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

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.

Methylated DNA markers for plasma detection of ovarian cancer: Discovery, validation, and clinical feasibility.

OBJECTIVE: Aberrant DNA methylation is an early event in carcinogenesis which could be leveraged to detect ovarian cancer (OC) in plasma. METHODS: DNA from frozen OC tissues, benign fallopian tube epithelium (FTE), and buffy coats from cancer-free women underwent reduced representation bisulfite sequencing (RRBS) to identify OC MDMs. Candidate MDM selection was based on receiver operating characteristic (ROC) discrimination, methylation fold change, and low background methylation among controls. Blinded biological validation was performed using methylated specific PCR on DNA extracted from independent OC and FTE FFPE tissues. MDMs were tested using Target Enrichment Long-probe Quantitative Amplified Signal (TELQAS) assays in pre-treatment plasma from women newly diagnosed with OC and population-sampled healthy women. A random forest modeling analysis was performed to generate predictive probability of disease; results were 500-fold in silico cross-validated. RESULTS: Thirty-three MDMs showed marked methylation fold changes (10 to >1000) across all OC subtypes vs FTE. Eleven MDMs (GPRIN1, CDO1, SRC, SIM2, AGRN, FAIM2, CELF2, RIPPLY3, GYPC, CAPN2, BCAT1) were tested on plasma from 91 women with OC (73 (80%) high-grade serous (HGS)) and 91 without OC; the cross-validated 11-MDM panel highly discriminated OC from controls (96% (95% CI, 89-99%) specificity; 79% (69-87%) sensitivity, and AUC 0.91 (0.86-0.96)). Among the 5 stage I/II HGS OCs included, all were correctly identified. CONCLUSIONS: Whole methylome sequencing, stringent filtering criteria, and biological validation yielded candidate MDMs for OC that performed with high sensitivity and specificity in plasma. Larger plasma-based OC MDM studies, including testing of pre-diagnostic specimens, are warranted.