six women with a history of high-grade cytology and subsequent excisional treatment, both as latent infections. CONCLUSIONS: HPV can be detected in cervical tissue specimens without any evidence of an active HPV infection, indicative of a latent, immunologically controlled infection. Modeling studies should consider including a latent state in their model when estimating the appropriate age to stop screening and when evaluating the impact of HPV vaccination.

Efficacy of AS04-Adjuvanted Vaccine Against Human Papillomavirus (HPV) Types 16 and 18 in Clearing Incident HPV Infections: Pooled Analysis of Data From the Costa Rica Vaccine Trial and the PATRICIA Study.

Clinical trial data and real-world evidence suggest that the AS04-adjuvanted vaccine targeting human papillomavirus types 16 and 18 (AS04-HPV-16/18) vaccine provides nearly 90% protection against cervical intraepithelial neoplasia grade 3 or higher irrespective of type, among women vaccinated before sexual debut. This high efficacy is not fully explained by cross-protection. Although AS04-HPV-16/18 vaccination does not affect clearance of prevalent infections, it may accelerate clearance of newly acquired infections. We pooled data from 2 large-scale randomized controlled trials to evaluate efficacy of the AS04-HPV-16/18 vaccine against clearance of nontargeted incident infections. Results of our analysis do not suggest an effect in expediting clearance of incident infections.

Risk Factors for Non-Human Papillomavirus (HPV) Type 16/18 Cervical Infections and Associated Lesions Among HPV DNA-Negative Women Vaccinated Against HPV-16/18 in the Costa Rica Vaccine Trial.

BACKGROUND: Factors that lead human papillomavirus (HPV) infections to persist and progress to cancer are not fully understood. We evaluated co-factors for acquisition, persistence, and progression of non-HPV-16/18 infections among HPV-vaccinated women. METHODS: We analyzed 2153 women aged 18-25 years randomized to the HPV-vaccine arm of the Costa Rica HPV Vaccine Trial. Women were HPV DNA negative for all types at baseline and followed for approximately 11 years. Generalized estimating equation methods were used to account for correlated observations. Time-dependent factors evaluated were age, sexual behavior, marital status, hormonally related factors, number of full-term pregnancies (FTPs), smoking behavior, and baseline body mass index. RESULTS: A total of 1777 incident oncogenic non-HPV-16/18 infections were detected in 12 292 visits (average, 0.14 infections/visit). Age and sexual behavior-related variables were associated with oncogenic non-HPV-16/18 acquisition. Twenty-six percent of incident infections persisted for ≥1 year. None of the factors evaluated were statistically associated with persistence of oncogenic non-HPV-16/18 infections. Risk of progression to Cervical Intraepithelial Neoplasia grade 2 or worst (CIN2+) increased with increasing age (P for trend = .001), injectable contraceptive use (relative risk, 2.61 [95% confidence interval, 1.19-5.73] ever vs never), and increasing FTPs (P for trend = .034). CONCLUSIONS: In a cohort of HPV-16/18-vaccinated women, age and sexual behavior variables are associated with acquisition of oncogenic non-HPV-16/18 infections; no notable factors are associated with persistence of acquired infections; and age, parity, and hormonally related exposures are associated with progression to CIN2+.

Cancer Risk Stratification of Anal Intraepithelial Neoplasia in Human Immunodeficiency Virus-Positive Men by Validated Methylation Markers Associated With Progression to Cancer.

BACKGROUND: High-grade anal intraepithelial neoplasia (HGAIN; AIN2-3) is highly prevalent in HIV+ men, but only a minority of these lesions progress towards cancer. Currently, cancer progression risk cannot be established; therefore, no consensus exists on whether HGAIN should be treated. This study aimed to validate previously identified host cell DNA methylation markers for detection and cancer risk stratification of HGAIN. METHODS: A large independent cross-sectional series of 345 anal cancer, AIN3, AIN2, AIN1, and normal control biopsies of HIV+ men was tested for DNA methylation of 6 genes using quantitative methylation-specific PCR. We determined accuracy for detection of AIN3 and cancer (AIN3+) by univariable and multivariable logistic regression analysis, followed by leave-one-out cross-validation. Methylation levels were assessed in a series of 10 anal cancer cases with preceding HGAIN at similar anatomic locations, and compared with the cross-sectional series. RESULTS: Methylation levels of all genes increased with increasing severity of disease (P < .05). HGAIN revealed a heterogeneous methylation pattern, with a subset resembling cancer. ZNF582 showed highest accuracy (AUC = 0.88) for AIN3+ detection, slightly improved by addition of ASCL1 and SST (AUC = 0.89), forming a marker panel. In the longitudinal series, HGAIN preceding cancer displayed high methylation levels similar to cancers. CONCLUSIONS: We validated the accuracy of 5 methylation markers for the detection of anal (pre-) cancer. High methylation levels in HGAIN were associated with progression to cancer. These markers provide a promising tool to identify HGAIN in need of treatment, preventing overtreatment of HGAIN with a low cancer progression risk.

