Background
Cervical cancer remains one of the main causes of death from cancer among women worldwide [
1]. Cytology-based screening has successfully reduced mortality associated with cervical cancer [
2]. However, the majority of cases of cervical cancer are still associated with absent or deficient screening. In previous studies, approximately 50 % of cervical cancers were diagnosed in women who were not screened [
3,
4]. Complete participation would achieve a greater improvement in screening effectiveness than intensifying screening policies [
3]. Therefore, it is important to improve participation rates among women with a history of non-attendance.
Epidemiological studies have emphasized that human papillomaviruses (HPVs) are the main etiological factor for cervical cancer and that these viruses are present in almost all cervical cancer tissues [
5]. Screening participation rates for cervical cancer can be improved by offering non-attending women the tools to collect a vaginal sample at home. Self-collection is an acceptable method to potentially increase participation [
6]. Studies have demonstrated that self-collected samples are suitable for HPV DNA testing and can increase participation rates in primary screening for cervical cancer [
6‐
11]. However, women whose self-collected specimens test positive for high-risk HPV (hrHPV) require additional triage testing because the specificity of assays for hrHPV is insufficient to justify direct referral for colposcopy in all cases [
11,
12]. Although cytology is an accepted and standard method of examination in triage for hrHPV-positive women [
13], cytological testing of self-collected samples does not yield reliable results and a visit to a physician is required [
14].
In normal, precancerous and cervical cancer tissues, the DNA methylation profiles of the host genome may indicate tissue-specific perturbations that occur during carcinogenesis [
15]. DNA methylation leaves a heritable record of such interactions and is an ideal biomarker for cancer detection [
16‐
20], which could be used to triage possible cases of cervical cancer [
21‐
24]. Previously, we used a CpG island microarray approach to identify novel genes that were silenced by methylation in cervical squamous cell carcinoma (SCC) [
25]. Quantitative analysis of the
PAX1 and
SOX1 genes can be used effectively for detection of cases of CIN that are grade 3 or worse (CIN3+) [
17]. Using methylated DNA sequence immunoprecipitation coupled with microarray analysis to identify other genes with clinical applications, we found that the gene for zinc finger protein 582 (ZNF582) was highly methylated in SCC [
18]. This gene is also highly methylated in adenocarcinoma (AC) of the cervix [
26]. In Taiwanese Gynecologic Oncology Group (TGOG) studies, we used a methylation biomarker and hrHPV tests to detect CIN3+ lesions in low grade squamous intraepithelial lesions (LSIL). ZNF582 methylation is implicated as a promising biomarker for use in the positive triage of cytological diagnoses of low grade squamous intraepithelial lesions [
27]. Combined parallel testing using Pap smears and PAX1 or SOX1 methylation tests may provide better performance than a combination of Pap smears with HPV-testing in detection of cervical neoplasm [
28]. The clinical performance of
PAX1,
SOX1, and
ZNF582 as biomarkers of cervical neoplasm has been validated in multi-center clinical trials; therefore, analysis of changes in the methylation status of these genes could be applied for self-collected vaginal samples.
The aim of this study was to validate the concordance and clinical performance of PAX1, SOX1, and ZNF582 methylation for detection of CIN3+ lesions in self-collected and physician-collected vaginal samples. Our hope is that these genes will serve as sensitive methylation biomarkers for clinical cervical cancer screening of self-collected samples.
Discussion
Our data showed reasonable to good concordance in the DNA methylation the
PAX1,
SOX1, and
ZNF582 genes for detection of CIN3+ lesions between self-collected vaginal samples and physician-collected cervical samples. These findings indicate that in the future, the requirement for patients to visit a physician for screening could be reduced by submitting self-collected samples. Compared with the current cytology-based call-recall programs, self-collected vaginal samples can increase access to cervical screening and may help to further reduce the incidence of cervical cancer by increasing the rate of participation in screening programs [
2,
11].
Our study for the comparison of the concordance of methylation status between self-collected vaginal samples and physician-collected cervical samples was conducted in a relatively large sample-size. Self-collection was found to be comparable with physician-collected samples for the detection of cervical methylation biomarkers. This is consistent with other recent reports, which have also shown high concordance in the results of methylation analysis of self-sampled vaginal material and physician-collected cervical scrapes [
31]. Furthermore, our study was conducted using a relatively large number of high-grade squamous intraepithelial neoplasm (HSIL, including CIN2, CIN3/CIS, SCC, AC/ASC) samples, including seven cases of ACs/ASCs. In addition, assessment of the methylation biomarkers in
ZNF582 provided the best clinical accuracy among self-collected samples (AUC: 0.83; sensitivity, 0.77 (95%CI, 0.65–0.87); specificity, 0.77 (95%CI, 0.66–0.86), using a cutoff of 0.0204). These results are comparable with those of well-performed cytological testing, indicating that methylation biomarker analysis of self-collected vaginal samples has the potential for use in population-based studies comparing the clinical performance of cytological testing for alternative methods of screening of cervical cancer.
