Evaluating PAX1 methylation for cervical cancer screening triage in non-16/18 hrHPV-positive women.

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 (PAX1m) 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 PAX1m analysis. The study assessed the statistical relationship between PAX1m levels and the presence of cervical lesions, comparing the diagnostic performance of PAX1m 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 PAX1m 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 PAX1m in identifying CIN2 + lesions was 0.948 (95% CI: 0.895-0.99). PAX1m 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 PAX1m, 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 PAX1m analysis as a reliable tool for triaging non-16/18 hrHPV-positive women in outpatient settings.

[Value of SOX1 and PAX1 Gene Methylation Detection in Secondary Triage of High-Grade Cervical Lesions].

Objective To evaluate the value of SOX1 and PAX1 gene methylation detection in the secondary triage of high-grade cervical lesions.Methods Exfoliated cervical cells were collected from 122 patients tested positive for human papilloma virus (HPV) and subjected to thin-prep cytologic test (TCT) and SOX1/PAX1 gene methylation tests.Results The HPV test combined with TCT showed the sensitivity of 95.24% and the specificity of 23.75% for detecting cervical intraepithelial neoplasia (CIN) grade 2 and above (CIN2+).After the addition of the SOX1/PAX1 gene methylation detection in secondary triage,the sensitivity for detecting CIN2+ was 83.33%,which had no statistically significant difference from the sensitivity of TCT combined with HPV test (P=0.078).However,the specificity reached 77.50%,which was significantly higher than that of HPV test combined with TCT (P<0.001).The SOX1/PAX1 gene methylation level in the CIN2+ group was higher than those in the normal cervical tissue and the CIN1 group(P<0.001).The cut-off values of SOX1 and PAX1 gene methylation for CIN2+ detection were -11.81 and -11.98,respectively.Conclusion Adding the detection of SOX1/PAX1 gene methylation in secondary triage significantly improves the efficiency and accuracy of CIN2+ detection.

Allelic loss of HLA class I facilitates evasion from immune surveillance in cervical intraepithelial neoplasia.

Loss of heterozygosity (LOH) has been reported to occur in HLA regions in cervical intraepithelial neoplasia (CIN) and cervical cancer. However, the details of how this is related to the progression of CIN have been unclear. In this study, we examined the human papillomavirus (HPV) antigen-presenting capacity of people with CIN and the significance of LOH of HLA class I in the progression of CIN. It was shown that differences in antigen-presenting capacity among each case depended on HLA types, not HPV genotypes. Focusing on the HLA type, there was a positive correlation between antigen-presenting capacity against HPV and the frequency of allelic loss. Furthermore, the lost HLA-B alleles had a higher HPV antigen-presenting capacity than intact alleles. In addition, frequency of LOH of HLA class I was significantly higher in advanced CIN (CIN2-3) than in cervicitis or early-stage CIN (CIN1): around half of CIN2-3 had LOH of any HLA class I. Moreover, the antigen-presenting capacity against E5, which is the HPV proteins that facilitate viral escape from this immune surveillance by suppressing HLA class I expression, had the most significant impact on the LOH in HLA-B. This study suggests that HPV evades immune surveillance mechanisms when host cells lose the capacity for antigen presentation by HLA class I molecules, resulting in long-term infection and progression to advanced lesions.

PAX1 methylation as a robust predictor: developing and validating a nomogram for assessing endocervical curettage (ECC) necessity in human papillomavirus16/18-positive women undergoing colposcopy.