Characterisation of anal intraepithelial neoplasia and anal cancer in HIV-positive men by immunohistochemical markers p16, Ki-67, HPV-E4 and DNA methylation markers.

Human papillomavirus (HPV)-induced anal intraepithelial neoplasia (AIN, graded 1-3) is highly prevalent in HIV-positive (HIV+) men who have sex with men (MSM), but only a minority of lesions progresses to cancer. Our study aimed to characterise comprehensively anal tissue samples from a cross-sectional series (n = 104) of HIV+ MSM and longitudinal series (n = 40) of AIN2/3 progressing to cancer using different biomarkers. The cross-sectional series consisted of 8 normal, 26 AIN1, 45 AIN2, 15 AIN3 and 10 anal squamous cell carcinoma. Tissue sections were immunohistochemically (IHC) stained for p16 (viral transformation marker), Ki-67 (cellular proliferation marker) and HPV-E4 (viral production marker). We evaluated the expression of IHC markers and compared it with DNA methylation, a marker for malignant transformation. E4 positivity decreased, whereas p16 and Ki-67 scores and methylation marker positivity increased (P values < .001) with increasing severity of anal lesions. Within AIN2, a heterogeneous biomarker pattern was observed concerning E4, p16 and methylation status, reflecting the biological heterogeneity of these lesions. In the longitudinal series, all AIN2/3 and carcinomas showed high p16 and Ki-67 expression, strong methylation positivity and occasional E4 positivity. We earlier showed that high methylation levels are associated with progression to cancer. The observed E4 expression in some AIN2/3 during the course of progression to cancer and absence of E4 in a considerable number of AIN1 lesions make the potential clinical significance of E4 expression difficult to interpret. Our data show that IHC biomarkers can help to characterise AIN; however, their prognostic value for cancer risk stratification, next to objective methylation analysis, appears to be limited.

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

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

Efficacy of the bivalent HPV vaccine against HPV 16/18-associated precancer: long-term follow-up results from the Costa Rica Vaccine Trial.

