Abstract
Introduction
Methylation assays have demonstrated potential as dependable and high-precision approaches for identifying or triaging individuals with cervical cancer (CA) or cervical intraepithelial neoplasia (CIN). Our investigation aimed to assess the efficacy of the diagnosis and triage of the PAX1/SOX1 methylation panel in detecting CIN or CA.
Methods
A total of 461 patients with abnormal high-risk human papillomavirus (hrHPV) or cytology test results were recruited for this study. Each patient underwent an assortment of assessments, comprising a cytology test, hrHPV test, colposcopy examination, and PAX1 and SOX1 methylation tests.
Results
The extent of methylation of both genes demonstrates a positive correlation with the severity of CIN lesions and CA. To determine the correlation for patients with CIN2 or worse (CIN2+), the area under curve was 0.821 (95% CI: 0.782–0.853) for PAX1 and 0.800 (95% CI: 0.766–0.838) for SOX1, while for CIN3 or worse (CIN3+), 0.881 (95% CI: 0.839–0.908) for PAX1 and 0.867 (95% CI: 0.830–0.901) for SOX1. The PAX1/SOX1 methylation marker panel performed sensitivity and specificity of 77.16% and 91.67% for CIN2+, 84.76% and 90.50% for CIN3+, respectively. Regarding triaging hrHPV+ patients, the PAX1/SOX1 methylation test only referred 11.83% of the patients who are unnecessary for colonoscopy examination, which is comparatively lower than cytology, thereby signifying a promising triage strategy for hrHPV-positive women. Furthermore, we observed that the positive PAX1/SOX1 methylation test result for untreated CIN1 or fewer patients would result in a higher likelihood of progression upon a 24-month follow-up visit.
Conclusion
The present investigation demonstrates that the PAX1/SOX1 methylation marker panel exhibits favorable diagnostic performance in CIN detection and holds the potential to be employed for individual CIN tests or hrHPV-positive triage.
Keywords: Cervical cancer, Cervical intraepithelial lesion, DNA methylation markers, Triage
Introduction
Cervical cancer (CA) is the fourth most prevalent cancer and the fourth leading cause of cancer-related fatalities among women on a global scale. The mortality associated with CA is disproportionately high in developing regions, primarily due to inadequate screening and treatment modalities [1]. CA screening programs are critical for the early detection and diagnosis of CA, or cervical intraepithelial neoplasia (CIN), which can lead to timely interventions and thereby enhance survival outcomes [1–3].
Historically, the Papanicolaou test, aka Pap smear, has been the conventional method for CA screening, significantly reducing morbidity and mortality from invasive CA over numerous decades [4]. However, the effectiveness of the Pap smear is hampered by its low sensitivity, reliance on highly skilled clinicians for proper execution and interpretation of results, and variability in criteria among various screening centers [3–5]. The advent of molecular testing and acknowledgment that persistent high-risk human papillomavirus (hrHPV) infections cause the majority of CA cases have led to the widespread adoption of HPV DNA testing as a screening approach [1, 2, 6]. Although HPV DNA testing is more sensitive and reproducible than the Pap smear, its specificity is relatively lower, resulting in unnecessary referrals for colposcopy and heightened patients’ anxiety [7, 8]. This lower specificity is largely due to the transient nature of most HPV infections and the fact that only a minority of these infections will progress to invasive disease [9, 10]. Additionally, HPV DNA testing cannot distinguish between different stages of CIN [6, 8]. Consequently, it is imperative to discover new biomarkers with high sensitivity and specificity for the detection of CIN and CA to refine screening and diagnostic methodologies, or as a supplement triage method of current detection method.
