High-risk human papillomavirus distribution according to human immunodeficiency virus status among women with cervical cancer in Abidjan, Côte d'Ivoire, 2018 to 2020.

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

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

Vulvar lichen sclerosus (LS) is an inflammatory dermatosis which 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 to 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 three-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 (ORs) were determined to identify whether the biomarkers were associated with progression to cancer in patients with LS. 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 three-marker panel tested positive in 70% of LS which progressed to VSCC versus only 17% of LS in patients without cancer development (p=0.002). Also, mutant p53 IHC was observed more frequently in LS with progression to VSCC as compared to non-progressive LS cases (42% versus 3%, respectively, p=0.001). Multivariable analysis identified a mutant p53 status as the only independent risk factor for cancer development in LS (OR 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.

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.

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.

Does "One Size Fits All"? Rethinking FIGO Depth of Invasion Measurements in Vulvar Cancer.

Depth of invasion (DOI) is an important diagnostic parameter in patients with vulvar carcinoma, where a cutoff value of 1 mm largely determines the tumor stage and the need for groin surgery. DOI measurement should be reproducible and straightforward. In light of the new recommendation on how to measure DOI in the International Federation of Gynecology and Obstetrics (FIGO) staging system 2021, an exploratory study was conducted on the current practice of DOI measurement in vulvar cancer. In this study of 26 selected cases, 10 pathologists with high exposure to vulvar cancer cases in daily practice assessed both the conventional (FIGO 2009) and alternative (FIGO 2021) DOI methods for applicability and preference. In this set of cases, the DOI measurement according to FIGO 2009 was generally considered easier to apply than the measurement according to FIGO 2021, with applicability being rated as "easy to reasonable" in 76.9% versus 38.5% of cases, respectively ( P =0.005). The preferred method was FIGO 2009 or tumor thickness in 14 cases and FIGO 2021 in 6 cases. No invasion was preferred in 1 case. For the remaining 5 cases, half of the pathologists opted for the FIGO 2009 method and half for the FIGO 2021 method. Although the FIGO 2009 method proved to be more readily applicable in most of the cases studied, the method may differ for each case. There may not be a "one size fits all" solution for all cases of vulvar cancer.

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.

Prevalence of prescribing topical corticosteroids to patients with lichen sclerosus following surgery for vulvar cancer: a survey among gynaecologic oncologists in The Netherlands.

BACKGROUND: Vulvar lichen sclerosus (LS) is a chronic inflammatory dermatosis which can progress to precursor lesion differentiated vulvar intraepithelial neoplasia (dVIN) and vulvar squamous cell carcinoma (VSCC). The risk of developing recurrent vulvar cancer following LS-associated VSCC is high. Evidence suggests that treatment of LS with topical corticosteroids (TCS) can prevent progression to dVIN, VSCC and recurrences. However, current guidelines do not give any recommendation on the management of LS following surgery for VSCC. The aim of this study was to conduct a survey among all registered gynaecologic oncologists (GOs) in the Netherlands to evaluate the current management of LS patients without a history of VSCC (LSnoVSCC) and patients with LS following surgery for VSCC (LSVSCC). METHODS: An online survey was distributed to all registered GOs in the Netherlands. Primary outcome measures were the frequency, type and duration of TCS treatment prescribed for LSnoVSCC and LSVSCC patients, separately. As a secondary outcome measure, reasons for treating or not treating patients with LSnoVSCC and LSVSCC with TCS were analysed. RESULTS: Forty-four GOs completed the survey, resulting in a response rate of 75%. TCS were prescribed more often to patients with LSnoVSCC as compared to patients with LSVSCC (86% versus 52%, respectively, p < 0.001). If treatment was initiated, ultra-potent (class IV) TCS were most commonly prescribed for an indefinite period of time for both patient groups. The most reported reason for treating patients in both groups with TCS was symptoms, followed by clinical aspects of the lesion and prevention of progression to dVIN and VSCC. CONCLUSION: The majority of GOs who participated in our study endorse the utilisation of long-term ultra-potent TCS therapy in both patients with LSnoVSCC and LSVSCC. Nevertheless, Dutch GOs are currently prescribing TCS more frequently to patients with LSnoVSCC than to patients with LSVSCC.