BACKGROUND: Oncogenic human papillomavirus (HPV) infections cause most cases of cervical cancer. Here, we report long-term follow-up results for the Costa Rica Vaccine Trial (publicly funded and initiated before licensure of the HPV vaccines), with the aim of assessing the efficacy of the bivalent HPV vaccine for preventing HPV 16/18-associated cervical intraepithelial neoplasia grade 2 or worse (CIN2+). METHODS: Women aged 18-25 years were enrolled in a randomised, double-blind, controlled trial in Costa Rica, between June 28, 2004, and Dec 21, 2005, designed to assess the efficacy of a bivalent vaccine for the prevention of infection with HPV 16/18 and associated precancerous lesions at the cervix. Participants were randomly assigned (1:1) to receive an HPV 16/18 AS04-adjuvanted vaccine or control hepatitis A vaccine. Vaccines were administered intramuscularly in three 0·5 mL doses at 0, 1, and 6 months and participants were followed up annually for 4 years. After the blinded phase, women in the HPV vaccine group were invited to enrol in the long-term follow-up study, which extended follow-up for 7 additional years. The control group received HPV vaccine and was replaced with a new unvaccinated control group. Women were followed up every 2 years until year 11. Investigators and patients were aware of treatment allocation for the follow-up phase. At each visit, clinicians collected cervical cells from sexually active women for cytology and HPV testing. Women with abnormal cytology were referred to colposcopy, biopsy, and treatment as needed. Women with negative results at the last screening visit (year 11) exited the long-term follow-up study. The analytical cohort for vaccine efficacy included women who were HPV 16/18 DNA-negative at vaccination. The primary outcome of this analysis was defined as histopathologically confirmed CIN2+ or cervical intraepithelial neoplasia grade 3 or worse associated with HPV 16/18 cervical infection detected at colposcopy referral. We calculated vaccine efficacy by year and cumulatively. This long-term follow-up study is registered with ClinicalTrials.gov, NCT00867464. FINDINGS: 7466 women were enrolled in the Costa Rica Vaccine Trial; 3727 received the HPV vaccine and 3739 received the control vaccine. Between March 30, 2009, and July 5, 2012, 2635 women in the HPV vaccine group and 2836 women in the new unvaccinated control group were enrolled in the long-term follow-up study. 2635 women in the HPV vaccine group and 2677 women in the control group were included in the analysis cohort for years 0-4, and 2073 women from the HPV vaccine group and 2530 women from the new unvaccinated control group were included in the analysis cohort for years 7-11. Median follow-up time for the HPV group was 11·1 years (IQR 9·1-11·7), 4·6 years (4·3-5·3) for the original control group, and 6·2 years (5·5-6·9) for the new unvaccinated control group. At year 11, vaccine efficacy against incident HPV 16/18-associated CIN2+ was 100% (95% CI 89·2-100·0); 34 (1·5%) of 2233 unvaccinated women had a CIN2+ outcome compared with none of 1913 women in the HPV group. Cumulative vaccine efficacy against HPV 16/18-associated CIN2+ over the 11-year period was 97·4% (95% CI 88·0-99·6). Similar protection was observed against HPV 16/18-associated CIN3-specifically at year 11, vaccine efficacy was 100% (95% CI 78·8-100·0) and cumulative vaccine efficacy was 94·9% (73·7-99·4). During the long-term follow-up, no serious adverse events occurred that were deemed related to the HPV vaccine. The most common grade 3 or worse serious adverse events were pregnancy, puerperium, and perinatal conditions (in 255 [10%] of 2530 women in the unvaccinated control group and 201 [10%] of 2073 women in the HPV vaccine group). Four women in the unvaccinated control group and three in the HPV vaccine group died; no deaths were deemed to be related to the HPV vaccine. INTERPRETATION: The bivalent HPV vaccine has high efficacy against HPV 16/18-associated precancer for more than a decade after initial vaccination, supporting the notion that invasive cervical cancer is preventable. FUNDING: US National Cancer Institute.

Molecular and pathological basis of HPV-negative cervical adenocarcinoma seen in a global study.

International surveys find HPV-negativity in up to 30% of cervical adenocarcinomas. We investigated the pathological diagnosis by expert consensus with immunohistochemistry and the presence of somatic mutations in recognised tumour genes in HPV-positive and negative cervical adenocarcinomas (CADC). A sample was selected of 45 paraffin-embedded cervical blocks diagnosed locally as usual cervical adenocarcinoma from a global study. These represented different diagnoses made at previous diagnostic review and HPV status. All were suitable for analysis for somatic tumour associated gene mutations. Three pathologists examined H/E slides and immunohistochemistry for p16, progesterone receptor and p53 and classified the cases. L1 genes from high-risk HPVs and low-risk HPVs were analysed by SPF10 PCR-DEIA-LiPA25 version 1 in whole tissue sections and microdissected tumour and retested by PCR for E6/E7 genes of hrHPVs if negative. Cases were analysed for microsatellite instability and next-generation sequencing mutation analysis. From the 45 cases, 20 cases of usual CADC were confirmed of which 17 (85%) were HPV-positive in tumour cells. The other 25 cases were reclassified as endometrial, serous, clear-cell and gastric-type adenocarcinomas and all were HPV-negative in tumour cells. Careful retesting for HPV DNA and IHC leads to more accurate identification of HPV-positive usual cervical adenocarcinomas. Endometrioid endometrial adenocarcinomas, other uterine adenocarcinoma with multiple somatic mutations were important in misclassification of HPV-negative cases locally managed as cervical adenocarcinoma, as was gastric-type adenocarcinoma with germline STK11 mutation in East Asia. Few consensuses confirmed HPV-negative usual cervical adenocarcinomas showed somatic tumorigenic mutations also seen in some HPV-positive usual CADC.

