Maaike CG Bleeker

DNA Methylation Markers for Endometrial Cancer Detection in Minimally Invasive Samples: A Systematic Review

Clinical indications for host-cell DNA methylation markers in cervical screening and management of cervical intraepithelial neoplasia: A review

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

Recurrent cervical cancer detection using DNA methylation markers in self‐collected samples from home

AbstractEarly detection of recurrent cervical cancer is important to improve survival rates. The aim of this study was to explore the clinical performance of DNA methylation markers and high‐risk human papillomavirus (HPV) in cervicovaginal self‐samples and urine for the detection of recurrent cervical cancer. Cervical cancer patients without recurrence (n = 47) collected cervicovaginal self‐samples and urine pre‐ and posttreatment. Additionally, 20 patients with recurrent cervical cancer collected cervicovaginal self‐samples and urine at time of recurrence. All samples were self‐collected at home and tested for DNA methylation and high‐risk HPV DNA by PCR. In patients without recurrent cervical cancer, DNA methylation levels decreased 2‐years posttreatment compared to pretreatment in cervicovaginal self‐samples (p < .0001) and urine (p < .0001). DNA methylation positivity in cervicovaginal self‐samples was more frequently observed in patients with recurrence (77.8%) than in patients without recurrence 2‐years posttreatment (25.5%; p = .0004). Also in urine, DNA methylation positivity was more frequently observed in patients with recurrence (65%) compared to those without recurrence (35.6%; p = .038). Similarly, high‐risk HPV positivity in both cervicovaginal self‐samples and urine was more frequent (52.6% and 55%, respectively) in patients with recurrence compared to patients without recurrence (14.9% and 8.5%, respectively) (p = .004 and p = .0001). In conclusion, this study shows the potential of posttreatment monitoring of cervical cancer patients for recurrence by DNA methylation and high‐risk HPV testing in cervicovaginal and urine samples collected at home. The highest recurrence detection rate was achieved by DNA methylation testing in cervicovaginal self‐samples, detecting 77.8% of all recurrences and, specifically, 100% of the local recurrences.

Evaluation of Six Methylation Markers Derived from Genome-Wide Screens for Detection of Cervical Precancer and Cancer

Diagnostic Performance of the ASCL1/ZNF582 Methylation Test for Detection of High-Grade Vulvar Intraepithelial Neoplasia and Vulvar Cancer

High-grade vulvar intraepithelial neoplasia (VIN), the precursor lesion to vulvar cancer, comprises human papillomavirus (HPV)-associated high-grade squamous intraepithelial lesion (HSIL) and HPV-independent VIN (HPVi-VIN), differing in pathogenesis and cancer risk. HSIL typically develops from low-grade squamous intraepithelial lesion (LSIL), and HPVi-VIN from lichen sclerosus (LS). The PreCursor-M AnoGYN Methylation test, targeting ASCL1/ZNF582, may improve diagnostic accuracy and risk stratification in high-grade VIN patients. This study assessed its diagnostic performance to detect high-grade VIN and cancer. ASCL1/ZNF582 methylation was analyzed in 170 vulvar formalin-fixed paraffin-embedded (FFPE) tissue samples from healthy controls, LS, LSIL, HSIL, HPVi-VIN and vulvar cancer patients by quantitative methylation-specific polymerase chain reaction (qMSP). Logistic regression analysis was used to evaluate its diagnostic performance and compare it to the previously established ZNF582/SST/miR124-2 marker panel. Methylation levels increased with disease severity, from low in controls, LS and LSIL to high in HSIL, HPVi-VIN and vulvar cancer. The ASCL1/ZNF582 marker panel detected 92% and 84% of HSIL at 70% and 80% specificity, respectively, and 96% of HPVi-VIN and 100% of vulvar cancer at both specificities. Both marker panels (ASCL1/ZNF582 and ZNF582/SST/miR124-2) showed comparable excellent diagnostic performance for high-grade VIN detection, with an area under the curve (AUC) of 0.93 (95% confidence interval [CI] 0.88-0.98) and AUC 0.91 (95% CI 0.86-0.97), respectively. In conclusion, the ASCL1/ZNF582 methylation assay accurately detects high-grade VIN and vulvar cancer, while minimizing the detection of benign and low-grade lesions, indicating its clinical value.

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% vs 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 vs 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% vs 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.

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

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 (LS 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 LS Forty-four GOs completed the survey, resulting in a response rate of 75%. TCS were prescribed more often to patients with LS The majority of GOs who participated in our study endorse the utilisation of long-term ultra-potent TCS therapy in both patients with LS

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

AimsAdequate 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 resultsBaseline 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.ConclusionImmunohistochemistry 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.

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

Abstract 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.

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

AbstractMethylation 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.

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

AbstractHigh‐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.

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

AbstractEndometrial 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.

