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

Detection of endometrial cancer using tampon-based collection and methylated DNA markers

Alterations in DNA methylation are early events in endometrial cancer (EC) development and may have utility in EC detection via tampon-collected vaginal fluid. For discovery, DNA from frozen EC, benign endometrium (BE), and benign cervicovaginal (BCV) tissues underwent reduced representation bisulfite sequencing (RRBS) to identify differentially methylated regions (DMRs). Candidate DMRs were selected based on receiver operating characteristic (ROC) discrimination, methylation level fold-change between cancers and controls, and absence of background CpG methylation. Methylated DNA marker (MDM) validation was performed using qMSP on DNA from independent EC and BE FFPE tissue sets. Women ≥45 years of age with abnormal uterine bleeding (AUB) or postmenopausal bleeding (PMB) or any age with biopsy-proven EC self-collected vaginal fluid using a tampon prior to clinically indicated endometrial sampling or hysterectomy. Vaginal fluid DNA was assayed by qMSP for EC-associated MDMs. Random forest modeling analysis was performed to generate predictive probability of underlying disease; results were 500-fold in-silico cross-validated. Thirty-three candidate MDMs met performance criteria in tissue. For the tampon pilot, 100 EC cases were frequency matched by menopausal status and tampon collection date to 92 BE controls. A 28-MDM panel highly discriminated between EC and BE (96% (95%CI 89-99%) specificity; 76% (66-84%) sensitivity (AUC 0.88). In PBS/EDTA tampon buffer, the panel yielded 96% (95% CI 87-99%) specificity and 82% (70-91%) sensitivity (AUC 0.91). Next generation methylome sequencing, stringent filtering criteria, and independent validation yielded excellent candidate MDMs for EC. EC-associated MDMs performed with promisingly high sensitivity and specificity in tampon-collected vaginal fluid; PBS-based tampon buffer with added EDTA improved sensitivity. Larger tampon-based EC MDM testing studies are warranted.

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.

A Simple Cervicovaginal Epigenetic Test for Screening and Rapid Triage of Women With Suspected Endometrial Cancer: Validation in Several Cohort and Case/Control Sets

PURPOSE Endometrial cancer (EC) incidence has been rising over the past 10 years. Delays in diagnosis reduce survival and necessitate more aggressive treatment. We aimed to develop and validate a simple, noninvasive, and reliable triage test for EC to reduce the number of invasive diagnostic procedures and improve patient survival. METHODS We developed a test to screen and triage women with suspected EC using 726 cervical smear samples from women with and without EC, and validated the test in 562 cervicovaginal samples using three different collection methods (cervical smear: n = 248; vaginal swab: n = 63; and self-collection: n = 251) and four different settings (case/control: n = 388; cohort of women presenting with postmenopausal bleeding: n = 63; a cohort of high-risk women with Lynch syndrome: n = 25; and a nested case/control setting from a screening cohort and samples taken up to 3 years before EC diagnosis: n = 86). RESULTS We describe the Women's cancer risk IDentification – quantitative polymerase chain reaction test for Endometrial Cancer (WID-qEC), a three-marker test that evaluates DNA methylation in gene regions of GYPC and ZSCAN12. In cervical, self-collected, and vaginal swab samples derived from symptomatic patients, it detected EC with sensitivities of 97.2% (95% CI, 90.2 to 99.7), 90.1% (83.6 to 94.6), and 100% (63.1 to 100), respectively, and specificities of 75.8% (63.6 to 85.5), 86.7% (79.3 to 92.2), and 89.1% (77.8 to 95.9), respectively. The WID-qEC identified 90.9% (95% CI, 70.8 to 98.9) of EC cases in samples predating diagnosis up to 1 year. Test performance was similar across menopausal status, age, stage, grade, ethnicity, and histology. CONCLUSION The WID-qEC is a noninvasive reliable test for triage of women with symptoms suggestive of ECs. Because of the potential for self-collection, it could improve early diagnosis and reduce the reliance for in-person visits.

Triage of human papillomavirus infected women by methylation analysis in first-void urine

AbstractHost cell DNA methylation analysis in urine provides promising triage markers for women diagnosed with a high-risk (HR) human papillomavirus (HPV) infection. In this study, we have investigated a panel of six host cell methylation markers (GHSR, SST, ZIC1, ASCL1, LHX8, ST6GALNAC5) in cervicovaginal secretions collected within the first part of the urine void (FVU) from a referral population. Cytology, histology, and HPV DNA genotyping results on paired FVU and cervical samples were available. Urinary median methylation levels from HR-HPV (n = 93) positive women were found to increase for all markers with severity of underlying disease. Significantly elevated levels were observed for GHSR and LHX8 in relation to high-grade cervical intraepithelial neoplasia (CIN2 +; n = 33), with area under de curve values of 0.80 (95% Confidence Interval (CI) 0.59–0.92) and 0.76 (95% CI 0.58–0.89), respectively. These findings are the first to support the assertion that methylation analysis of host cell genes is feasible in FVU and holds promise as molecular, triage strategy to discern low- from high-grade cervical disease in HR-HPV positive women. Molecular testing on FVU may serve to increase cervical cancer screening attendance in hard-to-reach populations whilst reducing loss to follow-up and await further optimization and validation studies.

