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

Abstract The majority of endometrial and cervical cancers present with abnormal vaginal bleeding but only a small proportion of women suffering from vaginal bleeding actually have such a cancer. A simple, operator‐independent and accurate test to correctly identify women presenting with abnormal bleeding as a consequence of endometrial or cervical cancer is urgently required. We have recently developed and validated the WID‐qEC test, which assesses DNA methylation of ZSCAN12 and GYPC via real‐time PCR, to triage women with symptoms suggestive of endometrial cancer using ThinPrep‐based liquid cytology samples. Here, we investigated whether the WID‐qEC test can additionally identify women with cervical cancer. Moreover, we evaluate the test's applicability in a SurePath‐based hospital‐cohort by comparing its ability to detect endometrial and cervical cancer to cytology. In a set of 23 cervical cancer cases and 28 matched controls the receiver operating characteristic (ROC) area under the curve (AUC) is 0.99 (95% confidence interval [CI]: 0.97‐1.00) with a sensitivity and specificity of 100% and 92.9%, respectively. Amongst the hospital‐cohort (n = 330), the ROC AUC is 0.99 (95% CI: 0.98‐1) with a sensitivity and specificity of 100% and 82.5% for the WID‐qEC test, respectively, and 33.3% and 96.9% for cytology (considering PAP IV/V as positive). Our data suggest that the WID‐qEC test detects both endometrial and cervical cancer with high accuracy.

with a sensitivity and specificity of 100% and 92.9%, respectively. Amongst the hospital-cohort (n = 330), the ROC AUC is 0.99 (95% CI: 0.98-1) with a sensitivity and specificity of 100% and 82.5% for the WID-qEC test, respectively, and 33.3% and 96.9% for cytology (considering PAP IV/V as positive). Our data suggest that the WID-qEC test detects both endometrial and cervical cancer with high accuracy. What's new?
While abnormal vaginal bleeding is a presenting symptom of endometrial and cervical cancers, only a small proportion of women who present with vaginal bleeding have endometrial or cervical cancer. Currently, the tests used to triage women with abnormal bleeding, such as ultrasound or cytology, are subjective and have modest accuracy. Here, the authors demonstrate that a real-time PCR-based test, which assesses DNA methylation at three gene regions using a cervical or vaginal sample, is able to identify 100% of women who have cervical or endometrial cancer with a high specificity (>80%), irrespective of the sample collection system.

| INTRODUCTION
Endometrial cancer is amongst the cancers with the steepest rise in incidence. 1,2 Abnormal vaginal bleeding represents the lead symptom of this disease. An estimated 0.33% and 5% to 10% of premenopausal and postmenopausal women with abnormal vaginal bleeding, respectively, are eventually diagnosed with endometrial cancer. 3,4 In contrast to the second cancer arising from the uterus, that is, cervical cancer, no screening methodology exists for endometrial cancer and cytology shows only modest sensitivity to detect endometrial cancer early. 5,6 In the past 5 years, global cervical cancer screening coverage has been 32%, ranging between 7% in South-East Asia and 75% in the European region, 7 indicating that the majority of global cervical cancer patients may unfortunately not be identified due to screening but may instead present with abnormal vaginal bleeding.
In addition to conventional cytology as the primary test method for cervical screening, the application of liquid-based cytology, utilizing either ThinPrep or SurePath systems, has been established in many countries with organized cervical cancer screening programs. 8 Unlike PreservCyt media, the liquid component of ThinPrep, SurePath contains formalin, which crosslinks DNA to proteins and therefore makes DNA-based tests more challenging.
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.

| MATERIALS AND METHODS
We have analyzed cervical smear samples from two different settings: 1. Case/control setting: Cervical smear samples collected in ThinPrep from women with cervical cancer (n = 23, mean age 50.3 years; 18, 4 and 1 squamous, adeno and small cell cancer, respectively) and matched cancer-free control women (n = 28, mean age 50.7 years). These samples were collected within the FORECEE study (for details see Refs. [9][10][11]). After signing an informed consent, cervical samples were collected at appropriate clinical venues by trained staff and the cervical sample procedures were performed by a small group of research midwives or physicians using the ThinPrep system (Hologic Inc., cat #70098-002) according to standard operating procedures. Cervical cells were sampled from the cervix using a Cervex brush (Rovers Medical Devices, cat #70671-001), which was rotated five times through 360 while in contact with the cervix to maximize cell sampling.
2. Cervical smear samples from a hospital-based cohort: Women who attended the gynecology department at the general district hospital in Hall in Tirol were invited to provide a written consent that their sample (collected exactly as described above but in SurePath), surplus to cytological assessment and human papilloma virus (HPV) testing can be utilized for research. A written informed consent was obtained from a total of 330 women. Cytological assessment and HPV testing were carried out in 329 women, of which 326 had sufficient residual sample available for DNA extraction. A total of 6/330 women were eventually diagnosed with a cancer (primary cervical cancers, n = 2; recurrent cervical cancer, n = 1; endometrial cancers, n = 3).
The remaining 320/330 were diagnosed with preneoplastic lesions [cervical intraepithelial neoplasia grade 2/3 (CIN2/3), n = 22], had cancers in the past but were currently disease-free, or had benign conditions (for details see Figure 1). Amongst controls, 34 had previously undergone a total hysterectomy, 25 had a cancer of the vulva, vagina, cervix or uterus, but were free of disease at sample collection, and 23 had previously had a cancer of other organs. Eight patients had prior radiotherapy.
DNA methylation-specific, quantitative real-time PCR (MethyLight) analysis was performed as previously described. 8,9 The final test result 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
Applying the same cutoff for the WID-qEC ΣPMR as previously defined for endometrial cancers (WID-qEC ΣPMR ≥0.03), 9 the sensitivity and specificity was 100% and 92.9%, respectively. These data suggested that the WID-qEC test identified women with cervical cancer with a very high sensitivity and specificity.
Amongst the hospital-based cohort (n = 330), the ROC AUC distinguishing endometrial or cervical cancers from all controls (including CIN2/CIN3) was 0.99 (95% CI: 0.98-1; Figure 2B). All six cervical and  Among 56/320 WID-qEC false positive cases, 10 women were subsequently diagnosed with CIN2 or CIN3, 2 women were subsequently diagnosed with complex atypical hyperplasia (CAH) and 1 woman was suspicious for endometrial cancer (polyp, thickened suspicious endometrium and enlarged lymph node) but refused procedures to obtain a histological diagnosis. HPV infections did not seem to drive false positive WID-qEC results: Amongst the 298 CIN2/3 F I G U R E 2 WID-qEC performance. The WID-qEC test is assessed in (A) the case-control set (23 cervical cancer cases and 28 age-matched control women) and (B) the hospital-cohort (six cancers-three endometrial and three cervical cancers-320 controls among which 22, two and one were subsequent to sample collection diagnosed with CIN2/3, complex atypical endometrial hyperplasia and highly suspicious endometrium Our data also confirm the low sensitivity of cytology to detect endometrial cancer. 5,6 Overall, our current results, in conjunction with recently published data, 9