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Abstract: Introduction: To evaluate the performance of hypermethylation analysis of ASCL1, LHX8 and ST6GALNAC5 in physician‐taken cervical scrapes for detection of cervical cancer and cervical intraepithelial neoplasia (CIN) grade 3 in women living with HIV (WLHIV) in South Africa. Methods: Samples from a prospective observational cohort study were used for these analyses. Two cohorts were included: a cohort of WLHIV who were invited for cervical screening (n = 321) and a gynaecologic outpatient cohort of women referred for evaluation of abnormal cytology or biopsy proven cervical cancer (n = 108, 60% HIV seropositive). Cervical scrapes collected from all subjects were analysed for hypermethylation of ASCL1, LHX8 and ST6GALNAC5 by multiplex quantitative methylation specific PCR (qMSP). Histology endpoints were available for all study subjects. Results: Hypermethylation levels of ASCL1, LHX8 and ST6GALNAC5 increased with severity of cervical disease. The performance for detection of CIN3 or worse (CIN3+) as assessed by the area under the receiver operating characteristic (ROC) curves (AUC) was good for ASCL1 and LHX8 (AUC 0.79 and 0.81 respectively), and moderate for ST6GALNAC5 (AUC 0.71). At a threshold corresponding to 75% specificity, CIN3+ sensitivity was 72.1% for ASCL1 and 73.8% for LHX8 and all samples from women with cervical cancer scored positive for these two markers. Conclusions: Hypermethylation analysis of ASCL1 or LHX8 in cervical scrape material of WLHIV detects all cervical carcinomas with an acceptable sensitivity and good specificity for CIN3+, warranting further exploration of these methylation markers as a stand‐alone test for cervical screening in low‐resource settings. [ABSTRACT FROM AUTHOR]

Background: Urine sampling may offer a less invasive solution than cervical sampling to test for human papillomavirus (HPV) for HPV vaccine impact monitoring. Methods: Paired samples of urine and exfoliated cervical cells were obtained for 89 women with history of high-risk (HR) HPV-positive normal cytology in Bhutan. Urine sampling protocol included self-collection of first-void urine immediately into a conservation medium and procedures to optimize DNA yield. Colposcopical abnormalities were biopsied. Two HPV assays were used: a multiplex type-specific PCR (E7-MPG) and a less analytically sensitive GP5+/6+ PCR followed by reverse line blot. Results: HPV positivity for 21 types common to both assays was similar in urine and cells by E7-MPG (62.9% and 57.3%, respectively, p = 0.32) but lower in urine by GP5+/6+ (30.3% and 40.4%, p = 0.05). HPV6/11/16/18 positivity did not significantly differ between urine and cells by either assay. Sensitivity of urine (using cells as gold standard) to detect 21 HPV types was 80% and 58% for E7-MPG and GP5+/6+, respectively, with specificity 61% and 89%. HPV type distribution in urine and cells was similar, regardless of assay. The 5 detected CIN3+ were HR-HPV positive in cells by both assays, compared to 4 and 3 by E7-MPG and GP5+/6+, respectively, in urine samples. Conclusion: For the monitoring of vaccine impact, we demonstrate validity of a urine sampling protocol to obtain HPV prevalence data that are broadly comparable to that from cervical cells. However, detection of HPV in urine varies according to assay sensitivity, presumably because low level infections are frequent. [ABSTRACT FROM AUTHOR]

Introduction: Primary HPV-testing has been shown to provide a superior detection of women at risk of cervical (pre)cancer compared to cytology-based screening. However, as most high-risk HPV infections are harmless, additional triage testing of HPV-positive women is necessary to identify those with cervical (pre)cancer. In this paper, we compare the performance, advantages and limitations of clinically relevant available triage strategies for HPV-positive women. Areas covered: Many different colposcopy triage strategies, comprising both microscopy-based and molecular (virus/host-related) markers, have been suggested: Pap cytology, p16/Ki-67 dual-stained cytology, HPV16/18 genotyping, viral DNA methylation and host cell DNA methylation. Literature search was limited to triage strategies that have achieved at least phase 2 of the five-phase framework for biomarker development and studies including large cohorts (≥100 hrHPV-positive women). Triage markers were stratified by sample type (cervical scrape, self-collected sample) and by study population (screening, non-attendee, referral). Expert commentary: At present, repeat Pap cytology and Pap cytology combined with HPV16/18 genotyping are the only triage strategies that have been robustly shown to be ready for implementation. Other strategies such as p16/Ki-67 dual-stained cytology and host cell DNA methylation analysis, with or without additional HPV16/18 genotyping, are attractive options for the near future. [ABSTRACT FROM PUBLISHER]