Studies have shown the critical role of epigenetic modifications in carcinogenesis, including DNA methylation, histone alteration, and microRNA (miRNA) expression [11]. For CA, researchers have identified numerous gene methylation sites linked to its advancement, positioning the methylation status of these loci as promising biomarkers for early detection [12, 13]. Furthermore, assessing the methylation levels of certain genes may aid in forecasting treatment outcomes, such as the response to cisplatin-based neoadjuvant chemotherapy [14, 15]. This can enable the selection of optimal treatment strategies for patients, minimize adverse effects, and identify new therapeutic targets.
The paired box gene 1 (PAX1) belongs to the PAX family, which is essential in tissue development and cell differentiation during embryonic growth. It has been shown to suppress malignant characteristics triggered by multiple pathways under oncogenic stimuli, suggesting its function as a putative tumor suppressor [16]. Likewise, sex-determining region Y-box 1 (SOX1) serves as another tumor suppressor, modulating the Wnt signaling pathway across various cancers [17]. The methylation levels of both PAX1 and SOX1 have been proven to be correlated with CA progression, highlighting their potential as diagnostic biomarkers for CIN [4, 18, 19].
In this study, we conducted a case-control study utilizing quantitative DNA methylation assays to evaluate the diagnostic efficacy of the PAX1 and SOX1 methylation panels in detecting CIN. Additionally, we sought to assess the accuracy of this panel in triaging hrHPV-positive patients relative to the conventional cytology tests’ performance.
Methods
Patient Selection
Between November 2018 and January 2020, a total of 658 patients from Guizhou Provincial People’s Hospital, displaying abnormal HPV or cytology test results and requiring colonoscopy examination, were enrolled in this study. The study collected results from colonoscopy examination, cytology test, hrHPV test, and PAX1/SOX1 methylation test for each patient. The patients were categorized into normal, CIN1, CIN2, CIN3, and CA groups based on their histological results. Eligibility criteria for enrollment included being over 18 years old, possessing an intact uterus, and having no history of treatment for CIN or CA. Exclusion criteria included (1) being pregnant, lactating, or having a history of cancer related to the reproductive tract; (2) having undergone any vaginal examination or being in the menstrual period within 2 days prior to the tests; and (3) having inadequate DNA concentration in cell samples or sample confusion. All patients were managed and treated according to the standard guidelines for cervical neoplasia.
Detection of Gene Methylation
Cervical scraping samples were preserved in a TCT cell preservation solution under −15°C. Genomic DNA was extracted from the cervical exfoliated cells using the QIAamp DNA Mini Kit (Qiagen GmbH, Hilden, Germany) following the manufacturer's recommendations. The DNA concentrations and 260/280 ratios were measured by a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA), and a 260/280 ratio of around 1.6–2.1 and a DNA concentration of no less than 30 ng/μL were required for all DNA samples. A 60 µL blank sample was also processed as a negative control through the extraction process. The subsequent bisulfite conversion was performed using the EZ DNA Methylation-GoldM Kit (Zymo Research, CA, USA).
The quantitative methylation-specific PCR was performed using the Applied Biosystems 7,500 (Thermo Fisher Scientific, USA) with the SOX1 and PAX1 gene methylation detection kit (HybriBro Pharmaceutical Technology Co. Ltd., Guangzhou, China), using the ACTB gene as an internal reference. The crossing point value of SOX1, PAX1, and ACTB was obtained for each sample, and the crossing point value of ACTB should be between 15 and 35; otherwise, it should be retested. The cut-off value for PAX1 is 38 and 38.6 for SOX1, and either of them tested as positive would be regarded as PAX1/SOX1 methylation positive.
HPV Detection
Cervical biopsies samples were proceeded to HPV (including types 6, 11, 16, 18, 31, 33, 35, 39, 42, 43, 44, 45, 51, 52, 56, 58, 59, and 68) test by using the Digene HC2 High-Risk HPV DNA Test (QIAGEN, Gaithersburg, USA) following the manufacturer’s instruction.