OBJECTIVE: The major challenge in routine endocervical curettage (ECC) among Human Papillomavirus (HPV) 16/18-positive patients is that only a small fraction benefit. Nevertheless, current reported models often overestimate the validity and necessity of ECC, making it difficult to improve benefits for patients. This research hypothesized that assessing paired boxed gene 1 methylation levels (PAX1m) and clinical characteristics could enhance the predictive accuracy of detecting additional high-grade squamous intraepithelial lesions or worse (HSIL +) through ECC that were not identified by colposcopy-directed biopsy (CDB). METHODS: Data from 134 women with HPV16/18 positivity undergoing CDB and ECC between April 2018 and April 2022 were collected and analyzed. Quantitative methylation-specific polymerase chain reaction (qMSP) was utilized to measure PAX1m, expressed as ΔCp. Univariate and multivariate regression analyses were conducted to screen variables and select predictive factors. A nomogram was constructed using multivariate logistic regression to predict additional HSIL + detected by ECC. The discrimination, calibration, and clinical utility of the nomogram were evaluated using receiver operating characteristic curves (ROC) and the calibration plot. RESULTS: Age (odds ratio [OR], 5.654; 95% confidence interval [CI], 1.131-37.700), cytology (OR, 24.978; 95% CI, 3.085-540.236), and PAX1 methylation levels by grade (PAX1m grade) (OR, 7.801; 95% CI, 1.548-44.828) were independent predictive factors for additional detection of HSIL + by ECC. In HPV16/18-positive women, the likelihood of additional detection of HSIL + through ECC increased with the severity of cytological abnormalities, peaking at 43.8% for high-grade cytological lesions. Moreover, when cytological findings indicated low-grade lesions, PAX1 methylation levels were positively correlated with the additional detection of HSIL + by ECC (P value < 0.001). A nomogram prediction model was developed (area under curve (AUC) = 0.946; 95% CI, 0.901-0.991), demonstrating high sensitivity (90.9%) and specificity (90.5%) at the optimal cutoff point of 107. Calibration analysis confirmed the model's strong agreement between predicted and observed probabilities. CONCLUSION: The clinical nomogram presented promising predictive performance for the additional detection of HSIL + through ECC among women with HPV16/18 infection. PAX1 methylation level could serve as a valuable tool in guiding individualized clinical decisions regarding ECC for patients with HPV 16/18 infection, particularly in cases of low-grade cytological findings.

Impact of CD44 genetic variants on clinicopathological characteristics of uterine cervical cancer patients.

CD44 genetic variants have been found to be related to various cancers. However, to date, no study has demonstrated the involvement of CD44 polymorphisms in uterine cervical cancer in Taiwanese women. Therefore, we conducted a retrospective study, consecutively recruiting 113 patients with invasive cancer, 92 patients with high-grade cervical intraepithelial neoplasias, and 302 control women to assess the relationships among CD44 polymorphisms, cervical carcinogenesis, and patient survival. Real-time polymerase chain reaction was used to determine the genotypic distributions of six polymorphisms: rs1425802, rs187115, rs713330, rs11821102, rs10836347, and rs13347. The results revealed that women with the mutant homozygous genotype CC exhibited a higher risk of invasive cancer compared to those with the wild homozygous genotype TT [p=0.035; hazard ratio (HR)=10.29, 95% confidence interval (95% CI)=1.18-89.40] and TT/TC [p=0.032; HR=10.66, 95% CI=1.23-92.11] in the CD44 polymorphism rs713330. No significant association was found between CD44 genetic variants and clinicopathological parameters. Among the clinicopathological parameters, only positive pelvic lymph node metastasis (p=0.002; HR=8.57, 95% CI=2.14-34.38) and the AG/GG genotype compared to AA (p=0.014; HR=3.30, 95% CI=1.28-8.49) in CD44 polymorphism rs187115 predicted a higher risk of poor five-year survival, according to multivariate analysis. In conclusion, an important and novel finding revealed that Taiwanese women with the AG/GG genotype in CD44 polymorphism rs187115 exhibited a higher risk of poor five-year survival.

TPP1 is associated with risk of advanced precursors and cervical cancer survival.

It is unclear how telomere-binding protein TPP1 interacts with human telomerase reverse transcriptase (hTERT) and influences cervical cancer development and progression. This study included all eligible 156 cervical cancers diagnosed during 2003-2008 and followed up through 2014, 102 cervical intraepithelial neoplasia (CIN) patients, and 16 participants with normal cervix identified at the same period. Correlation of expression of TPP1 and hTERT in these lesions was assessed using Kappa statistics. TPP1 was knocked down by siRNA in three cervical cancer cell lines. We assessed mRNA expression using quantitative real-time polymerase chain reaction and protein expression using tissue microarray-based immunohistochemical staining. We further analyzed the impact of TPP1 expression on the overall survival of cervical cancer patients by calculating the hazard ratio (HR) with 95% confidence intervals (CIs) using the multivariable-adjusted Cox regression model. Compared to the normal cervix, high TPP1expression was significantly associated with CIN 3 and cervical cancers (P<0.001 for both). Expressions of TPP1 and hTERT were highly correlated in CIN 3 (Kappa statistics = 0.50, P = 0.005), squamous cell carcinoma (Kappa statistics = 0.22, P = 0.011), and adenocarcinoma/adenosquamous carcinoma (Kappa statistics = 0.77, P = 0.001). Mechanistically, knockdown of TPP1 inhibited the expression of hTERT in both mRNA and protein levels. High expression of TPP1 (HR = 2.61, 95% CI 1.23-5.51) and co-high expression of TPP1 and hTERT (HR = 2.38, 95% CI 1.28-4.43) were independently associated with worse survival in cervical cancer patients. TPP1 and hTERT expression was correlated and high expression of TPP1 was associated with high risk of CIN 3 and cervical cancer and could predict a worse survival in cervical cancer.