Vulvar lichen sclerosus (LS) is a chronic skin condition which may progress to vulvar squamous cell carcinoma (VSCC) through differentiated vulvar intraepithelial neoplasia (dVIN). LS symptoms are treated with topical corticosteroids (TCS), which can also prevent progression to dVIN and VSCC. However, current international guidelines do not give any recommendation on the treatment of LS following surgery for VSCC. To evaluate the current management of LS patients without a history of VSCC (LS^noVSCC) and patients with LS following surgery for VSCC (LS^VSCC), a survey study was conducted among all gynaecologic oncologists (GOs) in The Netherlands. The findings of this study demonstrate that Dutch GOs prescribed TCS more often to patients with LS^noVSCC as compared to patients with LS^VSCC. However, when deciding to prescribe TCS, the majority of Dutch GOs prescribed ultra-potent TCS for an indefinite period of time for both LS^noVSCC and LS^VSCC patients.

Optimising follow-up strategy based on cytology and human papillomavirus after fertility-sparing surgery for early stage cervical cancer: a nationwide, population-based, retrospective cohort study.

BACKGROUND: The optimal follow-up strategy to detect recurrence after fertility-sparing surgery for early stage cervical cancer is unknown. Tailored surveillance based on individual risks could contribute to improved efficiency and, subsequently, reduce costs in health care. The aim of this study was to establish the predictive value of cervical cytology and high-risk human papillomavirus (HPV) testing to detect recurrent cervical intraepithelial neoplasia grade 2 or worse (CIN2+; including recurrent cervical cancer) after fertility-sparing surgery. METHODS: In this nationwide, population-based, retrospective cohort study, we used data from the Netherlands Cancer Registry and the Dutch Nationwide Pathology Databank. All patients aged 18-40 years with cervical cancer of any histology who received fertility-sparing surgery (ie, large loop excision of the transformation zone, conisation, or trachelectomy) between Jan 1, 2000, and Dec 31, 2020, were included. Pathology data from diagnosis, treatment, and during follow-up were analysed. The primary and secondary outcomes were the cumulative incidence of recurrent CIN2+ and recurrence-free survival, overall and stratified by results for cytology and high-risk HPV. FINDINGS: 1548 patients were identified, of whom 1462 met the inclusion criteria. Of these included patients, 19 568 pathology reports were available. The median age at diagnosis was 31 years (IQR 30-35). After a median follow-up of 6·1 years (IQR 3·3-10·8), recurrent CIN2+ was diagnosed in 128 patients (cumulative incidence 15·0%, 95% CI 11·5-18·2), including 52 patients (cumulative incidence 5·4%, 95% CI 3·7-7·0) with recurrent cervical cancer. The overall 10-year recurrence-free survival for CIN2+ was 89·3% (95% CI 87·4-91·3). By cytology at first follow-up visit within 12 months after fertility-sparing surgery, 10-year recurrence-free survival for CIN2+ was 92·1% (90·2-94·1) in patients with normal cytology, 84·6% (77·4-92·3) in those with low-grade cytology, and 43·1% (26·4-70·2) in those with high-grade cytology. By high-risk HPV status at first follow-up visit within 12 months after surgery, 10-year recurrence-free survival for CIN2+ was 91·1% (85·3-97·3) in patients who were negative for high-risk HPV and 73·6% (58·4-92·8) in those who were positive for high-risk HPV. Cumulative incidence of recurrent CIN2+ within 6 months after any follow-up visit (6-24 months) in patients negative for high-risk HPV with normal or low-grade cytology was 0·0-0·7% and with high-grade cytology was 0·0-33·3%. Cumulative incidence of recurrence in patients positive for high-risk HPV with normal or low-grade cytology were 0·0-15·4% and with high-grade cytology were 50·0-100·0%. None of the patients who were negative for high-risk HPV without high-grade cytology, at 6 months and 12 months, developed recurrence. INTERPRETATION: Patients who are negative for high-risk HPV with normal or low-grade cytology at 6-24 months after fertility-sparing surgery, could be offered a prolonged follow-up interval of 6 months. This group comprises 80% of all patients receiving fertility-sparing surgery. An interval of 12 months seems to be safe after two consecutive negative tests for high-risk HPV with an absence of high-grade cytology, which accounts for nearly 75% of all patients who receive fertility-sparing surgery. FUNDING: KWF Dutch Cancer Society.

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.

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.

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.

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.

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.

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.

The European Society of Gynaecological Oncology (ESGO), the International Society for the Study of Vulvovaginal Disease (ISSVD), the European College for the Study of Vulval Disease (ECSVD) and the European Federation for Colposcopy (EFC) consensus statements on pre-invasive vulvar lesions.