Statistical Analysis
Statistical analyses were performed and illustrated using IBM SPSS Statistics 26.0 and GraphPad Prism 9.0.0. The Kruskal-Wallis test was used to compare the mean ΔCt value between groups, with a p value <0.05 considered significant. The performance of each methylation biomarker was assessed by receiver operating characteristics (ROC) curve. The area under the ROC curves, sensitivities, specificities, and 95% confidence intervals were calculated for conclusive test results, and the Wilcoxon test was used to determine the significance of the CIN1 or less prediction results.
Results
Sample Characteristics
This study recruited a total of 651 patients, of whom 120 patients had inadequate cytology results, 32 patients lacked age information, and 33 patients had incomplete hrHPV test results. Consequently, 461 patients with complete cytology, HPV testing, and histology outcomes were subjected to further analysis. The demographic characteristics and clinical information of these patients are displayed in Table 1. The CA group contained 46 cases of squamous cell carcinoma and 1 case of adenocarcinoma.
Table 1.
Demographic and clinical characteristics of patients (%)
| Histological results | Total | |||||
|---|---|---|---|---|---|---|
| normal | CIN1 | CIN2 | CIN3 | CA | ||
| Number of subjects | ||||||
| All | 144 (31.24) | 120 (26.03) | 67 (14.53) | 83 (18.00) | 47 (10.20) | 461 |
| Age ≥30 years | 109 (29.95) | 81 (22.25) | 59 (16.21) | 70 (19.23) | 45 (12.36) | 364 |
| Age, years | ||||||
| Mean±SD | 38.4±11.4 | 40.3±11.7 | 38.6±10.2 | 42.6±11.1 | 51.7±11.7 | 41.1±11.9 |
| Range | 18–66 | 23–66 | 20–66 | 19–70 | 29–83 | 18–75 |
| Cytology test | ||||||
| NILM | 36 (42.86) | 25 (29.76) | 9 (10.72) | 8 (9.52) | 6 (7.14) | 84 |
| ASC-US | 78 (35.94) | 67 (30.88) | 20 (9.22) | 36 (16.59) | 16 (7.37) | 217 |
| LISL | 17 (25.76) | 20 (30.30) | 15 (22.73) | 11 (16.67) | 3 (4.54) | 66 |
| ASC-H/AGC/HSIL+ | 13 (13.83) | 8 (8.51) | 23 (24.47) | 28 (29.79) | 22 (23.40) | 94 |
| HPV test | ||||||
| 16/18 | 42 (21.32) | 41 (20.81) | 18 (9.14) | 57 (28.93) | 39 (19.80) | 197 |
| 12 high riska | 81 (36.32) | 66 (29.60) | 44 (19.73) | 25 (11.21) | 7 (3.14) | 223 |
| Mid-low riskb | 9 (40.91) | 9 (40.91) | 3 (13.64) | 0 (0) | 1 (4.54) | 22 |
| Negative | 12 (63.16) | 4 (21.05) | 2 (10.53) | 1 (5.26) | 0 (0) | 19 |
a12 high-risk HPV genotypes including HPV 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68.
bMid-low-risk HPV genotypes including HPV 6, 11, 42, 43, 44, 53, 73, 81, and 82.
Association between PAX1/SOX1 Methylation Level and CIN Level
The mean ΔCt value of PAX1/SOX1 methylation level at various lesion stages was computed to ascertain their correlation. The ΔCt value was defined as the Ct value of the target gene (PAX1 or SOX1) methylation level minus the Ct value of the internal reference gene (ACTB) methylation level. The 2−ΔΔCT method was used in the validation, using the reference gene as the normalizer to calculate relative gene expression [20]. A low ΔCt value of PAX1 or SOX1 indicated relatively high methylation levels.