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

Ultrasound detection of endometrial cancer in women with postmenopausal bleeding: Systematic review and meta-analysis

To assess the performance of endometrial thickness (ET) cut-offs for detecting endometrial cancer (EC) in women with postmenopausal bleeding (PMB) and evaluate the clinical utility of additional ultrasound measures such as endometrial volume (EV), vascular flow index (VFI), vascularization index (VI), and uterine artery flow index (FI). Clinicaltrials.gov and MEDLINE database via PubMed were queried for studies published between 1/1990 and 3/2016 using specific MeSH terms. Original, peer-reviewed cohort studies reporting EC outcomes and specific ultrasound findings by PMB status were included. Study design, country, clinical setting inclusion/exclusion criteria, aggregate study-level demographic and clinical data were extracted from 44 studies including 17,339 women with PMB and 1341 cases of EC (7.7%). In women with PMB and EC (n = 417), pooled mean ET was 16.4 mm (95% CI, 14.8-18.1 mm). In women with PMB without EC, pooled mean ET was 4.1 mm. 31 studies reported outcomes using different ET cut-off values ranging from 3 to 20 mm. Compared to ≥3 or 4 mm, a cutoff of ≥5 mm had similar sensitivity (96.2, 95%CI 92.3, 98.1) with improved specificity for EC (51.5, 95%CI 42.3-60.7), allowing to reduce the rate of invasive workup for PMB by 17%. EV, VI, VFI, and FI were significantly correlated with EC, but performance of specific cut-offs was not analyzed due to limited data. Among women with PMB mean ET is substantially higher in women with EC compared to those without EC. An ET cutoff of ≥5 mm shows an acceptable tradeoff between sensitivity and specificity for diagnosis of EC.

Endometrial Cancer

Endometrial cancer

Endometrial cancer is the most common gynaecological cancer in high income countries and its incidence is rising globally. Although an ageing population and fewer benign hysterectomies have contributed to this trend, the growing prevalence of obesity is the major underlying cause. Obesity poses challenges for diagnosis and treatment and more research is needed to offer primary prevention to high-risk women and to optimise endometrial cancer survivorship. Early presentation with postmenopausal bleeding ensures most endometrial cancers are cured by hysterectomy but those with advanced disease have a poor prognosis. Minimally invasive surgical staging and sentinel-lymph-node biopsy provides a low morbidity alternative to historical surgical management without compromising oncological outcomes. Adjuvant radiotherapy reduces loco-regional recurrence in intermediate-risk and high-risk cases. Advances in our understanding of the molecular biology of endometrial cancer have paved the way for targeted chemotherapeutic strategies, and clinical trials will establish their benefit in adjuvant, advanced, and recurrent disease settings in the coming years.

Alicia Hulbert

Beatriz Pelegrina

Birgit I. Lissenberg‐Witte

Birgit MM Wever

Chiara Herzog

David Cibula

Emma J Crosbie

Fátima Marin

Jenneke C. Kasius

Jip A van Trommel

Joakim Dillner

Joan Brunet

Johannes CF Ket

Medical Information Specialist/Literature Researcher since 2003 at Vrije Universiteit Amsterdam University Library and Amsterdam University Medical Centers, location De Boelelaan (Amsterdam UMC, formerly known as VU Medical Center (VUmc)), Health Sciences, Dentistry (ACTA) and Psychology, incl. Neuropsychology. Experience in other sciences as well, like Theology, Social Sciences, Philosophy and Economy.