The European Society of Gynaecological Oncology (ESGO), the International Society for the Study of Vulvovaginal Disease (ISSVD), the European College for the Study of Vulval Disease (ECSVD), and the European Federation for Colposcopy (EFC) developed consensus statements on pre-invasive vulvar lesions in order to improve the quality of care for patients with vulvar squamous intraepithelial neoplasia, vulvar Paget disease in situ, and melanoma in situ. For differentiated vulvar intraepithelial neoplasia (dVIN), an excisional procedure must always be adopted. For vulvar high-grade squamous intraepithelial lesion (VHSIL), both excisional procedures and ablative ones can be used. The latter can be considered for anatomy and function preservation and must be preceded by several representative biopsies to exclude malignancy. Medical treatment (imiquimod or cidofovir) can be considered for VHSIL. Recent studies favor an approach of using imiquimod in vulvar Paget's disease. Surgery must take into consideration that the extension of the disease is usually wider than what is evident in the skin. A 2 cm margin is usually considered necessary. A wide local excision with 1 cm free surgical margins is recommended for melanoma in situ. Following treatment of pre-invasive vulvar lesions, women should be seen on a regular basis for careful clinical assessment, including biopsy of any suspicious area. Follow-up should be modulated according to the risk of recurrence (type of lesion, patient age and immunological conditions, other associated lower genital tract lesions).

The European Society of Gynaecological Oncology (ESGO), the International Society for the Study of Vulvovaginal Disease (ISSVD), the European College for the Study of Vulval Disease (ECSVD) and the European Federation for Colposcopy (EFC) Consensus Statements on Pre-invasive Vulvar Lesions.

ABSTRACT: The European Society of Gynaecological Oncology (ESGO), the International Society for the Study of Vulvovaginal Disease (ISSVD), the European College for the Study of Vulval Disease (ECSVD), and the European Federation for Colposcopy (EFC) developed consensus statements on pre-invasive vulvar lesions in order to improve the quality of care for patients with vulvar squamous intraepithelial neoplasia, vulvar Paget disease in situ, and melanoma in situ. For differentiated vulvar intraepithelial neoplasia (dVIN), an excisional procedure must always be adopted. For vulvar high-grade squamous intraepithelial lesion (VHSIL), both excisional procedures and ablative ones can be used. The latter can be considered for anatomy and function preservation and must be preceded by several representative biopsies to exclude malignancy. Medical treatment (imiquimod or cidofovir) can be considered for VHSIL. Recent studies favor an approach of using imiquimod in vulvar Paget's disease. Surgery must take into consideration that the extension of the disease is usually wider than what is evident in the skin. A 2 cm margin is usually considered necessary. A wide local excision with 1 cm free surgical margins is recommended for melanoma in situ. Following treatment of pre-invasive vulvar lesions, women should be seen on a regular basis for careful clinical assessment, including biopsy of any suspicious area. Follow-up should be modulated according to the risk of recurrence (type of lesion, patient age and immunological conditions, other associated lower genital tract lesions).

Increased Angiogenesis and Lymphangiogenesis in Adenomyosis Visualized by Multiplex Immunohistochemistry.

There is evidence for increased angiogenesis in the (ectopic) endometrium of adenomyosis patients under the influence of vascular endothelial growth factor (VEGF). VEGF stimulates both angiogenesis and lymph-angiogenesis. However, information on lymph vessels in the (ectopic) endometrium of adenomyosis patients is lacking. In this retrospective matched case-control study, multiplex immunohistochemistry was performed on thirty-eight paraffin embedded specimens from premenopausal women who had undergone a hysterectomy at the Amsterdam UMC between 2001 and 2018 to investigate the evidence for (lymph) angiogenesis in the (ectopic) endometrium or myometrium of patients with adenomyosis versus controls with unrelated pathologies. Baseline characteristics of both groups were comparable. In the proliferative phase, the blood and lymph vessel densities were, respectively, higher in the ectopic and eutopic endometrium of patients with adenomyosis than in the endometrium of controls. The relative number of blood vessels without α-smooth muscle actinin (α SMA) was higher in the eutopic and ectopic endometrium of adenomyosis patients versus controls. The level of VEGF staining intensity was highest in the myometrium but did not differ between patients with adenomyosis or controls. The results indicate increased angiogenesis and lymphangiogenesis in the (ectopic) endometrium affected by adenomyosis. The clinical relevance of our findings should be confirmed in prospective clinical studies.

Vulvar intraepithelial neoplasia: Incidence and long-term risk of vulvar squamous cell carcinoma.