The risk of lymph node metastasis in the new FIGO 2018 stage IA cervical cancer with >7 mm diameter

In the 2018 FIGO staging system, cervical cancers with ≤5 mm depth of invasion (DOI) and a diameter of >7 mm, first classified as stage IB, are classified as stage IA. In this group, it is unclear what the risk of lymph node metastasis (LNM) is. This retrospective cohort study aims to determine the incidence of LNM and to study the association between disease-related characteristics and LNM. Women diagnosed with FIGO 2009 IB cervical cancer, with ≤5 mm DOI and a diameter >7 mm, treated with a radical hysterectomy and pelvic lymphadenectomy between 1985 and 2020 were selected from the databases of the Amsterdam University Medical Center and the University Medical Center Groningen. The specimens of patients with LNM were revised by expert pathologists. The incidence of LNM was calculated. The associations between LNM and DOI, diameter, histological type, clinical visibility and lymphovascular space invasion (LVSI) were evaluated by calculating odds ratios using logistic regression. Of the 389 patients included, 10 had pathologically confirmed LNM (2.6%, 95% confidence interval=1.3%-4.5%). In case of LVSI, univariate analysis showed an increased risk of LNM (p=0.003 and p=0.012, respectively). No difference in LNM was found between lesions diagnosed by microscopy and clinically visible lesions. No LNM were found in patients without LVSI and a DOI of ≤3 mm. For patients with stage IA cervical cancer with a diameter >7 mm, we recommend considering a pelvic lymph node assessment in case of DOI >3 mm and/or presence of LVSI.

Use of p53 immunohistochemistry can improve diagnostic agreement for differentiated vulvar intraepithelial neoplasia ( dVIN ): an international reproducibility study

Aims Differentiated or HPV‐independent vulvar intraepithelial neoplasia (dVIN) can progress rapidly to invasive cancer and accurate pathological diagnosis is essential to facilitate appropriate interventions. Histological similarities of dVIN with non‐neoplastic lesions, however, often make the diagnosis less reproducible. We investigated among a diverse group of pathologists whether the diagnostic agreement improves with the use of p53 immunohistochemistry (IHC) interpreted using the pattern‐based schema. Methods and results Fifty haematoxylin–eosin (HE) stained archival slides (30 dVIN and 20 non‐dysplastic vulvar lesions) were selected and p53‐IHC was performed. Twenty‐four board‐certified pathologists from eight countries first assessed the HE slides alone, and after a washout period, re‐evaluated them alongside the p53‐IHC slides. During both rounds, slides were diagnosed as dVIN, favour dVIN, favour no‐VIN or no‐VIN. p53‐IHC was scored as wild‐type or mutant (diffuse, basal, cytoplasmic or null). Kappa ( κ ) statistics and McNemar's test were used for statistical analyses. Overall diagnostic agreement for dVIN saw a significant increase in the Kappa value ( κ  = 0.6 vs. κ  = 0.4, P  = 0.002) when HE and p53‐IHC slides were assessed together compared with histology assessment alone, although the level of agreement remained moderate. For p53‐IHC assessment, overall agreement was substantial ( κ  = 0.7). Diagnoses changing from no‐VIN/favour no‐VIN to dVIN correlated significantly with the identification of a p53‐mutant pattern ( P  < 0.001). Conclusions Our findings indicate that p53‐IHC is a robust ancillary tool that can be reproducibly interpreted by pathologists with varying experience levels and supports the routine use of p53‐IHC in cases where dVIN is considered in the differential diagnosis.

Evaluation of DNA Methylation Markers for Endometrial Cancer Risk-stratification Using Patient-collected Urine and Vaginal Samples and Clinician-collected Cervical Samples From Women With Postmenopausal Bleeding

The goal of this observational study is to investigate the clinical utility of DNA-methylation testing in urine and vaginal samples collected by patients and cervical samples collected by clinicians, to determine the risk of endometrial cancer in symptomatic women with postmenopausal bleeding. The study aims to answer the following research questions: * What is the diagnostic accuracy of DNA methylation testing in urine, vaginal and cervical samples compared to traditional TVUS for endometrial cancer detection? * What is the 2-year risk of EC among women testing negative on TVUS and/or DNA methylation tests or those testing positive on methylation only? Researchers will compare DNA methylation testing in patient-collected urine and vaginal samples as well as in clinician-collected cervical samples, with the traditional diagnostic pathway for women with PMB, which includes TVUS evaluation, and when indicated by abnormal TVUS findings, endometrial biopsy according to clinical guidelines. Participants will * take a urine and vaginal sample * have a cervical sample collected by a clinician * undergo TVUS evaluation according to clinical guidelines * If TVUS shows thickened endometrium (≥ 5 mm) and/or irregularity, an endometrial biopsy will be collected according to clinical guidelines * fill out a questionnaire regarding acceptability and preferences of sampling methods and complete a lifestyle questionnaire.

The Clinical Utility of DNA Methylation Testing in Patient-collected Urine and Vaginal Samples to Detect Endometrial Cancer: a Case-control Study

The goal of this observational case-control study is to investigate the use of DNA-methylation testing in patient-collected urine and vaginal samples to detect endometrial cancer. The study aims to answer the following questions: * Can DNA methylation testing in vaginal and full-void urine samples distinguish endometrial cancer cases from healthy controls? Researchers will compare patient-collected urine and vaginal samples from patients with diagnosed endometrial cancer (cases) to gynaecologically and oncologically healthy controls (controls). Participants will * take a urine and vaginal sample at the hospital. * answer a questionnaire regarding acceptability and preferences of self-sampling methods. * answer a lifestyle questionnaire.