HPV integration and cervical cancer: a failed evolutionary viral trait.

Countless efforts have been made to eradicate cervical cancer worldwide, including improving disease screening and human papillomavirus (HPV) vaccination programs. Nevertheless, cervical cancer still claims the lives of more than 300 000 women every year. Persistent infections with high-risk HPV genotypes 16 and 18 are the main cause of cancer and may result in HPV integration into the host genome. The central dogma is that HPV integration is an important step in oncogenesis, but in fact, it impedes the virus from replicating and spreading. HPV causing cervical cancer can therefore be perceived as a failed evolutionary viral trait. Here we outline the occurrence and mechanisms of HPV integration and how this process results in oncogenic transformation.

Increased human papillomavirus viral load is correlated to higher severity of cervical disease and poorer clinical outcome: A systematic review.

Cervical cancer is the fourth most common cancer in women worldwide and is caused by persistent infection with high-risk types of human papillomavirus (HPV). HPV viral load, the amount of HPV DNA in a sample, has been suggested to correlate with cervical disease severity, and with clinical outcome of cervical cancer. In this systematic review, we searched three databases (EMBASE, PubMed, Web of Science) to examine the current evidence on the association between HPV viral load in cervical samples and disease severity, as well as clinical outcome. After exclusion of articles not on HPV, cervical cancer, or containing clinical outcomes, 85 original studies involving 173 746 women were included. The vast majority (73/85 = 85.9%) reported that a higher viral load was correlated with higher disease severity or worse clinical outcome. Several studies reported either no correlation (3/85 = 3.5%), or the opposite correlation (9/85 = 10.6%); possible reasons being different categorization of HPV viral load levels, or the use of specific sampling methods. Despite variations in study design and populations, the above findings suggest that HPV viral load is correlated to clinical outcome, and may become an important biomarker for treatment selection and response monitoring for cervical cancer.

DNA Methylation and P53 Immunohistochemistry as Prognostic Biomarkers for Vulvar Lichen Sclerosus.

Vulvar lichen sclerosus (LS) is an inflammatory dermatosis that can progress to human papillomavirus (HPV)-independent vulvar intraepithelial neoplasia (HPVi VIN) and vulvar squamous cell carcinoma (VSCC). Although LS has a much lower cancer risk compared with HPVi VIN (5% versus 50%, respectively), its incidence is significantly higher. Therefore, there is a clinical need to identify LS patients with an increased cancer risk. Our objective was to study the value of DNA methylation and p53 immunohistochemistry (IHC) as prognostic biomarkers for progression to cancer in patients with LS. Vulvar tissues from 236 patients were selected, including 75 LS and 68 HPVi VIN, both with and without cancer development, 32 VSCC, and 61 healthy vulvar controls. Samples were subjected to p53 IHC and DNA methylation analysis of a 3-gene marker panel containing ZNF582, SST, and miR124-2. Methylation levels and p53 IHC status (mutant or wild-type) were assessed and compared among all disease categories. Odds ratios were determined to identify whether the biomarkers were associated with progression to cancer in patients with LS. The highest methylation levels were found in HPVi VIN and VSCC, followed by LS and healthy vulvar controls. The largest heterogeneity in methylation levels was observed in LS cases. In fact, the 3-marker panel tested positive in 70% of LS, which progressed to VSCC versus only 17% of LS in patients without cancer development (P = .002). Also, mutant p53 IHC was observed more frequently in LS with progression to VSCC compared with nonprogressive LS cases (42% versus 3%, respectively, P = .001). Multivariable analysis identified a mutant p53 status as the only independent risk factor for cancer development in LS (odds ratio: 34.0, 95% CI: 1.4-807.4). In conclusion, DNA methylation testing and p53 IHC show strong potential as prognostic biomarkers for the identification of LS patients at high risk of progression to cancer.

Molecular analysis for ovarian cancer detection in patient-friendly samples.