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.

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

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

Clinical and Analytical Performance of the BD Onclarity HPV Assay with SurePath Screening Samples from the Danish Cervical Screening Program Using the VALGENT Framework.

The Validation of HPV Genotyping Tests (VALGENT) framework is an international cooperation designed to evaluate human papillomavirus (HPV) assays with genotyping capabilities. Here, we assessed the performance of the BD Onclarity assay using Danish SurePath cervical screening samples collected under the fourth VALGENT installment, consisting of 998 consecutive samples from a screening population and 297 enriched samples with abnormal cytology (100 with atypical squamous cells of undetermined significance [ASCUS], 100 with low-grade squamous intraepithelial lesions [LSIL], and 97 with high-grade squamous intraepithelial lesions [HSIL]). The Onclarity assay detects six HPV genotypes individually (genotypes 16, 18, 31, 45, 51, and 52) and eight genotypes in three bulks (genotypes 33 and 58; genotypes 56, 59, and 66; and genotypes 35, 39, and 68). The clinical performance of the Onclarity assay for the detection of cervical intraepithelial neoplasia of grade 2 or worse (≥CIN2) and of two consecutive cytology outcomes negative for intraepithelial lesion or malignancy (2×NILM) was assessed relative to that of the GP5+/6+ PCR-enzyme immunoassay (GP-EIA) by a noninferiority test. The relative sensitivity for ≥CIN2 was 1.00 (95% confidence interval [CI], 0.97 to 1.04), and the relative specificity for 2×NILM was 1.04 (95% CI, 1.02 to 1.06). The Onclarity assay was found to be noninferior to the GP-EIA in terms of both sensitivity (P = 0.0006) and specificity (P < 0.0001). The type-specific performance of the Onclarity assay was also assessed, using the GP5+/6+ PCR with Luminex genotyping (GP-LMNX) as the comparator. The Onclarity assay showed good concordance for almost all HPV genotype groups. A stability analysis of SurePath samples was also performed, where a SurePath aliquot was stored refrigerated for 7 months and the internal control of the Onclarity assay was used as a marker for cellularity. The threshold cycle (CT ) value was the same (24.8) in the first and second Onclarity runs, showing that a SurePath sample can be stored refrigerated for 7 months and still remain a valid test specimen.

Human papillomavirus type 16 causes a defined subset of conjunctival in situ squamous cell carcinomas.

Squamous cell carcinoma of the conjunctiva is associated with a number of risk factors, including HIV infection, iatrogenic immunosuppression and atopy. In addition, several studies have suggested an involvement of HPV, based on the presence of viral DNA, but did not establish whether there was active infection or evidence of causal disease association. In this manuscript, 31 cases of conjunctival in situ squamous cell carcinoma were classified as HPV DNA-positive or -negative, before being analysed by immunohistochemistry to establish the distribution of viral and cellular biomarkers of HPV gene expression. Our panel included p16INK4a, TP53 and MCM, but also the virally encoded E4 gene product, which is abundantly expressed during productive infection. Subsequent in situ detection of HPV mRNA using an RNAscope approach confirmed that early HPV gene expression was occurring in the majority of cases of HPV DNA-positive conjunctival in situ squamous cell carcinoma, with all of these cases occurring in the atopic group. Viral gene expression correlated with TP53 loss, p16INK4a elevation, and extensive MCM expression, in line with our general understanding of E6 and E7's role during transforming infection at other epithelial sites. A characteristic E4 expression pattern was detected in only one case. HPV mRNA was not detected in lower grades of dysplasia, and was not observed in cases that were HPV DNA-negative. Our study demonstrates an active involvement of HPV in the development of a subset of conjunctival in situ squamous cell carcinoma. No high-risk HPV types were detected other than HPV16. It appears that the conjunctiva is a vulnerable epithelial site for HPV-associated transformation. These cancers are defined by their pattern of viral gene expression, and by the distribution of surrogate markers of HPV infection.

Correction to: Human papillomavirus type 16 causes a defined subset of conjunctival in situ squamous cell carcinomas.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.