Jordi Ponce

Karin Sundström

Lena Schreiberhuber

Line Bjørge

Lisanne Verhoef

Lukas Dostalek

Álvaro Carmona

Álvaro Carmona Pestaña es Licenciado en Bioquímica (Universidad de Sevilla, 2008-2013), con un Máster en Neurociencias (Universidad Autónoma de Barcelona, ​​2013-2014), Máster en Investigación Médica Clínica y Experimental (Universidad de Sevilla, 2015-2016) y un Máster en Educación especializado en Biología y Geología (UCAM, 2020-2021). Obtuvo una beca internacional de doctorado en la Universidad de Roma, La Sapienza (2016-2020), donde su tesis abordó las modificaciones epigenéticas y los modificadores de histonas en la señalización Notch y la proliferación de células tumorales en leucemia linfoblástica aguda de células T (T-ALL). Posee experiencia en técnicas avanzadas de biología molecular y celular, como rtPCR, qPCR, inmunoprecipitación de cromatina y secuenciación de nueva generación. Ha trabajado en proyectos de investigación traslacional en oncología, incluyendo el desarrollo de nuevas moléculas con actividad antiproliferativa en colaboración con instituciones como el CSIC (Madrid), la Universidad de Milán y la Universidad de Siena. Posteriormente, fue investigador postdoctoral en el Institut Català d'Oncología (ICO-IDIBELL), donde lideró el ensayo clínico SCREENWIDE, enfocado en el diagnóstico molecular no invasivo de cáncer ginecológico. En este rol, gestionó la captación de pacientes, bases de datos y análisis bioinformático de biomarcadores genéticos. En el ámbito académico, ha desempeñado roles de coordinación e innovación docente. Actualmente es Coordinador Académico del Grado en Enfermería en la Universidad Loyola Andalucía, tras haber ocupado un puesto similar en la Universidad Alfonso X el Sabio, donde lideró estrategias de enseñanza con inteligencia artificial y simulación clínica. Su enfoque en innovación educativa le valió el Premio Carmen Domínguez Alcón por integrar el arte y la medicina en la enseñanza de la enfermería a través de obras del Museo del Prado. Ha dirigido numerosos Trabajos de Fin de Grado y Máster en oncología y salud digital, y ha participado en la evaluación de proyectos científicos. En investigación, su producción incluye publicaciones en revistas de alto impacto como Clinical Cancer Research, The Lancet - eBiomedicine y Journal of Clinical Oncology, con estudios sobre epigenética, cáncer y desinformación médica en redes sociales. También ha desarrollado estudios sobre el impacto de la inteligencia artificial en enfermería y alfabetización en salud. Como divulgador científico, ha participado en eventos como Naukas, Desgranando Ciencia y la Feria de la Ciencia de Jerez, además de colaborar en el programa No es un día cualquiera de RNE. Su trabajo en transferencia de conocimiento ha generado un impacto tangible en la educación, la biomedicina y la comunicación científica, consolidándolo como un referente en la intersección entre ciencia, docencia e innovación digital.

Maaike CG Bleeker

Marta Pineda

Martin Widschwendter

Prof Martin Widschwendter is Professor for Cancer Prevention and Screening and Director of the European Translational Oncology Prevention and Screening (EUTOPS) Institute, Leopold-Franzens-University of Innsbruck, Austria. Prof Widschwendter also holds the positions of Professor in Women’s Cancer at University College London (UCL), UK, (where he was the Head of Department of Women’s Cancer, UCL EGA Institute for Women's Health for almost 10 years until March 2020), Consultant Gynaecological Oncologist, LKH Hall in Tirol, Austria, Guest Professor at the Department of Women’s and Children’s Health, Karolinska Institutet, Sweden and FRCOG (Fellows ad eundem) of the Royal College of Obstetricians and Gynaecologists (RCOG), London, UK. In 2001, having completed his training in Gynaecology and Obstetrics in Austria, Prof Widschwendter worked at the Norris Comprehensive Cancer Centre in Los Angeles (USA) on translational epigenetics (Prof Peter A Jones group) and spent three years as the lead clinician and surgeon of a large breast cancer centre before embarking on a career at UCL/UCLH from 2005 where he undertook sub-speciality training in gynaecological oncology. As the Head of Department of Women’s Cancer within the UCL EGA Institute for Women’s Health, he established a research group focusing on the role of early detection, risk prediction, and prevention of breast and gynaecological cancers within several major research programmes (EpiFemCare, FORECEE, BRCA PROTECT and BRCA PREVENT). In 2016, Martin was awarded a Fellowship ad eundem of the Royal Academy of Obstetrics and Gynaecology (FRCOG) and since March 2020, he has been Professor for Cancer Prevention and Screening, as well as the Director of the European Translational Oncology Prevention & Screening (EUTOPS) Institute at the Leopold-Franzens-University of Innsbruck, Austria. In July 2020, Martin was conferred a Guest Professorship at the Karolinska Institutet, Stockholm, Sweden. Prof Widschwendter was the PI and Coordinator of several EU projects including the 7th Framework Programme (FP7), Horizon 2020 (H2020), and was also awarded a European Research Council (ERC) Advanced Grant. He is an author on more than 215 papers in high impact journals, has contributed to numerous textbooks, secured substantial grant income in the last 10 years, lectured widely on his research and clinical experience in the UK and abroad and is an Editor for the International Journal of Gynecological Cancer.

Michael Wong

Consultant Obstetrician & Gynaecologist

Michal Zikan

Nienke E van Trommel

Paula Peremiquel-Trillas

Renske DM Steenbergen

Rianne van den Helder

Sonia Paytubi

W. Marchien van Baal