BACKGROUND: High ovarian cancer mortality rates motivate the development of effective and patient-friendly diagnostics. Here, we explored the potential of molecular testing in patient-friendly samples for ovarian cancer detection. METHODS: Home-collected urine, cervicovaginal self-samples, and clinician-taken cervical scrapes were prospectively collected from 54 patients diagnosed with a highly suspicious ovarian mass (benign n = 25, malignant n = 29). All samples were tested for nine methylation markers, using quantitative methylation-specific PCRs that were verified on ovarian tissue samples, and compared to non-paired patient-friendly samples of 110 age-matched healthy controls. Copy number analysis was performed on a subset of urine samples of ovarian cancer patients by shallow whole-genome sequencing. RESULTS: Three methylation markers are significantly elevated in full void urine of ovarian cancer patients as compared to healthy controls (C2CD4D, P = 0.008; CDO1, P = 0.022; MAL, P = 0.008), of which two are also discriminatory in cervical scrapes (C2CD4D, P = 0.001; CDO1, P = 0.004). When comparing benign and malignant ovarian masses, GHSR shows significantly elevated methylation levels in the urine sediment of ovarian cancer patients (P = 0.024). Other methylation markers demonstrate comparably high methylation levels in benign and malignant ovarian masses. Cervicovaginal self-samples show no elevated methylation levels in patients with ovarian masses as compared to healthy controls. Copy number changes are identified in 4 out of 23 urine samples of ovarian cancer patients. CONCLUSIONS: Our study reveals increased methylation levels of ovarian cancer-associated genes and copy number aberrations in the urine of ovarian cancer patients. Our findings support continued research into urine biomarkers for ovarian cancer detection and highlight the importance of including benign ovarian masses in future studies to develop a clinically useful test.

Ovarian cancer is often found late with limited treatment options. Currently, it is difficult to diagnose ovarian cancer correctly and no recommended early detection or screening methods exist. Our aim was to explore the use of DNA-based tests in patient-friendly samples for ovarian cancer detection. Patient-friendly samples are patient materials that can be collected from home without pain or discomfort, such as self-collected vaginal swabs and urine. Using DNA-based tests, we found that urine of women with ovarian cancer contains ovarian cancer-associated signals. Our findings encourage further development of a potential urine test for ovarian cancer detection. This approach could aid early detection and guide women with ovarian masses to appropriate specialist care.

Accurate detection of copy number aberrations in FFPE samples using the mFAST-SeqS approach.

BACKGROUND: Shallow whole genome sequencing (Shallow-seq) is used to determine the copy number aberrations (CNA) in tissue samples and circulating tumor DNA. However, costs of NGS and challenges of small biopsies ask for an alternative to the untargeted NGS approaches. The mFAST-SeqS approach, relying on LINE-1 repeat amplification, showed a good correlation with Shallow-seq to detect CNA in blood samples. In the present study, we evaluated whether mFAST-SeqS is suitable to assess CNA in small formalin-fixed paraffin-embedded (FFPE) tissue specimens, using vulva and anal HPV-related lesions. METHODS: Seventy-two FFPE samples, including 36 control samples (19 vulva;17 anal) for threshold setting and 36 samples (24 vulva; 12 anal) for clinical evaluation, were analyzed by mFAST-SeqS. CNA in vulva and anal lesions were determined by calculating genome-wide and chromosome arm-specific z-scores in comparison with the respective control samples. Sixteen samples were also analyzed with the conventional Shallow-seq approach. RESULTS: Genome-wide z-scores increased with the severity of disease, with highest values being found in cancers. In vulva samples median and inter quartile ranges [IQR] were 1[0-2] in normal tissues (n = 4), 3[1-7] in premalignant lesions (n = 9) and 21[13-48] in cancers (n = 10). In anal samples, median [IQR] were 0[0-1] in normal tissues (n = 4), 14[6-38] in premalignant lesions (n = 4) and 18[9-31] in cancers (n = 4). At threshold 4, all controls were CNA negative, while 8/13 premalignant lesions and 12/14 cancers were CNA positive. CNA captured by mFAST-SeqS were mostly also found by Shallow-seq. CONCLUSION: mFAST-SeqS is easy to perform, requires less DNA and less sequencing reads reducing costs, thereby providing a good alternative for Shallow-seq to determine CNA in small FFPE samples.

miR-129-5p inhibits anchorage-independent growth through silencing of ACTN1 and the ELK4/c-FOS axis in HPV-transformed keratinocytes.