Comparison of HPV-positive triage strategies combining extended genotyping with cytology or p16/ki67 dual staining in the Italian NTCC2 study.

BACKGROUND: Each high-risk HPV genotype has different oncogenic potential, and the risk of CIN3+ varies according to genotype. We evaluated the performance of different strategies of HPV-positivity triage combining cytology, p16/ki67 dual staining (DS), and extended genotyping. METHODS: Samples from 3180 consecutive women from the NTCC2 study (NCT01837693) positive for HPV DNA at primary screening, were retrospectively analyzed by the BD Onclarity HPV Assay, which allows extended genotyping. Genotypes were divided into three groups based on the risk of CIN3+. HPV DNA-positive women were followed up for 24 months or to clearance. FINDINGS: Combining the three groups of genotypes with cytology or DS results we identify a group of women who need immediate colposcopy (PPV for CIN3+ from 7.8 to 20.1%), a group that can be referred to 1-year HPV retesting (PPV in those HPV-positive at retesting from 2.2 to 3.8), and a group with a very low 24-month CIN3+ risk, i.e. 0.4%, composed by women cytology or DS negative and positive for HPV 56/59/66 or 35/39/68 or negative with the Onclarity test, who can be referred to 3-year retesting. INTERPRETATION: Among the baseline HPV DNA positive/cytology or DS negative women, the extended genotyping allows to stratify for risk of CIN3+, and to identify a group of women with a risk of CIN3+ so low in the next 24 months that they could be referred to a new screening round after 3 years. FUNDING: Italian Ministry of Health (grant number RF-2009-1536040). Hologic-Genprobe, Roche Diagnostics, and Becton & Dickinson provided financial and non-financial support.

[A multicenter study on the accuracy of PAX1/JAM3 dual genes methylation testing for screening cervical cancer].

Objective: To explore the value of cervical cytologic DNA methylation for screening cervical cancer. Methods: This study was a prospective multicenter study conducted from May to October 2022 in Peking Union Medical College Hospital, Zhejiang Provincial People's Hospital, and the Second Affiliated Hospital of Zhejiang University School of Medicine. Women who accepted opportunistic cervical cancer screening in gynecological outpatient clinics were subjected to liquid-based thin-layer cytology testing (TCT), high-risk human papillomavirus (hrHPV) DNA testing and PAX1/JAM3 dual-genes methylation testing (PAX1m/JAM3m). Colposcopy evaluation and biopsy were offered to women according to current guidelines. The accuracies of various testing methods and their combinations were compared based on histological diagnosis. Results: A total of 1 184 samples diagnosed by histopathology were included in this study, consisting of 541 cases (45.7%) of benign cervical tissue or chronic cervicitis, 273 (23.1%) of cervical intraepithelial neoplasia (CIN) 1, 168 (14.2%) of CIN2, 140 (11.8%) of CIN3, and 62 (5.2%) of cervical cancer. The sensitivity and specificity of PAX1m/JAM3m testing for detecting CIN2 or more severe lesions (CIN2+) were 74.1% and 95.9%, respectively. The sensitivity and specificity of PAX1m/JAM3m testing for detecting CIN3+were 87.6% and 86.8%, respectively. Receiver operating characteristic curve analysis showed that, for detecting CIN3+, the area under curve of PAX1m/JAM3m testing (0.872, 95%CI: 0.847-0.897) was significantly superior to TCT testing (0.580, 95%CI: 0.551-0.610) or hrHPV testing (0.503, 95%CI: 0.479-0.515) (all P values<0.05). Conclusions: The PAX1m/JAM3m test in cervical exfoliated cells has excellent accuracy for the diagnosis of both CIN2+and CIN3+, which is superior to traditional screening protocols and screening strategies.

Analysis of the diagnostic performance of PAX1/SOX1 gene methylation in cervical precancerous lesions and its role in triage diagnosis.

