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

Abstract

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.

FIG 1.

Overview of the study and selection of WID-qEC targets. (A) Schematic workflow of WID-qEC development from sample to assessment. The test was developed by epigenome-wide analysis of cervicovaginal samples from cancer cases and controls, and thresholds were fixed in a small pilot set. The test was then validated in five independent validation sets using two predefined thresholds: ^aa high-sensitivity threshold was applied in high-risk and/or symptomatic settings (FORECEE Validation, Barcelona Validation, PMB Cohort, and Lynch Cohort), whereas ^ba high-specificity threshold was applied in lower-risk settings (Karolinska Cohort). (B) Example plots of selected CpG beta (methylation) values in control samples and EC cases versus immune cell proportion, and (C) CpGs in their proximity (within ±500 bp). (D) AUC values of individual MethyLight reactions for discrimination of controls and cancer cases in the FORECEE Pilot set. The top three reactions, ranked by AUC, were selected for further analysis. AUC, area under the curve; bp, base pair; CpG, cytosine nucleotide followed by guanine nucleotide; EC, endometrial cancer; PMB, postmenopausal bleeding; WID-qEC, Women's cancer risk IDentification – quantitative polymerase chain reaction test for Endometrial Cancer.

FIG 2.

WID-qEC performance in comparison with alternative detection methods and time to diagnosis. (A) The WID-qEC exhibits a higher area under the curve than DNA mutation analysis (P = .002 in DeLong's test) in the Barcelona Validation set. Comparison with ultrasound offers a higher AUC point estimate and improved receiver operating characteristic curve profile (Data Supplement). Data on WID-qEC, ultrasound, and DNA mutation analysis are numerical (Σ PMR, millimeter thickness, and number of mutations, respectively). (B) The WID-qEC identifies a majority of ECs from cancer-free controls in cervical samples taken up to 3 years before diagnosis or follow-up in the Karolinska Cohort (Data Supplement). The applied threshold is the high-specificity threshold (threshold 2). AUC, area under the curve; DNAmut, DNA mutation; EC, endometrial cancer; PMR, percentage methylated reference; US, ultrasound; WID-qEC, Women's cancer risk IDentification – quantitative polymerase chain reaction test for Endometrial Cancer.