A persistent infection with human papillomavirus (HPV) can induce precancerous lesions of the cervix that may ultimately develop into cancer. Cervical cancer development has been linked to altered microRNA (miRNA) expression, with miRNAs regulating anchorage-independent growth being particularly important for the progression of precancerous lesions to cancer. In this study, we set out to identify and validate targets of miR-129-5p, a previously identified tumor suppressive miRNA involved in anchorage-independent growth and HPV-induced carcinogenesis. We predicted 26 potential miR-129-5p targets using online databases, followed by KEGG pathway enrichment analysis. RT-qPCR and luciferase assays confirmed that 3'UTR regions of six genes (ACTN1, BMPR2, CAMK4, ELK4, EP300, and GNAQ) were targeted by miR-129-5p. Expressions of ACTN1, CAMK4, and ELK4 were inversely correlated to miR-129-5p expression in HPV-transformed keratinocytes, and their silencing reduced anchorage-independent growth. Concordantly, miR-129-5p overexpression decreased protein levels of ACTN1, BMPR2, CAMK4 and ELK4 in anchorage-independent conditions. Additionally, c-FOS, a downstream target of ELK4, was downregulated upon miR-129-5p overexpression, suggesting regulation through the ELK4/c-FOS axis. ACTN1 and ELK4 expression was also upregulated in high-grade precancerous lesions and cervical cancers, supporting their clinical relevance. In conclusion, we identified six targets of miR-129-5p involved in the regulation of anchorage-independent growth, with ACTN1, BMPR2, ELK4, EP300, and GNAQ representing novel targets for miR-129-5p. For both ACTN1 and ELK4 functional and clinical relevance was confirmed, indicating that miR-129-5p-regulated ACTN1 and ELK4 expression contributes to HPV-induced carcinogenesis.

Unlocking the potential of tumor-derived DNA in urine for cancer detection: methodological challenges and opportunities.

High cancer mortality rates and the rising cancer burden worldwide drive the development of innovative methods in order to advance cancer diagnostics. Urine contains a viable source of tumor material and allows for self-collection from home. Biomarker testing in this liquid biopsy represents a novel approach that is convenient for patients and can be effective in detecting cancer at a curable stage. Here, we set out to provide a detailed overview of the rationale behind urine-based cancer detection, with a focus on non-urological cancers, and its potential for cancer diagnostics. Moreover, evolving methodological challenges and untapped opportunities for urine biomarker testing are discussed, particularly emphasizing DNA methylation of tumor-derived cell-free DNA. We also provide future recommendations for technical advancements in urine-based cancer detection and elaborate on potential mechanisms involved in the transrenal transport of cell-free DNA.

DNA methylation as a triage tool for cervical cancer screening - A meeting report.

Introduction: DNA methylation is proposed as a novel biomarker able to monitor molecular events in human papillomavirus (HPV) infection pathophysiology, enabling the distinction between HPV-induced lesions with regression potential from those that may progress to HPV-related cancer. Methods: This meeting report summarises the presentations and expert discussions during the HPV Prevention and Control Board-focused topic technical meeting on DNA methylation validation in clinician-collected and self-collected samples, novel DNA methylation markers discovery, implementation in cervical cancer screening programs, and their potential in women living with human immunodeficiency virus (HIV). Results: Data presented in the meeting showed that HPV-positive, baseline methylation-negative women have a lower cumulative cervical cancer incidence than baseline cytology-negative women, making DNA methylation an attractive triage strategy. However, additional standardised data in different settings (low- versus high-income settings), samples (clinician-collected and self-collected), study designs (prospective, modelling, impact) and populations (immunocompetent women, women living with HIV) are needed. Conclusion: Establishing international validation guidelines were identified as the way forward towards accurate validation and subsequent implementation in current screening programs.

Incidence and Risk Factors for Recurrence and Progression of HPV-Independent Vulvar Intraepithelial Neoplasia.