Background: Cervical cancer is the most common female cancer in Rwanda that, in 2011, became the first African country to implement a national vaccination programme against human papillomavirus (HPV).Methods: To provide a robust baseline for future evaluations of vaccine effectiveness, cervical cell specimens were obtained from 2508 women aged 18-69 years from the general population in Kigali, Rwanda, during 2013/14. 20 % of women were HIV-positive. Samples were used for liquid-based cytology and HPV testing (44 types) with GP5+/6+ PCR.Results: HPV prevalence was 34 %, being highest (54 %) in women ≤19 years and decreasing to 20 % at age ≥50. Prevalence of high risk (HR) HPV and cytological abnormalities was 22 and 11 % respectively (including 2 % with high-grade squamous intraepithelial lesions, HSIL) decreasing with age. Age-standardised prevalence of HR HPV was 22 % (or 19 % among HIV-negative women), and HPV16 was the most common type. Prevalence of HPV and cytological abnormalities were significantly higher in HIV-positive than HIV-negative women, and the difference increased with age. Other significant risk factors for HPV positivity in multivariate analyses were high lifetime number of sexual partners, receiving cash for sex, and being a farmer. 40 % of women with HSIL were infected with HPV16/18 and there was no significant difference between HIV-positive and HIV-negative women.Conclusions: This study confirms Rwanda to be a setting of high prevalence of HPV and cervical disease that is worsened by HIV. These data will serve as a robust baseline for future evaluations of HPV vaccine programme effectiveness. [ABSTRACT FROM AUTHOR]

Background: Cervical cancer is the most common female cancer in Bhutan, the first low/middle-income country to implement a national human papillomavirus (HPV) vaccination programme. Methods: To provide a robust baseline for future evaluations of vaccine effectiveness, cervical cell specimens were obtained from 2,505 women aged 18–69 years from the general population, and biopsies from 211 cervical intraepithelial neoplasia grade 3 (CIN3) and 112 invasive cervical cancer (ICC) cases. Samples were tested for HPV using GP5+/6+ PCR. Results: Among the general population, HPV prevalence was 26%, being highest (33%) in women ≤24 years, but remaining above 15% in all age-groups. Determinants of HPV included age, marital status, and number of sexual partners. Among the eight percent with cytological abnormalities, 24 CIN3 and 4 ICC were histologically confirmed. Even after additional testing with a sensitive E7 PCR, no infections with vaccine-targeted HPV types were detected in the few vaccinated women (n = 34) compared to 6% prevalence in unvaccinated women of similar age (p = 0 · 215). Conclusion: Based upon type-specific prevalence among biopsies, at least 70% of ICC in Bhutan are theoretically preventable by HPV16/18 vaccination, but screening programmes should be expanded among older women, who have an important underlying burden of CIN3 and ICC. [ABSTRACT FROM AUTHOR]

