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.

The WID‐qEC test: Performance in a hospital‐based cohort and feasibility to detect endometrial and cervical cancers

“…DNA methylation‐specific, quantitative real‐time PCR (MethyLight) analysis was performed as previously described 8,9 . The final test result (WID‐qEC ∑PMR) is defined as the sum of the percentage of fully methylated reference (PMR) values of the three regions tested ( ZSCAN12 and two regions in GYPC ).…”

“…Bisulfite modified DNA was amplified using the ×1 Luna Universal Probe qPCR Master Mix (NEB, cat. #M3004G) and primer‐probe sets as described 9 . All PCR reactions, including the three marker regions in the ZSCAN12 and GYPC (two independent regions) genes as well as the reference gene COL2A1 , were performed in technical duplicates.…”

“…PCR reactions were run on the QuantStudio 7 Pro (Applied Biosystems) and results further extracted via the Design and Analysis Software 2.5.0 (Applied Biosystems). The PMR molecules at the target locus were standardized using an R script, dividing the TARGET : COL2A1 input amount ratio (derived using the COL2A1 standard curve) of a sample by the TARGET : COL2A1 input amount ratio of gBlocks Gene Fragments DNA and multiplying by 100 9 …”

“…The PMR molecules at the target locus were standardized using an R script, dividing the TARGET:COL2A1 input amount ratio (derived using the COL2A1 standard curve) of a sample by the TARGET:COL2A1 input amount ratio of gBlocks Gene Fragments DNA and multiplying by 100. 9 All statistical analyses were carried out in R version 4.1.2…”

“…Recently, we reported the WID-qEC test, a real-time PCR-based test assessing methylated alleles of regions within the ZSCAN12 and GYPC genes. 9 The WID-qEC test, based on ThinPrep samples, detects 90% to 100% of endometrial cancers independent of collection devices, menopausal status, age, stage, grade, ethnicity and histology and was able to diagnose 91% of endometrial cancers 1 year prior to diagnosis. 9 Here, we have employed a case/control and a hospital-based cohort study to assess whether the WID-qEC test (a) is also able to detect cervical cancer, (b) is also applicable for SurePath samples utilizing the same threshold as defined in our previous studies, (c) is superior to cytology and (d) retains a low false-positive rate when assessed within a hospital-based cohort.…”