Non-invasive detection of endometrial cancer by DNA methylation analysis in urine. van den Helder Rianne,Wever Birgit M M,van Trommel Nienke E,van Splunter Annina P,Mom Constantijne H,Kasius Jenneke C,Bleeker Maaike C G,Steenbergen Renske D M Clinical epigenetics BACKGROUND:The incidence of endometrial cancer is rising, and current diagnostics often require invasive biopsy procedures. Urine may offer an alternative sample type, which is easily accessible and allows repetitive self-sampling at home. Here, we set out to investigate the feasibility of endometrial cancer detection in urine using DNA methylation analysis. RESULTS:Urine samples of endometrial cancer patients (n = 42) and healthy controls (n = 46) were separated into three fractions (full void urine, urine sediment, and urine supernatant) and tested for three DNA methylation markers (GHSR, SST, ZIC1). Strong to very strong correlations (r = 0.77-0.92) were found amongst the different urine fractions. All DNA methylation markers showed increased methylation levels in patients as compared to controls, in all urine fractions. The highest diagnostic potential for endometrial cancer detection in urine was found in full void urine, with area under the receiver operating characteristic curve values ranging from 0.86 to 0.95. CONCLUSIONS:This feasibility study demonstrates, for the first time, that DNA methylation analysis in urine could provide a non-invasive alternative for the detection of endometrial cancer. Further investigation is warranted to validate its clinical usefulness. Potential applications of this diagnostic approach include the screening of asymptomatic women, triaging women with postmenopausal bleeding symptoms, and monitoring women with increased endometrial cancer risk. 10.1186/s13148-020-00958-7

The endometrial cancer detection using non-invasive hypermethylation of and genes in women with postmenopausal bleeding in Northwest China. CytoJournal Objective:The objective of this study was to verify the clinical predictive performance of methylated cysteine dioxygenase type 1 () and CUGBP Elav-like family member 4 () in endometrial cancer (EC) women with postmenopausal bleeding (PMB). Material and Methods:A single-center, prospective, and case-control study was conducted in the Gansu Provincial Maternity and Child-care Hospital with 138 female postmenopausal patients enrolled in 2022. All patients underwent body mass index (BMI) detection, transvaginal ultrasonography (TVUS) detection, carbohydrate antigen 125 detection, and the cervical exfoliated cell gene methylation detection to analyze the sensitivity, specificity, and accuracy of different screening tests statistically with the biopsy and/or dilation and curettage (D&C) pathological diagnosis under hysteroscopy as the gold standard. Results:There was no significant difference in age between the EC group and the non-EC group, = 0.492. Using quantitative polymerase chain reaction (qPCR) technology, we validated the and methylation detection with 87.5% sensitivity and 95.9% specificity as a useful strategy for the triage of women with PMB for the detection of EC. In addition, 100% of type II EC ( = 6) were positively detected by the or methylation test. Conclusion:The and methylation test with high specificity as an auxiliary diagnostic tool or alternative method provides physicians with a reference to distinguish between benign and malignant tumors in women with postmenopausal bleeding, to justify the necessity of using invasive methods to confirm diagnosis. 10.25259/Cytojournal_78_2023

Detection of endometrial cancer using tampon-based collection and methylated DNA markers. Gynecologic oncology OBJECTIVE:Alterations in DNA methylation are early events in endometrial cancer (EC) development and may have utility in EC detection via tampon-collected vaginal fluid. METHODS: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. RESULTS: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). CONCLUSION: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. 10.1016/j.ygyno.2023.04.014

Combining copy number, methylation markers, and mutations as a panel for endometrial cancer detection via intravaginal tampon collection. Sangtani Ajleeta,Wang Chen,Weaver Amy,Hoppman Nicole L,Kerr Sarah E,Abyzov Alexej,Shridhar Viji,Staub Julie,Kocher Jean-Pierre A,Voss Jesse S,Podratz Karl C,Wentzensen Nicolas,Kisiel John B,Sherman Mark E,Bakkum-Gamez Jamie N Gynecologic oncology OBJECTIVE:We aimed to assess whether endometrial cancer (EC) can be detected in shed DNA collected with vaginal tampon by analyzing copy number, methylation markers, and mutations. METHODS:Tampons were collected prior to hysterectomy from 38 EC patients and 28 women with benign indications. Extracted tampon DNA underwent the following: 1) low-coverage whole genome sequencing (LC-WGS) to assess copy number, 2) pyrosequencing to measure percent promotor methylation of HOXA9, RASSF1, and CDH13 and 3) next generation sequencing (NGS) to identify mutations in 19 genes associated with EC identified through The Cancer Genome Atlas. Sensitivity and specificity for each test and test combinations were calculated. RESULTS:Methylation analysis yielded the highest specificities but lowest sensitivities (37-40% sensitivity; 100% specificity for HOXA9, RASSF1 and HTR1B) while mutation analysis had improved sensitivity (50% sensitivity; 83% specificity). Only one "false positive" result for copy number variants was identified among women with benign surgical indications, which was based on detection of copy number changes, and associated with a leiomyosarcoma that was only recognized at hysterectomy. Considering any of the 3 biomarker classes as a positive, resulted in a sensitivity of 92% and specificity of 86%. Mutation analysis did not add sensitivity to the combination of analysis of copy number and methylation. CONCLUSIONS:This study demonstrates a proof-of-principle for non-invasive yet precise detection of endometrial cancer. We propose that with improved biomarker testing, it may be possible to develop a clinically useful test for detecting EC. 10.1016/j.ygyno.2019.11.028