Background: High-risk human papillomavirus (hrHPV) infections are causally related to cervical cancer development. The additional (epi)genetic alterations driving malignant transformation of hrHPV-infected cells however, are not yet fully elucidated. In this study we experimentally assessed the role of the PI3-kinase pathway and its regulator PIK3CA, which is frequently altered in cervical cancer, in HPV-induced transformation. Methods: Cervical carcinomas and ectocervical controls were assessed for PIK3CA mRNA and protein expression by quantitative RT-PCR and immunohistochemical staining, respectively. A longitudinal in vitro model system of hrHPVtransfected keratinocytes, representing the immortal and anchorage independent phenotype, was assayed for PI3- kinase activation and function using chemical pathway inhibition i.e. LY294002 treatment, and PIK3CA RNA interference. Phenotypes examined included cellular viability, migration, anchorage independent growth and differentiation. mRNA expression of hTERT and HPV16 E6E7 were studied using quantitative RT-PCR and Northern blotting. Results: Cervical carcinomas showed significant overexpression of PIK3CA compared to controls. During HPVinduced transformation in vitro, expression of the catalytic subunit PIK3CA as well as activation of downstream effector PKB/AKT progressively increased in parallel. Inhibition of PI3-kinase signalling in HPV16-transfected keratinocytes by chemical interference or siRNA-mediated silencing of PIK3CA resulted in a decreased phosphorylation of PKB/AKT. Moreover, blockage of PI3-kinase resulted in reduced cellular viability, migration, and anchorage independent growth. These properties were accompanied with a downregulation of HPV16E7 and hTERT mRNA expression. In organotypic raft cultures of HPV16- and HPV18-immortalized cells, phosphorylated PKB/ AKT was primarily seen in differentiated cells staining positive for cytokeratin 10 (CK10). Upon PI3-kinase signalling inhibition, there was a severe impairment in epithelial tissue development as well as a dramatic reduction in p- PKB/AKT and CK10. Conclusion: The present data indicate that activation of the PI3-kinase/PKB/AKT pathway through PIK3CA regulates various transformed phenotypes as well as growth and differentiation of HPV-immortalized cells and may therefore play a pivotal role in HPV-induced carcinogenesis. [ABSTRACT FROM AUTHOR]

Snijders PJF, Heideman DAM, Meijer CJLM. Methods for HPV detection in exfoliated cell and tissue specimens. APMIS 2010; 118: 520–528. Given the causal involvement of high-risk human papillomaviruses (HPVs) in cervical cancer and a subset of squamous cell carcinomas of other anogenital regions as well as the oropharynx, much attention has been focused on the development and application of HPV detection assays. HPV detection assays are almost exclusively based on the detection of viral nucleic acids, mostly viral DNA. The HPV detection methods that are nowadays in use can broadly be subdivided into target amplification methods and signal amplification methods. In this review, several principles of various methodologies are explained and examples of some commonly used HPV detection assays are given. In addition, attention is paid to the use of HPV assays for detecting clinically meaningful HPV infections, i.e. infections related to (pre)cancerous lesions, e.g. cervical cancer screening purposes. For the latter, it is important that HPV tests are clinically validated according to validation strategies as outlined in guidelines. [ABSTRACT FROM AUTHOR]

Background: Concerns have been raised that the proportion of cervical cancer preventable by human papillomavirus (HPV) 16/18 vaccines might be lower in sub-Saharan Africa than elsewhere. Method: In order to study the relative carcinogenicity of HPV types in Nigeria, as well as to estimate the vaccinepreventable proportion of invasive cervical cancer (ICC) in the country, we compared HPV type prevalence among 932 women from the general population of Ibadan, Nigeria, with that among a series of 75 ICC cases diagnosed in the same city. For all samples, a GP5+/6+ PCR based assay was used for the detection of 44 genital HPV types. Results: In the general population, 245 (26.3%, 95% confidence interval (CI) 23.5% - 29.2%) women were HPVpositive, among whom the prevalence of HPV35 and HPV16 were equally frequent (12.2%, 95% CI 8.4% - 17.0%). In ICC, however, HPV16 predominated strongly (67.6% of 68 HPV-positive cases), with the next most common types being 18 (10.3%, 95% CI 4.2% - 20.1%), 35, 45 and 56 (each 5.9%, 95% CI 1.6% - 14.4%). Comparing among HPVpositive women only, HPV16 and 18 were over-represented in ICC versus the general population (prevalence ratios 5.52, 95% CI 3.7 - 8.3 and 1.4, 95% CI 0.6 - 3.3, respectively). Other high-risk HPV types, as well as low-risk and multiple HPV infections were less common in HPV-positive women with ICC than from the general population. Conclusions: Our study confirms that in Nigeria, as elsewhere, women infected with HPV16 and 18 are at higher risk of developing ICC than those infected with other high-risk types, and that current HPV16/18 vaccines have enormous potential to reduce cervical cancer in the region. [ABSTRACT FROM AUTHOR]