The risk of vulvar squamous cell carcinoma (VSCC) in patients with high-grade vulvar intraepithelial neoplasia (VIN) is considered lower in high-grade squamous intraepithelial lesion (HSIL) compared to differentiated VIN (dVIN), but studies are limited. Our study investigated both the incidence of high-grade VIN and the cumulative incidence of VSCC in patients with HSIL and dVIN separately. A database of women diagnosed with high-grade VIN between 1991 and 2011 was constructed with data from the Dutch Pathology Registry (PALGA). The European standardized incidence rate (ESR) and VSCC risk were calculated, stratified for HSIL and dVIN. The effects of type of VIN (HSIL vs dVIN), age and lichen sclerosis (LS) were estimated by Cox regression. In total, 1148 patients were diagnosed with high-grade VIN between 1991 and 2011. Between 1991-1995 and 2006-2011, the ESR of HSIL increased from 2.39 (per 100 000 woman-years) to 3.26 and the ESR of dVIN increased from 0.02 to 0.08. The 10-year cumulative VSCC risk was 10.3%; 9.7% for HSIL and 50.0% for dVIN (log rank P < .001). Type of VIN, age and presence of LS were independent risk factors for progression to VSCC, with hazard ratios of 3.0 (95% confidence interval [CI] 1.3-7.1), 2.3 (95% CI 1.5-3.4) and 3.1 (95% CI 1.8-5.3), respectively. The incidence of high-grade VIN is rising. Because of the high cancer risk in patients with dVIN, better identification and timely recognition are urgently needed.

DNA methylation markers for cancer risk prediction of vulvar intraepithelial neoplasia.

Current clinical and histological classifications are unable to determine the risk of vulvar squamous cell carcinoma (VSCC) in high-grade vulvar intraepithelial neoplasia (VIN), making prognostic biomarkers highly needed. We studied host-cell DNA methylation markers in high-grade squamous intraepithelial lesion (HSIL) and differentiated VIN (dVIN) without VSCC, in HSIL and dVIN adjacent to VSCC and in human papillomavirus (HPV) positive and negative VSCC, relative to control vulvar tissues. A series of 192 formalin-fixed paraffin-embedded vulvar samples, including VSCC (n = 58), VIN adjacent to VSCC (n = 30), VIN without VSCC during follow-up (n = 41) and normal vulvar tissues (n = 63), were tested for 12 DNA methylation markers with quantitative multiplex methylation-specific PCR (qMSP). HPV status was determined by p16INK4A immunohistochemistry and high-risk HPV PCR analysis. Logistic regression analyses were used to determine methylation patterns and methylation marker performance for VIN and VSCC detection. Methylation markers showed significantly higher methylation levels with increasing severity of disease. VIN adjacent to VSCC showed a similar methylation-high pattern as VSCC, while VIN without VSCC displayed a heterogeneous methylation pattern. Vulvar carcinogenesis is associated with increased DNA methylation. Higher DNA methylation levels in VIN seem to reflect higher cancer risk, emphasizing the high potential of DNA methylation biomarkers in the diagnostic workup of VIN. As a next step, longitudinal studies are needed to verify the prognostic value of methylation biomarkers as a clinical tool for stratification of cancer risk in women with VIN.

Classification of high-grade cervical intraepithelial neoplasia by p16ink4a , Ki-67, HPV E4 and FAM19A4/miR124-2 methylation status demonstrates considerable heterogeneity with potential consequences for management.

High-grade cervical intraepithelial neoplasia (CIN2 and CIN3) represents a heterogeneous disease with varying cancer progression risks. Biomarkers indicative for a productive human papillomavirus (HPV) infection (HPV E4) and a transforming HPV infection (p16ink4a , Ki-67 and host-cell DNA methylation) could provide guidance for clinical management in women with high-grade CIN. This study evaluates the cumulative score of immunohistochemical expression of p16ink4a (Scores 0-3) and Ki-67 (Scores 0-3), referred to as the "immunoscore" (IS), in 262 CIN2 and 235 CIN3 lesions derived from five European cohorts in relation to immunohistochemical HPV E4 expression and FAM19A4/miR124-2 methylation in the corresponding cervical scrape. The immunoscore classification resulted in 30 lesions within IS group 0-2 (6.0%), 151 lesions within IS group 3-4 (30.4%) and 316 lesions within IS group 5-6 (63.6%). E4 expression decreased significantly from CIN2 to CIN3 (P < .001) and with increasing immunoscore group (Ptrend < .001). Methylation positivity increased significantly from CIN2 to CIN3 (P < .001) and with increasing immunoscore group (Ptrend < .001). E4 expression was present in 9.8% of CIN3 (23/235) and in 12.0% of IS group 5-6 (38/316). Notably, in a minority (43/497, 8.7%) of high-grade lesions, characteristics of both transforming HPV infection (DNA hypermethylation) and productive HPV infection (E4 expression) were found simultaneously. Next, we stratified all high-grade CIN lesions, based on the presumed cancer progression risk of the biomarkers used, into biomarker profiles. These biomarker profiles, including immunoscore and methylation status, could help the clinician in the decision for immediate treatment or a "wait and see" policy to reduce overtreatment of high-grade CIN lesions.