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Background In China, the national cervical cancer screening protocol involves initial testing for high-risk human papillomavirus (hrHPV), followed by cytology for hrHPV-positive cases. This study evaluates the effectiveness of PAX1 methylation ( PAX1 m ) analysis in identifying precancerous or cancerous lesions in cervical samples from Chinese women positive for non-16/18 hrHPV strains. Methods Between February 2022 and March 2023, 281 cervical samples from non-16/18 hrHPV-positive women underwent cytological examination and PAX1 m analysis. The study assessed the statistical relationship between PAX1 m levels and the presence of cervical lesions, comparing the diagnostic performance of PAX1 m to conventional cytology. Results A significant association was found between PAX1 methylation levels and the risk of CIN2 + and CIN3 + lesions, with 47 instances of CIN2 + detected. Odds ratios (ORs) for moderate and high PAX1 m levels were 8.86 (95% CI: 2.24–42.17) and 166.32 (95% CI: 47.09-784.97), respectively. The area under the ROC curve for PAX1 m in identifying CIN2 + lesions was 0.948 (95% CI: 0.895–0.99). PAX1 m demonstrated similar sensitivity and negative predictive value (NPV) to cytology but reduced the colposcopy referral rate from 47.7% with cytology alone to 25.6% with PAX1 m , showing superior specificity and positive predictive value across age groups. Conclusions PAX1 methylation is a strong indicator of CIN2 + and CIN3 + risk, offering diagnostic performance comparable to cytology with the added benefit of reduced unnecessary colposcopy referrals. These findings support the use of PAX1 m analysis as a reliable tool for triaging non-16/18 hrHPV-positive women in outpatient settings.

Background The risk of cervical cancer progression in high-risk human papillomavirus (HR-HPV)-positive women is associated with cervical lesion severity and molecular heterogeneity. Classification systems based on p16 and Ki67 expression cumulative scores (0–3 each)—p16/Ki67 collectively known as an immunoscore [IS]—are an accurate and reproducible method for grading cervical intraepithelial neoplasia (CIN) lesions. Meanwhile, DNA methylation is an early event in the development of cervical cancer. Hence, this study evaluated the relationship among CIN, p16/Ki-67 IS, and PAX1 / ZNF582 methylation. Methods In this study, 414 HPV-positive paraffin-embedded specimens were collected, and PAX1 / ZNF582 methylation and the p16/ki67 IS were determined. A total of 43 invalid samples were excluded and 371 were included in the statistical analyses. There were 103 cervicitis, 95 CIN1, 71 CIN2, 89 CIN3, and 13 squamous cell carcinoma (SCC) cases. The association between PAX1 / ZNF582 methylation and p16/Ki6 immunohistochemical staining scores was analyzed. Results The ΔCp of PAX1 m ( PAX1 methylation) and ZNF582 m ( ZNF582 methylation) decreased with cervical lesion severity (Cuzick trend test, all P  < 0.001). The severity of the cervical lesions and p16, Ki67, and p16/Ki67 IS showed an increasing trend (Multinomial Cochran-Armitage trend test, all P  < 0.001). The prevalence of PAX1 m / ZNF582 m increased with an increase in the IS of p16, Ki67, and p16/Ki67 (Cochran-Armitage trend test, all P  < 0.001). In cervical SCC, the IS was 5–6, and the PAX1 m / ZNF582 m was positive. Meanwhile, heterogeneity was observed in CIN lesions: 10 cases had an IS of 3–4 and were PAX1 m / ZNF582 m -positive in ≤ CIN1; 1 case had an IS of 0–2 and was PAX1 m / ZNF582 m -positive in CIN2/3. Conclusions Significant heterogeneity was observed in CIN lesions for p16 and Ki67 immunohistochemical staining scores and PAX1 / ZNF582 methylation. This may help clinicians personalize the management of CIN based on the predicted short-term risk of cancer progression, minimizing the rate of missed CIN1 diagnoses and incorrect treatment of CIN2/3.