OBJECTIVES: Human papillomavirus (HPV)-independent vulvar intraepithelial neoplasia (VIN) is a rare yet aggressive precursor lesion of vulvar cancer. Our objectives were to estimate its long-term incidence, the risk of recurrent disease and progression to vulvar cancer, and risk factors thereof. MATERIALS AND METHODS: Patients with HPV-independent VIN between 1991 and 2019 in a selected region were identified from the Dutch Nationwide Pathology Databank (Palga). Data were collected from the pathology reports. Crude and European age-standardized incidence rates were calculated for 10-year periods. Kaplan-Meier analyses were performed to determine the cumulative recurrence and cancer incidence, followed by Cox regression analyses to identify associated risk factors. RESULTS: A total of 114 patients were diagnosed with solitary HPV-independent VIN without prior or concurrent vulvar cancer. The European age-standardized incidence rate increased from 0.09 to 0.69 per 100,000 women-years between 1991-2010 and 2011-2019. A cumulative recurrence and cancer incidence of 29% and 46% were found after 8 and 13 years of follow-up, respectively. Nonradical surgery was identified as the only independent risk factor for recurrent HPV-independent VIN. Risk factors associated with progression to cancer were increasing age and a mutant p53 immunohistochemical staining pattern. CONCLUSIONS: The incidence of detected HPV-independent VIN has substantially increased the last decade and the subsequent recurrence and vulvar cancer risks are high. Although HPV-independent VIN may present as a wide morphologic spectrum, surgical treatment should aim for negative resection margins followed by close surveillance, especially for p53 mutant lesions.

High-grade vulvar intraepithelial neoplasia: comprehensive characterization and long-term vulvar carcinoma risk.

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.

Analytical validation and diagnostic performance of the ASCL1/ZNF582 methylation test for detection of high-grade anal intraepithelial neoplasia and anal cancer.

DNA methylation testing on biopsies can detect high-grade anal intraepithelial neoplasia (HGAIN) in need of treatment and anal cancer. This study aimed to analytically validate and determine the diagnostic performance of a newly developed multiplex quantitative methylation-specific PCR, PreCursor-M AnoGYN (RUO), combining ASCL1, ZNF582 and a reference (ACTB) in one assay. Analytical validation was performed on two qPCR devices using predefined quality criteria. Diagnostic performance was determined on a cross-sectional series of 111 anal biopsies covering all stages of anal disease. Differences in methylation levels were assessed using the Kruskal-Wallis test. Area under the curve was determined using logistic regression analysis. Detection rates were calculated at predefined specificities for the cross-sectional and an additional longitudinal series of 23 HGAIN biopsies preceding anal cancer (i.e., progressive HGAIN). For both devices analytical quality criteria were met. ASCL1 and ZNF582 methylation levels increased with increasing severity of disease (p < 6*10-8). Diagnostic performance for AIN3+ was 0.81. All cancers and virtually all progressive HGAIN were detected at 70% and 80% specificity. In conclusion, the ASCL1/ZNF582 methylation test (PreCursor-M AnoGYN (RUO)) was demonstrated to be highly robust and reproducible. Moreover, it had excellent diagnostic accuracy to detect AIN3+ and can potentially be used to guide HGAIN management.

Methylation testing for the detection of recurrent cervical intraepithelial neoplasia.

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.

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.

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

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

DNA methylation testing for endometrial cancer detection in urine, cervicovaginal self-samples and cervical scrapes.

Endometrial cancer incidence is rising and current diagnostics often require invasive biopsy procedures. DNA methylation marker analysis of minimally- and non-invasive sample types could provide an easy-to-apply and patient-friendly alternative to determine cancer risk. Here, we compared the performance of DNA methylation markers to detect endometrial cancer in urine, cervicovaginal self-samples and clinician-taken cervical scrapes. Paired samples were collected from 103 patients diagnosed with stage I to IV endometrial cancer. Urine and self-samples were collected at home. All samples were tested for nine DNA methylation markers using quantitative methylation-specific PCR. Methylation levels measured in endometrial cancer patients were compared to unpaired samples of 317 healthy controls. Diagnostic performances were evaluated by univariable and multivariable logistic regression analysis, followed by leave-one-out cross-validation. Each methylation marker showed significantly higher methylation levels in all sample types of endometrial cancer patients compared to healthy controls (P < .01). Optimal three-marker combinations demonstrated excellent diagnostic performances with area under the receiver operating curve values of 0.95 (95% CI: 0.92-0.98), 0.94 (0.90-0.97) and 0.97 (0.96-0.99), for endometrial cancer detection in urine, self-samples and scrapes, respectively. Sensitivities ranged from 89% to 93% at specificities of 90% to 92%. Virtually equal performances were obtained after cross-validation and excellent diagnostic performances were maintained for stage I endometrial cancer detection. Our study shows the value of methylation analysis in patient-friendly sample types for endometrial cancer detection of all stages. This approach has great potential to screen patient populations at risk for endometrial cancer.