Background: Activation of telomerase resulting from deregulated hTERT expression is a key event during high-risk human papillomavirus (hrHPV)-induced cervical carcinogenesis. In the present study we examined hTERT promoter activity and its relation to DNA methylation as one of the potential mechanisms underlying deregulated hTERT transcription in hrHPV-transformed cells. Methods: Using luciferase reporter assays we analyzed hTERT promoter activity in primary keratinocytes, HPV16- and HPV18-immortalized keratinocyte cell lines and cervical cancer cell lines. In the same cells as well as cervical specimens we determined hTERT methylation by bisulfite sequencing analysis of the region spanning -442 to +566 (relative to the ATG) and quantitative methylation specific PCR (qMSP) analysis of two regions flanking the hTERT core promoter. Results: We found that in most telomerase positive cells increased hTERT core promoter activity coincided with increased hTERT mRNA expression. On the other hand basal hTERT promoter activity was also detected in telomerase negative cells with no or strongly reduced hTERT mRNA expression levels. In both telomerase positive and negative cells regulatory sequences flanking both ends of the core promoter markedly repressed exogenous promoter activity. By extensive bisulfite sequencing a strong increase in CpG methylation was detected in hTERT positive cells compared to cells with no or strongly reduced hTERT expression. Subsequent qMSP analysis of a larger set of cervical tissue specimens revealed methylation of both regions analyzed in 100% of cervical carcinomas and 38% of the high-grade precursor lesions, compared to 9% of low grade precursor lesions and 5% of normal controls. Conclusions: Methylation of transcriptionally repressive sequences in the hTERT promoter and proximal exonic sequences is correlated to deregulated hTERT transcription in HPV-immortalized cells and cervical cancer cells. The detection of DNA methylation at these repressive regions may provide an attractive biomarker for early detection of cervical cancer. [ABSTRACT FROM AUTHOR]

Background: A substantial number of microRNAs (miRNAs) is subject to epigenetic silencing in cancer. Although epigenetic silencing of tumour suppressor genes is an important feature of cervical cancer, little is known about epigenetic silencing of miRNAs. Since DNA methylation-based silencing of hsa-miR-124 occurs in various human cancers, we studied the frequency and functional effects of hsa-miR-124 methylation in cervical carcinogenesis. Results: Quantitative MSP analysis of all 3 loci encoding the mature hsa-miR-124 (hsa-miR-124-1/-2/-3) showed methylation in cervical cancer cell lines SiHa, CaSki and HeLa as well as in late passages of human papillomavirus (HPV) type 16 or 18 immortalised keratinocytes. Treatment of SiHa cells with a demethylating agent reduced hsa-miR-124 methylation levels and induced hsa-miR-124 expression. In HPV-immortalised keratinocytes increased methylation levels were related to reduced hsa-miR-124 expression and higher mRNA expression of IGFBP7, a potential hsa-miR-124 target gene. Ectopic hsa-miR-124 expression in SiHa and CaSki cells decreased proliferation rates and migratory capacity. Combined hsa-miR-124-1 and/or hsa-miR-124-2 methylation analysis of 139 cervical tissue specimens showed an increasing methylation frequency from 0% in normal tissues up to 93% in cervical carcinomas. Increased methylation levels of hsa-miR-124-1 and hsa-miR-124-2 were significantly correlated with reduced hsa-miR-124 expression in cervical tissue specimens. Combined hsa-miR-124-1 and/or hsa-miR-124-2 methylation analysis of 43 cervical scrapes of high-risk HPV positive women was predictive of underlying high-grade lesions. Conclusions: DNA methylation-based silencing of hsa-miR-124 is functionally involved in cervical carcinogenesis and may provide a valuable marker for improved detection of cervical cancer and its high-grade precursor lesions. [ABSTRACT FROM AUTHOR]