The limitation of this study is the restricted sample set, especially in the normal controls, because we focused on investigating the concordance between physician-collected samples and self-collected samples obtained using a cytobrush. This device was designed for physician sampling and is not particularly practical for use in self-collection sampling. Consequently, the level of compliance and success in obtaining a sample using this method was low among the women in the control group and few samples were obtained. In addition, this was a hospital-based study and so the results may not be representative of the general population. A standardized education program and user-friendly tools for self-collection are also warranted.
In developed countries with extensive infrastructure for conducting cytological examinations, Pap smears combined with methylation tests may perform better than a combination of Pap smears with HPV-testing in the detection of cervical neoplasm [
28]. The reasons underlying the lack of participation in screening programs among women in developed countries are complex. Some examples of the barriers that have been reported are practical, such as appointment times and embarrassment [
32]; therefore, creative and sensitive methods that take into consideration these barriers to participation in cervical cancer screening are required [
32]. Self-collected sampling is time-saving and avoids embarrassment. For a non-attendee, self-collection of samples for molecular screening of hrHPV could be a suitable method for primary cervical cancer screening followed by cytology-based triage. Although the detection rate for CIN2+ or CIN3+ lesions is promising, cervical cytology sampling still requires intervention by a clinician [
21,
23,
24]. Recent studies have used methylation biomarkers to triage patients who screened positive for hrHPV. The sensitivity of direct triage by combined analysis of the promoter methylation of miR-124-2 and the MAL genes in self-collected cervicovaginal material was similar to that of triage with cytological analysis of an additional physician-collected smear [
20]. The search for complete methylation markers for use in triage of hrHPV-positive women or in primary screening of cervical cancer alone may further revolutionize cervical cancer screening.
Although self-collection of samples for hrHPV testing is an acceptable method screening for hrHPV infection, insufficient specificity will lead false-positive results in many patients in the absence of cervical neoplasm. Triage of these patients is required to confirm a true cervical intraepithelial neoplasm. Given the lack of infrastructure for conducting cytological examinations in low-resource areas, cytological screening is not ideal for triage; furthermore, the sensitivity of this method varies from 30 % to 87 % [
33]. In contrast, only a few neoplastic cells are required for the detection of promoter methylation within a gene of interest using the QMSP assay. We determined that the highest sensitivity values for the detection of CIN3+ lesions by determining the methylation status of
PAX1,
SOX1, and
ZNF582 in self-collected samples was 0.73, 0.73, and 0.77, respectively (Table
3). The clinical performance of this type of assay resembled that of a traditional cytological examination. The potential use of these new biomarkers as tools for cervical cancer screening as well as their possible use in the developing world to triage hrHPV-positive women during primary screening warrants further validation.
We used CIN3+ rather than CIN2+ as the cutoff in our study because of the equivocal nature of CIN2 lesions when diagnosed and the heterogeneity of their DNA methylation profiles [
17,
34]. While only 5 % of CIN2 lesions progress to invasive cancer and approximately 40 % regress, the corresponding percentages for CIN3 lesions are 33 % and 12 %, respectively [
35]. The pathology of CIN2 lesions is not clearly defined and these are the most difficult for pathologists to confirm among all Pap smear diagnoses [
36,
37]. The clinical management of patients with CIN2 lesions should be reassessed using the most accurate techniques. The incorporation of molecular markers, such as DNA methylation profiles, into cervical cancer screening might help to decrease the number of unnecessary referrals and repeat diagnostic procedures, which are not only a drain on financial resources but also inflict an unnecessary burden on the patient. Additional studies are required to define the nature of CIN2 lesions with or without DNA methylation in longitudinal studies.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
Conception and design of the experiments: C-CC, H-CL. Performance of the experiments: H-CW, P-HS and Y-WH. Analyses of the data: R-LH. Contribution of reagents/materials/analysis tools: Y-PL, Y-WL. Drafting and editing of the manuscript: C-CC, R-L and HH-CL. Recruitment of participants and collection of clinical samples: C-CC, H-CL, C-YT, M-HY. All authors read and approved the final manuscript.