Effects of 2 and 3 Vaccinations With the Bivalent Human Papillomavirus (HPV) Vaccine on the Prevalence and Load of HPV in Clearing and Persistent Infections in Young Women.

BACKGROUND: Human papillomavirus (HPV) viral load (VL) is associated with persistence, which increases cervical cancer risk. The bivalent vaccine protects against oncogenic HPV-16/18 and cross-protects against several nonvaccine types. We examined the effect of 2-dose (2D) and 3-dose (3D) vaccination on HPV prevalence and VL in clearing infections and persistent infections, 6 years and 12 years postvaccination, respectively. METHODS: Vaginal swabs collected from the "HPV Amongst Vaccinated and Non-vaccinated Adolescents" study (HAVANA, 3D-eligible) and HAVANA-2 (2D-eligble) participants were genotyped for HPV with the SPF10-DEIA-LiPA25 system. HPV VL was measured with type-specific quantitative polymerase chain reaction (qPCR). RESULTS: HPV-16, -18, -31, -33, and -45 clearing and/or persistent infection prevalence and HPV-16, -18, and -31 VLs in clearing infections were significantly reduced in 3D-vaccinated women compared to unvaccinated women. Except for HPV-11 and -59 clearing infections, no significant VL differences were observed among vaccinated women, ≤6 and >6 years post-vaccination. Infection numbers were low in 2D-eligible women, with no HPV-16/18 in vaccinated women. No VL differences for the remaining types were found. CONCLUSIONS: 3D vaccination reduces HPV prevalence in clearing infections and persistent infections and decreases HPV VLs in clearing infections, 12 years post-vaccination for vaccine and several nonvaccine types. 2D-eligible women had low infection numbers, with no HPV-16/18 among vaccinated women.

Downregulation of miR-193a/b-3p during HPV-induced cervical carcinogenesis contributes to anchorage-independent growth through PI3K-AKT pathway regulators.

Cervical cancer is caused by a persistent infection with high-risk types of human papillomavirus (HPV) and an accumulation of (epi)genetic alterations in the host cell. Acquisition of anchorage-independent growth represents a critical hallmark during HPV-induced carcinogenesis, thereby yielding the most valuable biomarkers for early diagnosis and therapeutic targets. In a previous study, we found that miR-193a-3p and miR-193b-3p were involved in anchorage-independent growth. This study aimed to delineate the role of miR-193a/b-3p in HPV-induced carcinogenesis and to identify their target genes related to anchorage-independent growth. Cell viability and colony formation were assessed in SiHa cancer cells and HPV-16 and -18 immortalized keratinocytes upon miR-193a/b-3p overexpression. Both microRNAs reduced cell growth of all three cell lines in low-attachment conditions and showed a minor effect in adherent conditions. Online target-predicting programs and publicly available expression data were used to find candidate messenger RNA (mRNA) targets of miR-193a/b-3p. Seven targets showed reduced mRNA expression upon miR-193a/b-3p overexpression. For three targets, Western blot analysis was also performed, all showing a reduced protein expression. A direct interaction was confirmed using luciferase assays for six genes: LAMC1, PTK2, STMN1, KRAS, SOS2, and PPP2R5C, which are phosphatidylinositol 3-kinase/protein kinase B (PI3K-AKT) regulators. All six targets were overexpressed in cervical cancers and/or precursor lesions. Together with an observed downregulation of phosphorylated-AKT upon miR-193a/b-3p overexpression, this underlines the biological relevance of miR-193a/b-3p downregulation during HPV-induced cervical carcinogenesis. In conclusion, the downregulation of miR-193a-3p and miR-193b-3p is functionally involved in the acquisition of HPV-induced anchorage independence by targeting regulators of the PI3K-AKT pathway.

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.

Validation of ASCL1 and LHX8 Methylation Analysis as Primary Cervical Cancer Screening Strategy in South African Women with Human Immunodeficiency Virus.

