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.

BACKGROUND:

Endometrial cancer (EC) incidence is raising and is the fourth most common cancer type in women worldwide with about 417,000 new cases and 90,000 deaths annually. Moreover, EC is the most common gynaecological cancer in developed countries including Denmark with about 800 new cases and 90 deaths yearly. Early diagnosis is essential since the prognosis for women with an advanced EC stage is poor. No screening exists for EC and the most commonly observed clinical symptom is post-menopausal bleeding (PMB), which is a common condition affecting about 11% of postmenopausal women. Because 90% of EC cases are preceded by PMB, referral for specialized gynaecological care through cancer patient pathways is indicated for all Danish women with PMB, causing anxiety among women and high health-care costs. However, only 5-10% of women presenting with PMB have EC.

Worldwide, transvaginal ultrasound (TVUS) evaluation of the endometrium (cut-off: thickness ≥4mm and/or irregular lining of the uterine cavity) is used to identify women with PMB who have the highest cancer risk and therefore require an endometrial biopsy with or without hysteroscopy. While TVUS evaluation is highly sensitive (94.8%) it suffers from low specificity (51%) causing high numbers of unnecessary invasive procedures in healthy women. Moreover, the diagnostic program depends on skilled specialists for ultrasound evaluation, endometrial sampling as well as histopathological examination. This reinforces the clinical urgent and unmet need for a simpler, non-invasive, user-provided and more specific test to aid EC diagnosis.

This study aims to determine whether DNA methylation testing in patient-collected urine and vaginal samples is a clinically safe non-invasive alternative to determine EC risk. Testing for elevated DNA hypermethylation levels of cancer-specific genes has been demonstrated to provide a promising biomarker for detection of early-stage cancers, including cervical and endometrial cancer. DNA hypermethylation has been linked to silencing of tumour suppressor genes, thereby contributing to cancer development. Biologically, the ability to use vaginal and urine material for EC detection by DNA methylation testing is explained by local shedding of EC-derived DNA and cell fragments into the vaginal debris and urine as well as transrenal excretion of tumour-shedded circulating DNA into the urine.

Mainly small proof-of-concepts and case-controls studies have shown feasibility to detect EC using DNA methylation testing in plasma, self-collected vaginal samples, and clinician-collected cervical scrapes. However, the first paper reporting on the diagnostic value of DNA methylation analysis in vaginal samples and urine as a liquid biopsy for EC was just published by co-supervisor Prof. Steenbergen last year. Here, case-control data revealed excellent diagnostic performance of EC-specific DNA methylation markers in patient-collected urine and vaginal samples with sensitivities of 89-90% and specificities of 90-92% for EC detection using markers panels including: CDH13, GHSR, SST, CDO1, and ZIC1.

MATERIALS AND METHODS The vaginal and urine samples will be stored in a biobank at the Dept. of Pathology, Randers Regional Hospital (RHR) until shipment for DNA methylation testing analysis using quantitative methylation-specific PCR (qMSP) in Prof. Steenbergens' laboratory at Amsterdam University Medical Centre, The Netherlands. All samples will be tested for methylation markers, three for vaginal samples (CDO1, GHSR, ZIC1) and three for urine (GHSR, CDH13, SST). Results on endometrial biopsies and hysterectomies including molecular biomarkers and histological type will be retrieved from the nationwide Danish Pathology Databank and the electronic patient journal linked to the patient through the Civil Registration System in Denmark, where all residents have a unique 10-cipher code (CPR-number), to which all health data is linked.

AIM:

1) To determine the diagnostic accuracy of DNA methylation testing in patient-collected full-void urine and vaginal samples to detect EC among cancer cases and healthy controls

RESEARCH PLAN AND FEASIBILITY The study will be conducted in collaboration between University Research Clinic for Cancer Screening, Dept. of Public Health Programmes, Dept. of Pathology, RHR and with gynaecological departments in Central Denmark (Randers and Aarhus) and Southern Denmark Region (Odense) working as inclusion sites. State-of-the-art laboratory facilities are in place at Prof. Steenbergen's laboratory at Amsterdam UMC, supporting the feasibility of the studies.

PERSPECTIVES AND COMMUNICATION If successful, the research will have international impact and result in a novel diagnostic tool that will differentiate between women with PMB who do or do not need specialist referral through cancer patients' pathways to have invasive endometrial biopsies. This will facilitate timely diagnosis, lower the anxiety among patients, and reducing the pressure on the health-care system. Results will be published in international peer-reviewed journals and presented at conferences and will be concluded with a PhD dissertation. The results will be distributed to relevant clinical fora and implemented in national as well as international guidelines.

Patients with endometrial cancer (cases): Inclusion Criteria:

• Women ≥ 60 years
• Diagnosed with cancer corpus uteri
• Living in Central Denmark Region or Southern Denmark Region, Denmark
• Able to provide written consent for participation
• Able to read and understand Danish

Patients with endometrial cancer (cases): Exclusion Criteria:

• Other concurrent cancer diagnosis besides cancer corpus uteri
• Withdrawal of consent

Healthy controls: Inclusion Criteria:

• Postmenopausal women ≥ 60 years
• Living in Central Denmark Region, Denmark
• Able to provide written consent for participation
• Able to read and understand Danish

Healthy controls: Exclusion Criteria:

• Diagnosed with cancer or undergoing diagnostic assessment herfore without the last 5 years.
• Vaginal bleeding within the last 3 months.
• Known gynaecological diseases such as cervical dysplasia or atypical endomatrial hyperplasia within the last 5 years.
• Previously hysterectomized
• Withdrawal of consent.

Cases will be recruited from Department of Obstetrics and Gynaecology, Aarhus University Hospital, Central Denmark Region, and Odense University Hospital, Southern Denmark Region, Denmark.

Controls will recruited from Department of Orthopaedics, Randers Regional Hospital, Central Denmark Region, Denmark.