The mean ΔCt value of PAX1 methylation level was 13.84 in the normal group, 14.18 in CIN1, 11.17 in CIN2, 7.58 in CIN3, and 3.80 in CA. For SOX1 methylation level, the mean ΔCt value was 14.70 in the normal group, 14.83 in CIN1, 12.26 in CIN2, 8.25 in CIN3, and 4.87 in CA. Significant differences were found between each group except Normal versus CIN1 (Fig. 1). These findings suggest that the methylation levels of PAX1 and SOX1 were considerably higher in CIN2+ patients than in CIN1 or normal cervical tissues. Moreover, a higher methylation level was significantly associated with more severe lesion or cancer. The raw Ct values of all subjects are listed in online supplementary data 1 (for all online suppl. material, see https://doi.org/10.1159/000538464).
Fig. 1.
Distribution of ΔCt value of PAX1/SOX1 methylation in different levels of CIN or cancer, with mean ΔCt value and its 95% CI in each group. ns, not significant. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Performance of PAX1/SOX1 Methylation Level Detection for CIN Diagnosis
The ROC curves were drawn to determine the cut-off value of PAX1/SOX1 methylation level in different CIN grades, as shown in Figures 2 and 3. The area under the ROC curve was 0.821 for PAX1 methylation and 0.800 for SOX1 methylation in the group of CIN2+, while 0.881 for PAX1 and 0.867 for SOX1 were in the group of CIN3+. The positive cut-off value for PAX1 was Ct ≤38 and, for SOX1, was Ct ≤38.6. By applying the cut-off value, the sensitivity and specificity of PAX1 and SOX1 methylation panels in detecting CIN2+ and CIN3+ were 77.16% and 91.67%, 90.00% and 82.78%, respectively. For patients at or above 30 years old, the sensitivity and specificity in detecting CIN2+ and CIN3+ were 84.76% and 90.50%, 94.78% and 80.32%, respectively. The diagnostic performance of the PAX1/SOX1 methylation test in different CIN grades is shown in Table 2.
Fig. 2.
ROC curve of the PAX1/SOX1 methylation panel in detecting CIN2 or worse.
Fig. 3.
ROC curve of the PAX1/SOX1 methylation panel in detecting CIN3 or worse.
Table 2.
Diagnostic performance of PAX1/SOX1 methylation test
| Sensitivity | Specificity | PPV | NPV | Accuracy | |
|---|---|---|---|---|---|
| Overall, % | |||||
| ≥CIN1 | 51.42 | 92.36 | 93.68 | 46.34 | 64.21 |
| ≥CIN2 | 77.16 | 91.67 | 87.36 | 84.32 | 85.47 |
| ≥CIN3 | 90.00 | 82.78 | 67.24 | 95.47 | 84.82 |
| Age ≥30, % | |||||
| ≥CIN1 | 58.43 | 91.74 | 94.30 | 48.54 | 68.41 |
| ≥CIN2 | 84.76 | 90.50 | 87.97 | 87.86 | 87.91 |
| ≥CIN3 | 94.78 | 80.32 | 68.99 | 97.09 | 84.89 |
Comparison of Diagnostic Performance and hrHPV+ Triage Performance
Compared to the cytology test, the PAX1/SOX1 methylation test demonstrated significantly higher specificity (91.30% in the CIN2+ group and 81.51% in the CIN3+ group), PPV (88.17% in the CIN2+ group and 68.05% in the CIN3+ group), NPV (83.67% in the CIN2+ group and 94.82% in the CIN3+ group), and accuracy (85.48% in the CIN2+ group and 84.05% in the CIN3+ group) in detecting CIN2+ lesions from hrHPV+ patients (Table 3). Furthermore, we compared the triage performance of different testing methods for hrHPV+ patients. Considering the efficacy of triaging CIN1 or fewer patients, the PAX1/SOX1 methylation test showed an unnecessary referral rate of 11.83% while 42.13% for the HPV16/18 genotyping test, and 52.15% for the cytology test (Table 4).
Table 3.