Methylation panels, tools for investigating epigenetic changes associated with diseases like cancer, can identify DNA methylation patterns indicative of disease, providing diagnostic or prognostic insights. However, the application of methylation panels focusing on the sex-determining region Y-box 1 (SOX1) and paired box gene 1 (PAX1) genes for diagnosing cervical lesions is under-researched. This study aims to examine the diagnostic performance of PAX1/SOX1 gene methylation as a marker for cervical precancerous lesions and its potential application in triage diagnosis. From September 2022 to April 2023, 181 patients with abnormal HPV-DNA tests or cytological exam results requiring colposcopy were studied at Hubei Maternal and Child Health Hospital, China. Data were collected from colposcopy, cytology, HPV-DNA tests, and PAX1/SOX1 methylation detection. Patients were categorized as control, cervical intraepithelial neoplasia Grade 1 (CIN1), Grade 2 (CIN2), Grade 3 (CIN3), and cervical cancer (CC) groups based on histopathology. We performed HPV testing, liquid-based cytology, and PAX1/SOX1 gene methylation testing. We evaluated the diagnostic value of methylation detection in cervical cancer using DNA methylation positivity rate, sensitivity, specificity, and area under the curve (AUC), and explored its potential for triage diagnosis. PAX1/SOX1 methylation positivity rates were: control 17.1%, CIN1 22.5%, CIN2 100.0%, CIN3 90.0%, and CC 100.0%. The AUC values for PAX1 gene methylation detection in diagnosing CIN1+, CIN2+, and CIN3+ were 0.52 (95% confidence interval [CI]: 0.43-0.62), 0.88 (95% CI: 0.80-0.97), and 0.88 (95% CI: 0.75-1.00), respectively. Corresponding AUC values for SOX1 gene methylation detection were 0.47 (95% CI: 0.40-0.58), 0.80 (95% CI: 0.68-0.93), and 0.92 (95% CI: 0.811-1.00), respectively. In HPV16/18-negative patients, methylation detection showed sensitivity of 32.4% and specificity of 83.7% for CIN1+. For CIN2+ and CIN3+, sensitivity was all 100%, with specificities of 83.0% and 81.1%. Among the patients who underwent colposcopy examination, 166 cases had cytological examination results ≤ASCUS, of which 37 cases were positive for methylation, and the colposcopy referral rate was 22.29%. PAX1/SOX1 gene methylation detection exhibits strong diagnostic efficacy for cervical precancerous lesions and holds significant value in triage diagnosis.

PAX1 and SOX1 Gene Methylation as a Detection and Triage Method for Cervical Intraepithelial Neoplasia Diagnosis.

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.

The importance of combined HPV and CINtec® PLUS genotyping testing for p16 in women with cervical squamous cell carcinoma.

INTRODUCTION: Immunohistochemistry (IHC) for p16INK4A (p16) is a reliable surrogate test for the presence of a high-risk, potentially transformative human papillomavirus (HPV) infection in precursor and malignant lesions of the cervix. The purpose of this study was to evaluate changes in cervical cells caused by persistent HPV infection, by IHC (p16 protein) by comparison with HPV genotyping. PATIENTS, MATERIALS AND METHODS: The study included female patients aged between 26 and 57 years who presented to a public hospital, with complaints related to the genital area, namely vaginal bleeding and dyspareunia. After selecting the patients, samples were subjected to cytological testing and IHC for p16 and for the determination of HPV types. RESULTS: The relationship between HPV status and p16 status was statistically significant (p=0.0001), of the 41 patients, 53.7% were HPV positive, respectively 56.1% were p16 positive, the agreement relationship between the two indicators was very high (weighted kappa: 0.951). The clinical performance of CINtec® PLUS triage for p16 shows a high positive predictive value (PPV) and a high negative predictive value (NPV) of 95.7% and 100%, respectively, as regards HPV. CONCLUSIONS: The p16 marker (CINtec® PLUS) can be used as a prognostic biomarker and provides clinical usefulness through increased sensitivity (Se) and specificity (Sp) in the triage of women at risk of developing precancerous lesions, compared to cytology that is based on morphology, but has a rather low Se and high Sp, while HPV testing is very sensitive but slightly more specific.

YAP/TAZ-TEAD activity promotes the malignant transformation of cervical intraepithelial neoplasia through enhancing the characteristics and Warburg effect of cancer stem cells.

