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

Abstract

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.

Keywords: Cell-free nucleic acids; DNA methylation; Endometrial cancer; Endometrial neoplasm/diagnosis; Endometrial neoplasm/prevention & control; Liquid biopsy.

Figure 1.

Overall study flow diagram. Endometrial cancer (EC), benign endometrium (BE), benign cervicovaginal tissue (BCV), reduced representation bisulfite sequencing (RRBS), quantitative methylation-specific PCR (qMSP), formalin fixed paraffin embedded (FFPE), atypical endometrial hyperplasia (AEH).

Figure 2.

Heatmatrix for tissue-based biological validation. Increasing deciles of relative methylation intensity above a 95% cutoff for each methylated DNA marker (MDM) in benign endometrium (BE) samples are depicted on a yellow-red color spectrum with red depicting the highest intensity above the cutoff. Each row is a candidate MDM, each column is a patient tissue sample. Endometrial hyperplasia without atypia (HPw/oA), atypical endometrial hyperplasia (AEH), uterine carcinosarcoma (CS), clear cell endometrial cancer (CC), grade 1/2 endometrioid endometrial cancer (EG1/2), grade 3 endometrioid endometrial cancer (EG3).

Figure 3.

A) Heatmatrix of 28 EC MDMs tested via qMSP on DNA from vaginal fluid collected via the tampon pilot. The tampon pilot included women with histologic confirmation of benign endometrium (BE) (N=92), endometrial hyperplasia without atypia (HPw/oA) (N= 25), atypical endometrial hyperplasia (AEH) (N=11), and endometrial cancer (EC) (N=100). CDH4, LYPLAL1, c17orf64, and MAX.chr12.52652301 are endometrium specific MDMs as they are similarly methylated in both EC and BE, but unmethylated in benign cervicovaginal tissues. B) Area under the receiver operating characteristics curve (AUC) for a reduced 3-MDM panel (SFMBT2, NBPF8, MAX.chr10.22624479).