Background: It is well known that a persistent infection with high-risk human papillomavirus (hrHPV) is causally involved in the development of squamous cell carcinomas of the uterine cervix (CxSCCs) and a subset of SCCs of the head and neck (HNSCCs). The latter differ from hrHPV-negative HNSCCs at the clinical and molecular level. Methods: To determine whether hrHPV-associated SCCs arising from different organs have specific chromosomal alterations in common, we compared genome-wide chromosomal profiles of 10 CxSCCs (all hrHPV-positive) with 12 hrHPV-positive HNSCCs and 30 hrHPV-negative HNSCCs. Potential organ-specific alterations and alterations shared by SCCs in general were investigated as well. Results: Unsupervised hierarchical clustering resulted in one mainly hrHPV-positive and one mainly hrHPV-negative cluster. Interestingly, loss at 13q and gain at 20q were frequent in HPV-positive carcinomas of both origins, but uncommon in hrHPV-negative HNSCCs, indicating that these alterations are associated with hrHPV-mediated carcinogenesis. Within the group of hrHPV-positive carcinomas, HNSCCs more frequently showed gains of multiple regions at 8q whereas CxSCCs more often showed loss at 17p. Finally, gains at 3q24-29 and losses at 11q22.3-25 were frequent (>50%) in all sample groups. Conclusion: In this study hrHPV-specific, organ-specific, and pan-SCC chromosomal alterations were identified. The existence of hrHPV-specific alterations in SCCs of different anatomical origin, suggests that these alterations are crucial for hrHPV-mediated carcinogenesis. [ABSTRACT FROM AUTHOR]

Aims. We investigated the effect of HR-HPV infection on the capacity of the cytokine network in whole blood cultures during carcinogenesis of cervical carcinoma. Methods. Thirty-nine women with moderate dysplasia, severe dysplasia, cervical carcinoma, or without dysplasia formed the study group. The control group consisted of 10 HR-HPV-negative women without CIN. Whole blood cultures were stimulated with phytohemagglutinin (PHA) and concentrations of tumour necrosis factor α (TNFα), interferon γ (IFNγ), interleukin 2 (IL-2), interleukin 12 (IL-12), interleukin 4 (IL-4), and interleukin 10 (IL-10) were determined by ELISAS. Results. A significant increase in cytokine release was detected in HR-HPV-positive women without dysplasia. In women with cervical cancer, release of IFNγ and IL-12 was of the same magnitude as in HR-HPV-positive women without clinical manifestations. Most Th1-type/Th2-type ratios decreased form CIN II to CIN III, and increased from CIN III to invasive carcinoma. Conclusions. (1) Infection with HR-HPV without expression of cervical dysplasia induces activation of the cytokine network. (2) Increases in ratios of Th1-type to Th2-type cytokines at the stage of cervical carcinoma were found by comparison with stage CIN III. (3) Significant changes in the kinetics of cytokine release to a Th2-type immune response in blood of women with cervical dysplasia occurred progressively from CIN II to CIN III. [ABSTRACT FROM AUTHOR]

Various high-risk HPV (HR HPV) DNA assays have been developed that allow detection of a broad spectrum of HR HPVs. Two of these assays [Hybrid Capture 2 (hc2) and GP5+/6+-PCR] have shown in large clinical trials a superior clinical sensitivity for cervical (we)cancer compared to cytology and an optimal balance between clinical sensitivity and specificity. Comparative studies showed that an increased sensitivity for HR HPV relative to GP5+/6+-PCR and/or hc2 results in a dramatic decrease in clinical specificity, whereas on the other hand a decreased sensitivity for virus leads to a decrease in sensitivity for (pre)cancer. These data argue for guidelines on HR H PV test requirements for cervical screening purposes. [ABSTRACT FROM AUTHOR]

The article deals with a study which investigated the efficacy of a quadrivalent vaccine against human papillomaviruses (HPV) for preventing high-grade cervical lesions, adenocarcinoma in situ and cervical cancer. Background on the prevalence and cause of HPV is offered. Study population were women aged 15-26 years old enrolled in the Females United to Unilaterally Reduce Endo/Ectocervical Disease (FUTURE) II trial. It describes the observed effects of the vaccine on the patients. Also noted is a commentary on the research findings.