Diagnostic performance of PAX1/SOX1 methylation test and cytology in hrHPV+
| Sensitivity | Specificity | PPV | NPV | Accuracy | |
|---|---|---|---|---|---|
| CIN2+, % | |||||
| Cytology | 87.96 | 20.94 | 47.59 | 68.06 | 47.07% |
| PAX1/SOX1m | 78.42 | 91.30 | 88.17 | 83.67 | 85.48% |
| CIN3+, % | |||||
| Cytology | 89.06 | 19.53 | 32.29 | 80.56 | 37.31 |
| PAX1/SOX1m | 89.84 | 81.51 | 68.05 | 94.82 | 84.05 |
Table 4.
Unnecessary referral of different test methods in triaging hrHPV+ patients (%)
| Normal | CIN1 | CIN2 | CIN3 | CA | Unnecessary referral rate (<CIN2) | |
|---|---|---|---|---|---|---|
| 16/18 HPV+ | 42 (21.32) | 41 (20.81) | 18 (9.14) | 57 (28.93) | 39 (19.80) | 42.13 |
| Methylation+ | 10 (5.92) | 10 (5.92) | 34 (20.11) | 69 (40.83) | 46 (27.22) | 11.83 |
| Cytology (≥ASC-US) | 95 (27.22) | 87 (24.93) | 53 (15.19) | 74 (21.20) | 40 (11.46) | 52.15 |
PAX1/SOX1 Methylation in Predicting Progression of Untreated CIN1 or less
From December 2022 to January 2023, we conducted a follow-up visit for patients who were diagnosed with CIN1 or less and did not receive any treatment during the baseline visit, following the standard follow-up protocol. A total of 117 patients attended the follow-up visit. Of these 17 patients who initially tested positive for methylation detection, 6 progressed to CIN2 or worse (progress). The remaining 100 patients who had negative methylation test results, either remained stable (persist) or recover (reverse). A significant difference was found between the meth-positive and meth-negative groups, suggesting that CIN1 patients with a positive result on the PAX1/SOX1 methylation test may have a relatively high risk of progression (Table 5).
Table 5.
Summary of prognosis result of patients with CIN1 or less (%)
| Group | Progress | Persist | Reverse | Total |
|---|---|---|---|---|
| Meth-positive | 6* (35.29) | 2 (11.76) | 9 (52.95) | 17 |
| Meth-negative | 0 (0) | 19 (19.00) | 81 (81.00) | 100 |
| Total | 6 (5.13) | 21 (17.95) | 90 (76.92) | 117 |
* p < 0.0001.
Discussion
Methylation marker tests are gaining popularity in the screening, diagnosis, and prognosis of cancer, as supported by numerous studies [11, 21, 22]. Multiple panels of methylation markers have been reported for the diagnosis and screening triage of CIN or CA, with potential diagnostic applications [23, 24]. In this study, we investigated and confirmed that the PAX1/SOX1 methylation level is associated with the progression of CIN levels and CA. The PAX1/SOX1 methylation test demonstrated 77.16% sensitivity and 91.67% specificity in detecting the CIN2+ group and a sensitivity of 90.00% and a specificity of 82.78% in detecting the CIN3+ group. For patients over 30 years old, the sensitivity can increase to 84.76% and 94.78% in detecting CIN2+ and CIN3+. The overall performance suggested that PAX1/SOX1 methylation test is non-inferior to current testing methods and could be used for detecting CIN lesions or CA.
The hrHPV test is widely employed for the detection of potential CIN or CA patients due to its high sensitivity, but its low specificity results in numerous unnecessary colposcopy referrals [25]. Therefore, an alternative test method to triage hrHPV-positive subjects is necessary to improve triage accuracy and reduce false referral rates. We assessed the triage accuracy of the PAX1/SOX1 methylation test, cytology test, and HPV16/18 genotyping in the hrHPV-positive group. Among these triage methods, the PAX1/SOX1 methylation test exhibited the lowest rate of unnecessary referrals.