Background Primary screening for high-risk human papillomavirus (hrHPV) with cytological triage for women with non-16/18 hrHPV-positive status has become popular in China. However, cytology relies on the subjective judgment of pathologists, leading to inconsistent clinical performance. Methods A total of 657 hrHPV-positive women aged 25–64 years were enrolled in this cross-sectional study. All participants underwent colposcopic biopsy after cytology triage, with cytology residual specimens undergoing DNA methylation testing. CIN2+ and CIN3+ sensitivity and specificity were compared between the different triage strategies ( n =487): PAX1 methylation ( PAX1 m ) , Glycophorin C methylation ( GYPC m ), cytology, and combinations between them or with HPV16/18. Results The area under the receiver operating characteristic curves (AUCs) for PAX1 m and GYPC m in detecting CIN2 or worse (CIN2+) were 0.867 (95% confidence interval [CI]: 0.796–0.937) and 0.873 (95% CI: 0.808–0.938), respectively. The sensitivities of PAX1 m and GYPC m were consistent with those of cytology for both CIN2+ and CIN3+ detection. The relative specificities of PAX1 m and GYPC m for CIN2+ detection compared to cytology were 2.83 (95% CI: 2.33–2.45) and 3.09 (95% CI: 2.40–3.98), respectively. The relative specificities of combining HPV 16/18 with PAX1 m and GYPC m for CIN2+ detection compared to cytology were 3.38 (95% CI: 2.96–3.86) and 3.67 (95% CI: 3.15–4.27), respectively. Compared to low levels of DNA methylation, high levels of PAX1 m and GYPC m resulted in odd ratios (ORs) of 57.66 (95% CI: 13.57–409.12, p < 0.001) and 23.87 (95% CI: 6.49–115.42, p < 0.001) for CIN3+, adjusted for HPV 16/18 and cytology results. Conclusions PAX1 m and GYPC m demonstrated superior ability to identify cervical precancerous lesions and cervical cancer, with AUC values exceeding 0.85. For detecting CIN2+/CIN3+ in women with hrHPV-positive status, DNA methylation (combined with HPV 16/18) showed higher specificity than cytology (combined with HPV 16/18) and is a potential molecular biomarker for detecting cervical (pre)cancer.

The aim of this study is to assess the diagnostic and screening performance of a standardized methylation-specific real-time PCR assay targeting and genes for cervical cancer in a Chinese cohort. Genomic DNA was extracted from cervical exfoliated cells and converted by sodium bisulfite and then analyzed by qMSP assay. Ct values were collected for and as target genes and as an endogenous reference gene. The samples included 295 cervicitis, 111 LSIL (low-grade squamous intraepithelial lesion), 51 HSIL (high-grade squamous intraepithelial lesion) and 30 cervical cancer. The Ct values decreased with the progression of cervical cancer from cervicitis, through LSIL and HSIL to cancer. The difference in Ct values between cytological grades was highly significant (p≤0.01) between grades either for or for except the difference between cervicitis and LSIL of . With the Ct cut-off values of gene and gene 38.6 and 38 and with the / in combination, the positive rate of methylation in invasive cancer tissues was 100%, in contrast to 11.5% (95% CI: 8.67%-14.33%) in cervicitis tissues, 45.1% (95% CI: 40.68%-49.52%) in LSIL tissues, and 68.5% (95% CI: 64.37%-72.63%) in HSIL tissues. The specificity and sensitivity of differentiating tumors from cervicitis were 0.957 (95% CI: 0.939-0.975) and 1.00, respectively. The specificity and sensitivity of differentiation between cervicitis+LSIL and HSIL+cervical cancer were 0.881 (95% CI: 0.852-0.91) and 0.748 (95% CI: 0.709-0.787), respectively. / methylation could be translated into clinical practice for cervical neoplasia detection.

Objective Referring all women who tested positive for human papillomavirus (HPV) 16/18 to colposcopy may lead to potential over-referral issues. Triage tests based on cytology results face challenges in achieving accurate diagnoses. Our study aims to assess the clinical effectiveness of PAX1 / JAM3 methylation (CISCER) test as a triage method for HPV 16/18-positive women. Methods From November 2021 to December 2022, a total of 334 women who tested positive for HPV 16/18 and were referred to colposcopy at The Second Affiliated Hospital of Zhejiang University School of Medicine were studied. The clinical utility of the CISCER test, cytology, and the combination of CISCER with cytology as potential triage tests was compared. Results We observed a significant increase in the methylation levels of PAX1 gene and JAM3 gene in women with cervical intraepithelial neoplasia (CIN) grade 2 or severe (CIN2+). The CISCER test demonstrated superior triage performance over cytology, even when used in combination with cytology, showing a high sensitivity of 89.0% (95% confidence interval [CI] 82.9–95.1%) and specificity of 95.3% (95% CI 92.6–98.0%). It achieved an area under the curve of 0.921 (95% CI 0.877–0.966) and an odds ratio of 164.02 (95% CI 68.64–391.95). The immediate CIN2+ risk based on positive CISCER results would be 89.0% (95% CI 80.8–94.1%), with an estimated average of 1.12 referrals needed to detect one CIN2+ case. Moreover, CISCER triaging successfully identified all cancer patients and did not miss any CIN3+ cases among women aged ≥ 30. Conclusions The PAX1 / JAM3 methylation detection exhibited excellent accuracy in identifying cervical precancerous lesions in HPV 16/18-positive women and could be considered as a triage tool to reduce excessive referrals for colposcopy and overtreatment.