Background Histomorphological grading of cervical intraepithelial neoplasia (CIN) is crucial for clinical management. CIN grading is however subjective and affected by substantial rates of discordance among pathologists, which may lead to overtreatment. To minimise this problem, a histology review of CIN lesions by a consensus panel of pathologists is often used. Diffuse strong p16INK4a immunostaining has been proposed to aid the identification of true high-grade cervical lesions (ie, CIN2/3). Aim To assess the value of additional interpretation of p16INK4a immunostains for making a more reproducible diagnosis of CIN2/3 lesions. Methods The authors used a series of 406 biopsies of cervical lesions, with known HPV status, stained for both H&E- and p16INK4a. First, in a randomly selected set of 49 biopsies, we examined the effect of additional interpretation of p16INK4a immunostained slides, on the agreement of CIN diagnosis among three pathologists. Second, the full series of samples was used to assess the accuracy of p16INK4a-supported lesion grading by a single pathologist, by evaluating the degree of diagnostic agreement with the consensus diagnosis of expert pathologists based on H&E-stained sections only. Results The study shows that the interobserver agreement between three pathologists for the routine H&E-based diagnosis ranged from fair (weighted kappa 0.44 (95% CI 0.19 to 0.64)) to moderate (weighted kappa 0.66 (95% CI 0.47 to 0.79)). The concordance increased substantially for p16INK4a-supported grading (mean weighted kappa 0.80 (95% CI 0.66 to 0.89)). Furthermore, an almost perfect agreement was found between the p16INK4a-supported diagnosis of a single pathologist and the consensus diagnosis of an expert pathology panel (kappa 0.88 (95% CI 0.85 to 0.89)). Conclusions These data demonstrate that additive use of p16INK4a immunohistochemistry significantly improves the accuracy of grading CIN lesions by a single pathologist, equalling an expert consensus diagnosis. Hence, the authors advocate the combined use of p16INK4a-stained slides and conventional H&E sections in routine histopathology to improve accuracy of diagnosis. [ABSTRACT FROM AUTHOR]

Summary: Background: Screening for human papillomavirus (HPV) infection is more effective in reducing the incidence of cervical cancer than screening using Pap smears. Moreover, HPV testing can be done on a vaginal sample self-taken by a woman, which offers an opportunity to improve screening coverage. However, the clinical accuracy of HPV testing on self-samples is not well-known. We assessed whether HPV testing on self-collected samples is equivalent to HPV testing on samples collected by clinicians. Methods: We identified relevant studies through a search of PubMed, Embase, and CENTRAL. Studies were eligible for inclusion if they fulfilled all of the following selection criteria: a cervical cell sample was self-collected by a woman followed by a sample taken by a clinician; a high-risk HPV test was done on the self-sample (index test) and HPV-testing or cytological interpretation was done on the specimen collected by the clinician (comparator tests); and the presence or absence of cervical intraepithelial neoplasia grade 2 (CIN2) or worse was verified by colposcopy and biopsy in all enrolled women or in women with one or more positive tests. The absolute accuracy for finding CIN2 or worse, or CIN grade 3 (CIN3) or worse of the index and comparator tests as well as the relative accuracy of the index versus the comparator tests were pooled using bivariate normal models and random effect models. Findings: We included data from 36 studies, which altogether enrolled 154 556 women. The absolute accuracy varied by clinical setting. In the context of screening, HPV testing on self-samples detected, on average, 76% (95% CI 69–82) of CIN2 or worse and 84% (72–92) of CIN3 or worse. The pooled absolute specificity to exclude CIN2 or worse was 86% (83–89) and 87% (84–90) to exclude CIN3 or worse. The variation of the relative accuracy of HPV testing on self-samples compared with tests on clinician-taken samples was low across settings, enabling pooling of the relative accuracy over all studies. The pooled sensitivity of HPV testing on self-samples was lower than HPV testing on a clinician-taken sample (ratio 0·88 [95% CI 0·85–0·91] for CIN2 or worse and 0·89 [0·83–0·96] for CIN3 or worse). Also specificity was lower in self-samples versus clinician-taken samples (ratio 0·96 [0·95–0·97] for CIN2 or worse and 0·96 [0·93–0·99] for CIN3 or worse). HPV testing with signal-based assays on self-samples was less sensitive and specific than testing on clinician-based samples. By contrast, some PCR-based HPV tests generally showed similar sensitivity on both self-samples and clinician-based samples. Interpretation: In screening programmes using signal-based assays, sampling by a clinician should be recommended. However, HPV testing on a self-sample can be suggested as an additional strategy to reach women not participating in the regular screening programme. Some PCR-based HPV tests could be considered for routine screening after careful piloting assessing feasibility, logistics, population compliance, and costs. Funding: The 7th Framework Programme of the European Commission, the Belgian Foundation against Cancer, the International Agency for Research on Cancer, and the German Guideline Program in Oncology. [ABSTRACT FROM AUTHOR]