BACKGROUND: Compared with women who are human immunodeficiency virus (HIV) negative, women with human immunodeficiency virus (WWH) have a higher human papillomavirus (HPV) prevalence and increased cervical cancer risk, emphasizing the need for effective cervical cancer screening in this population. The present study aimed to validate methylation markers ASCL1 and LHX8 for primary screening in a South African cohort of WWH. METHODS: In this post hoc analysis within the DIAgnosis in Vaccine And Cervical Cancer Screen (DiaVACCS) study, a South African observational multicenter cohort study, cervical scrape samples from 411 HIV-positive women were analyzed for hypermethylation of ASCL1 and LHX8 genes, HPV DNA, and cytology. Sensitivities, specificities, and positive and negative predictive values of primary methylation-based, HPV-based and cytology-based screening were calculated for the detection of cervical intraepithelial neoplasia of grade 3 or higher. RESULTS: Single markers ASCL1 and LHX8 resulted in a good performance for the detection of cervical intraepithelial neoplasia of grade 3 or higher, with sensitivities of 85.9% (95% confidence interval [CI], 78.2%-93.6%) and 89.7% (83.0%-96.5%), respectively, and specificities of 72.9% (67.3%-78.5%) and 75.0% (69.5%-80.5%). Combining markers ASCL1 and LHX8 resulted in a lower sensitivity compared with HPV testing (84.6% vs 93.6%, respectively; ratio, 0.90 [95% CI, .82-.99]) and a higher specificity (86.7% vs 78.3%; ratio 1.11 [1.02-1.20]) and reduced the referral rate from 46.8% to 33.4%. ASCL1/LHX8 methylation had a significantly higher sensitivity than cytology (threshold, high-grade intraepithelial squamous lesion or worse), (84.6% vs 74.0%, respectively; ratio, 1.16 [95% CI, 1.01-1.32]) and similar specificity (86.7% vs 91.0%; ratio, 0.95 [.90-1.003]). CONCLUSIONS: Our results validate the accuracy of ASCL1/LHX8 methylation analysis for primary screening in WWH, which offers a full-molecular alternative to cytology- or HPV-based screening, without the need for additional triage testing.

DNA Methylation Analysis to predict Regression of high-grade anal Intraepithelial Neoplasia in HIV+ men (MARINE): a cohort study protocol.

INTRODUCTION: Anal cancer precursors, or high-grade anal intraepithelial neoplasia (HGAIN), are highly prevalent in HIV-seropositive (HIV+) men who have sex with men (MSM). Around 30% of lesions regress within 1 year, but current histopathological assessment is unable to distinguish between HGAIN likely to regress and HGAIN likely to persist or progress to cancer. We aim to assess if host cell DNA methylation markers can predict regression of HGAIN, thus determining the need for immediate treatment or active surveillance. This could reduce overtreatment and the associated anal and psycho-sexual morbidity. METHODS AND ANALYSIS: This is an active surveillance cohort study in three centres located in Amsterdam, the Netherlands, in 200 HIV+ MSM diagnosed with HGAIN. Participants will not be treated, but closely monitored during 24 months of follow-up with 6 monthly visits including cytology, and high-resolution anoscopy with biopsies. The primary study endpoint is histopathological regression of each baseline HGAIN lesion at the end of the study. Regression is defined as ≤low grade anal intraepithelial neoplasia in the exit biopsy at 24 months. Regression proportions in lesions with low versus high methylation levels (ASCL1, ZNF582), other biomarkers (HPV genotype, HPV-E4, p16INK4A, Ki-67) and immunological markers at baseline will be compared. Main secondary endpoints are the histological and clinical outcome (ie, the number of octants affected by HGAIN) of each baseline HGAIN lesion and overall HGAIN disease (i.e., all lesions combined) after each visit. The health-related quality of life of the study group will be compared with that of a control group of 50 HIV+ MSM receiving regular HGAIN treatment. ETHICS AND DISSEMINATION: Ethics approval was obtained from the Institutional Review Board of the Academic Medical Center (Amsterdam, The Netherlands; reference no. 2021_099). Participants are required to provide written informed consent. Findings will be disseminated through publication in peer-reviewed scientific journals and presentations at international scientific conferences; dissemination to policy makers and the target patient group will be achieved through our (inter-)national network, professional associations and collaboration with a patient representative organisation. TRIAL REGISTRATION NUMBER: NL9664.