To explore the effect of PAX1 and JAM3 gene methylation on pathological upgrading before conization. A total of 549 patients who underwent colposcopy at our hospital were enrolled for analysis from December 2020 to April 2022. PAX1 and JAM3 gene methylation results in preoperative cervical exfoliated cells were collected. Univariate analysis and multivariate logistic regression analysis were conducted to identify the independent risk factors influencing the pathological upgrading of conization, aiming to establish a prediction model. A total of 88 patients were finally included for statistical analysis according to the inclusion and exclusion criteria. Based on univariate analysis and multivariate logistic regression analysis, ∆Ct PAX1 ( P  = 0.016, OR: 0.784, 95%CI 0.644–0.956) and cervical canal lesions ( P  = 0.048, OR: 3.469, 95%CI 1.014–11.870) were identified as independent risk factors for pathological upgrading for conization. Using the above results, we established a prediction model for pathological upgrading and plotted the receiver operator characteristic curve (ROC). The area under the curve (AUC) was calculated when the Youden index was maximized with an AUC value of 0.818 (95%CI 0.720–0.916), specificity of 94.4%, sensitivity of 60%. The cut-off value for ∆Ct PAX1 was determined as 4.34 when maximizing the Youden index. PAX1 could be a promising triage marker in predicting the pathological upgrading of CIN before conization. We found that if the ∆Ct PAX1 cut-off value is lower than 4.34, it is highly suggestive of pathological upgrading.

The relationship of PAX1/JAM3 methylation as well as HPV viral load (VL) with cervical lesions has been reported, but their role in persistent HPV infection without cervical high-grade lesions has not been fully elucidated. A total of 231 females diagnosed with persistent HPV infection and pathologically confirmed absence of high-grade cervical lesions were selected from the Colposcopy Outpatient Clinic of Peking University People’s Hospital, from March 2023 to December 2023. They were categorized into two groups based on the duration of HPV infection: the HPV persistent less than 3 years group and the more than 3 years group. PAX1/JAM3 methylation and HPV VL were determined by real-time PCR and BioPerfectus Multiplex Real-Time (BMRT)-HPV reports type-specific VL/10,000 cells, respectively. The average age of individuals with HPV infection lasting more than 3 years was higher compared to those with less than 3 years (48.9 vs. 45.1 years), with a statistically significant difference. Among the participants, 81.8% (189/231) had no previous screening. The methylation levels of JAM3 and PAX1 were significantly higher in individuals with HPV infection persisting for more than 3 years compared to those with less than 3 years, with a statistically significant difference (p < 0.05). There was a significant correlation between PAX1 and JAM3 methylation (p < 0.001), which could be used as cumulative evidence of HPV infection duration before the occurrence of precancerous lesions. The incidence of vaginal intraepithelial lesions was higher in individuals with HPV infection persisting for more than 3 years compared to those with less than 3 years, and HPV VL can be used as an indicative biomarker for concurrent cervical–vaginal lesions, especially for HPV other than 16/18 genotypes.