Objectives Methylation marker analysis using bi-marker panel MAL/miR-124-2 is a promising triage test for identifying cervical (pre)cancer in high-risk human papillomavirus (hrHPV) positive women. Bi-marker panel MAL/miR-124-2 can be applied directly on self-sampled cervico-vaginal material and its sensitivity is non-inferior to that of cytology, yet at the cost of more colposcopy referrals. Our objective was to increase specificity of MAL/miR-124-2 methylation analysis by varying the assay thresholds and adding HPV16/18 genotyping. Methods 1019 hrHPV-positive women were selected from a randomized controlled self-sampling trial (PROHTECT-3; 33-63 years, n = 46,001) and nine triage strategies with methylation testing of MAL/miR-124-2 and HPV16/18 genotyping were evaluated. The methylation assay threshold was set at four different predefined levels which correspond with clinical specificities for end-point cervical intra-epithelial grade 3 or worse (CIN3 +) of 50%, 60%, 70%, and 80%. Results The CIN3 + sensitivity of methylation analysis decreased (73.5 to 44.9%) while specificity increased (47.2 to 83.4%) when increasing the assay threshold. CIN3 + sensitivity and specificity of HPV16/18 genotyping were 68.0% and 65.6%, respectively. Combined methylation analysis at threshold-80 and HPV16/18 genotyping yielded similar CIN3 + sensitivity as that of methylation only at threshold-50 (77.6%) with an increased specificity (54.8%). Conclusions Combined triage by MAL/miR-124-2 methylation analysis with threshold-80 and HPV16/18 genotyping reaches high CIN3 + sensitivity with increased specificity to identify women with cervical (pre)cancer among HPV self-sample positive women. The combined strategy is attractive as it is fully molecular and identifies women at the highest risk of cervical (pre)cancer because of strongly elevated methylation levels and/or HPV16/18 positivity. [ABSTRACT FROM AUTHOR]

Human papillomavirus 45 (HPV45) is a member of the HPV18-related alpha-7 species and accounts for approximately 5% of all cervical cancer cases worldwide. This study evaluated the genetic diversity of HPV45 and the association of HPV45 variants with the risk of cervical cancer by sequencing the entire E6 and E7 open reading frames of 300 HPV45-positive cervical samples from 36 countries. A total of 43 HPV45 sequence variants were identified that formed 5 phylogenetic sublineages, A1, A2, A3, B1, and B2, the distribution of which varied by geographical region. Among 192 cases of cervical cancer and 101 controls, the B2 sublineage was significantly overrepresented in cervical cancer, both overall and in Africa and Europe separately. We show that the sequence analysis of E6 and E7 allows the classification of HPV45 variants and that the risk of cervical cancer may differ by HPV45 variant sublineage. [ABSTRACT FROM AUTHOR]