The cytology test’s performance in triaging hrHPV-positive patients in this study presented relatively high sensitivity and low specificity, particularly in triaging CIN3+ patients, which may be due to the patient selection bias [26, 27]. Additionally, prior knowledge of the hrHPV status may result in defining more patients as having abnormal cytology test results [28]. We demonstrated that the PAX1/SOX1 methylation test exhibited higher accuracy and a lower unnecessary referral rate compared to the cytology test. Furthermore, the results of the methylation test are more objective, providing greater stability and reliability which will not highly depend on the experienced medical staff to improve accuracy.
Multiple studies have evaluated the diagnostic performance of the PAX1/SOX1 methylation panel. A meta-analysis illustrates that the PAX1/SOX1 methylation panel had 0.72 for sensitivity and 0.77 for specificity in distinguishing CIN3+ patients, and PAX1/HPV DNA could reach 0.75 for sensitivity and 0.81 for specificity [18]. The results of our study generally match the trend, while we showed that the PAX1/SOX1/HPV DNA parallel test could also be a convincing method for diagnosis.
Another potential area for investigation is the performance of methylation detection in predicting CIN progression. Studies have shown that more than half of CIN1 or CIN2 patients will regress spontaneously [29, 30]. As hypermethylation of PAX1/SOX1 is an early event in the development of CA [31, 32], a positive result of the PAX1/SOX1 methylation test from a CIN2 or lower level patient may indicate a higher likelihood of progression, providing crucial information for clinical or treatment management. We performed a follow-up visit for CIN1 and fewer patients and found all 6 patients with progression status had initially tested positive for PAX1/SOX1 methylation, suggesting a potential association between a positive result of the PAX1/SOX1 methylation test and a higher possibility of progression. In this follow-up, we did not include CIN2 or above patients due to project management issues, but a study involving the FAM19A4/miR124-2 methylation panel supported the idea that CIN2/3 patients with negative methylation test results were more likely to regress without clinical treatment [33].
Another limitation of this study is that the study cohort did not include sufficient negative control subjects, i.e., patients with both negative HPV and cytology results, which did not fully reflect the composition of patients in the clinic. A more comprehensive study is required to confirm the specificity of the PAX1/SOX1 methylation panel in detecting patients without lesions, as well as its predictive value in CIN prognosis.
Conclusion
Our study confirms that the PAX1/SOX1 methylation panel test is an objective and highly specific method for detecting CA or CIN. It demonstrated a triage performance comparable to cytology and HPV16/18 genotyping, with superior accuracy as a triage tool for CIN2+ in hrHPV-positive women. Additionally, it exhibited potential in predicting the prognosis of untreated patients with CIN1 or less, though further evidence is required to confirm such application.
Statement of Ethics
This study was approved by the Ethic Committee in Guizhou Provincial People’s Hospital in September 2018, with approval number 2018-130. All patients were informed and signed the written consent form. The study was performed following the guidelines of the Declaration of Helsinki.
Conflict of Interest Statement
The authors have no conflicts of interest to declare.
Funding Sources
This study was supported by Guizhou Provincial Science and Technology Projects (No. 2021-450) and GZSYQN (No. 2021-06). The founders had no role in the design, data collection, data analysis, and reporting of this study.
Author Contributions
Y.G. and D.Z. made the design of the research. W.L., F.Q., and X.X. acquired the samples and clinical data and performed the experiments. J.Z. and E.J. made the analysis and interpretation of the data. Y.X. wrote the main manuscript text. Y.G. and W.L. reviewed and approved the manuscript.
Funding Statement
This study was supported by Guizhou Provincial Science and Technology Projects (No. 2021-450) and GZSYQN (No. 2021-06). The founders had no role in the design, data collection, data analysis, and reporting of this study.
Data Availability Statement
The raw data generated in this study are available from the corresponding author on reasonable request.
Supplementary Material.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
The raw data generated in this study are available from the corresponding author on reasonable request.