Background: The DNA methylation statuses of PAX1 and ZNF582 show great promise as biomarkers for the detection of oral squamous cell carcinoma (OSCC). This study aims to investigate the distribution of PAX1 or ZNF582 methylation in the exfoliated oral epithelial cells (OECs) of OSCC. Methods: Methylation data from 528 tumors and 50 adjacent nontumor tissues were acquired from The Cancer Genome Atlas and analyzed using UALCAN database. Sixty-one OSCC cases from Peking University School and Hospital of Stomatology were included in this study and the exfoliated OECs collected by oral swabs were collected from the cancerous lesion (CL), adjacent normal (AN), and contralateral normal (CN) sites. The methylation levels of these 2 genes in different sites were evaluated. Results: PAX1 and ZNF582 were both hypermethylated in OSCC compared with nontumor sites but showed different methylation patterns within the oral environment. Generally, a CL-centric methylation pattern of PAX1 where methylation levels decrease gradually from CL through AN to CN was observed, suggesting a field cancerization effect. ZNF582 methylation levels are significantly higher at lesion sites compared with normal sites, but no significant difference is observed between AN and CN. Coexistence of ZNF582 methylation in CL and AN or CN sites was also observed in some patients with OSCC. Furthermore, ZNF582 methylation was more sensitive among patients with OSCC. Conclusions: DNA methylation detection of PAX1 and ZNF582 in the exfoliated OECs is helpful for OSCC diagnosis. Hypermethylated PAX1 and ZNF582 show different methylation patterns in the oral cavity of patients with OSCC.

Background Atypical squamous cells of undetermined significance (ASC-US) often present diagnostic challenges with cytology-based results, leading to potential underdiagnosis or overdiagnosis. An effective triage method is essential for managing these cases to reduce unnecessary referrals and treatment. Methods A total of 322 women diagnosed with ASC-US were tested for HPV-DNA and the PAX1 and JAM3 methylation ( PAX1 m / JAM3 m ) test in the study. Results Methylation levels of PAX1 and JAM3 were significantly elevated in cervical lesions classified as CIN2 or more severe lesions (CIN2+). The methylation assay demonstrated a sensitivity of 83.8% and a specificity of 95.8%, outperforming HPV-DNA testing in differentiating high-grade cervical lesions among women with ASC-US. Moreover, PAX1 m / JAM3 m testing significantly reduced the colposcopy referral rate for further diagnostic procedures in high-risk HPV-positive women by 79.5%. Conclusions PAX1 m / JAM3 m testing shows promise as a reliable supplemental method to HPV-DNA testing for the triage of women with cytologic ASC-US. In addition, the molecular triage based on the CISCER assay or single PAX1 or JAM3 methylation, had better effects in the women with non-HPV16/18 group. This approach could potentially minimize overtreatment and unnecessary referrals in clinical practice, enhancing patient management and resource utilization.

This study aims to systematically evaluate the application value of gene methylation detection in cervical lesion screening and its potential advantages in the triage of non-16/18 high-risk human papillomavirus (HR-HPV) positive patients. This study enrolled 1,619 HPV-positive female patients who visited the Affiliated Hospital of Qingdao University from December 2023 to March 2025, with 989 patients ultimately meeting the inclusion criteria. All subjects underwent HPV-DNA testing, cytological examination, colposcopy, and gene methylation detection, with results analyzed in conjunction with histopathological evaluations. HPV-DNA detection was performed using fluorescence quantitative PCR methodology capable of identifying 17 high-risk HPV genotypes. Cytological examination results were classified according to the International Society of Cytology standards. gene methylation detection employed fluorescence quantitative PCR technology with ACTB as the internal reference gene, determining methylation levels through calculation of ΔCT values. Statistical analyses included ROC curve assessment of diagnostic performance, with intergroup comparisons conducted using one-way analysis of variance and Pearson's chi-squared test. The results demonstrated that PAX1 gene methylation detection showed significantly better diagnostic performance compared to cytological examination for the detection of CIN2+ and CIN3+ lesions. The AUC values for gene methylation detection in diagnosing CIN2+and CIN3+ were 0.934 (95% confidence interval [CI]: 0.916-0.948) with sensitivity of 93.49% and specificity of 93.24%and0.875 (95% confidence interval [CI]: 0.853-0.895)with sensitivity of 95.31% and specificity of 79.77%. Among non-16/18 HR-HPV(in women positive for high-risk HPV types other than 16/18) positive patients, gene methylation detection demonstrated higher sensitivity and specificity than cytological examination, enabling more accurate identification of patients requiring further intervention and reducing unnecessary colposcopy referrals. Furthermore, in HR-HPV positive patients with cytology results ≤ASCUS, gene methylation detection significantly decreased colposcopy referral rates (22.29%), thus alleviating patients' medical burden. gene methylation detection exhibits strong diagnostic efficiency for cervical lesions and holds significant value in triage diagnosis of non-16/18 HR-HPV positive.