A number of studies have confirmed that Yes-associated protein (YAP)/transcriptional co-activator with PDZ-binding motif (TAZ)-transcriptional enhanced associate domain (TEAD) activity is the driver of cancer development. However, the role and mechanism of the YAP/TAZ-TEAD pathway in cervical intraepithelial neoplasia (CIN) remain to be clarified. Therefore, this study was designed to observe the effect of YAP/TAZ-TEAD activity on the development of CIN and provide new ideas for the diagnosis and treatment of CIN. Firstly, cervical tissues were collected from CIN patients in different stages [CIN grade 1 (CIN1) tissue, CIN grade 2/3 (CIN 2/3) and squamous cell carcinoma (SCC)] and healthy volunteers. Next, the expression levels of YAP, TAZ and TEAD in cervical tissues and cells were observed by immunohistochemistry, qRT-PCR and western blot. Besides, Z172 and Z183 cells were transfected with siRNA-YAP/TAZ (si-YAP/TAZ) and YAP/TAZ overexpression vector (YAP-5SA). Also, Z172 cells were co-transfected with YAP-5SA and si-TEAD2/4. Subsequently, the stemness characteristics, glycolysis level and malignant transformation of cells in each group were observed by sphere-formation assay, commercial kit, MTT, Transwell, scratch experiment, xenotransplantation and western blot.The expression of YAP, TAZ and TEAD increased significantly in cervical cancer tissue and cell line at the stage of CIN2/3 and SCC. When YAP/TAZ was knocked down, the stemness characteristics, glycolysis level and malignant transformation of cancer cells were notably inhibited; while activating YAP/TAZ exhibited a completely opposite result. In addition, activating YAP/TAZ and knocking down the TEAD expression at the same time significant weakened the effect of activated YAP/TAZ signal on precancerous cells and reduced inhibitory effect of knocking down TEAD alone. YAP/TAZ-TEAD signal activates the characteristics and Warburg effect of cancer stem cells, thereby promoting the malignant transformation of CIN.

Long-term prediction by DNA methylation of high-grade cervical intraepithelial neoplasia: Results of the ARTISTIC cohort.

Methylation markers have shown potential for triaging high-risk HPV-positive (hrHPV+) women to identify those at increased risk of invasive cervical cancer (ICC). Our aim was to assess the performance of the S5 DNA methylation classifier for predicting incident high-grade cervical intraepithelial neoplasia (CIN) and ICC among hrHPV+ women in the ARTISTIC screening trial cohort. The S5 classifier, comprising target regions of tumour suppressor gene EPB41L3 and L1 and L2 regions of HPV16, HPV18, HPV31, and HPV33, was assayed by pyrosequencing in archived hrHPV+ liquid-based samples from 343 women with high-grade disease (139 CIN2, 186 CIN3, and 18 ICC) compared to 800 hrHPV+ controls. S5 DNA methylation correlated directly with increasing severity of disease and inversely with lead time to diagnosis. S5 could discriminate between hrHPV+ women who developed CIN3 or ICC and hrHPV+ controls (p <.0001) using samples taken on average 5 years before diagnosis. This relationship was independent of cytology at baseline. The S5 test showed much higher sensitivity than HPV16/18 genotyping for identifying prevalent CIN3 (93% vs. 61%, p = .01) but lower specificity (50% vs. 66%, p <.0001). The S5 classifier identified most women at high risk of developing precancer and missed very few prevalent advanced lesions thus appearing to be an objective test for triage of hrHPV+ women. The combination of methylation of host and HPV genes enables S5 to combine the predictive power of methylation with HPV genotyping to identify hrHPV-positive women who are at highest risk of developing CIN3 and ICC in the future.

PCDHGB7 hypermethylation-based Cervical cancer Methylation (CerMe) detection for the triage of high-risk human papillomavirus-positive women: a prospective cohort study.

BACKGROUND: Implementation of high-risk human papillomavirus (hrHPV) screening has greatly reduced the incidence and mortality of cervical cancer. However, a triage strategy that is effective, noninvasive, and independent from the subjective interpretation of pathologists is urgently required to decrease unnecessary colposcopy referrals in hrHPV-positive women. METHODS: A total of 3251 hrHPV-positive women aged 30-82 years (median = 41 years) from International Peace Maternity and Child Health Hospital were included in the training set (n = 2116) and the validation set (n = 1135) to establish Cervical cancer Methylation (CerMe) detection. The performance of CerMe as a triage for hrHPV-positive women was evaluated. RESULTS: CerMe detection efficiently distinguished cervical intraepithelial neoplasia grade 2 or worse (CIN2 +) from cervical intraepithelial neoplasia grade 1 or normal (CIN1 -) women with excellent sensitivity of 82.4% (95% CI = 72.6 ~ 89.8%) and specificity of 91.1% (95% CI = 89.2 ~ 92.7%). Importantly, CerMe showed improved specificity (92.1% vs. 74.9%) in other 12 hrHPV type-positive women as well as superior sensitivity (80.8% vs. 61.5%) and specificity (88.9% vs. 75.3%) in HPV16/18 type-positive women compared with cytology testing. CerMe performed well in the triage of hrHPV-positive women with ASC-US (sensitivity = 74.4%, specificity = 87.5%) or LSIL cytology (sensitivity = 84.4%, specificity = 83.9%). CONCLUSIONS: PCDHGB7 hypermethylation-based CerMe detection can be used as a triage strategy for hrHPV-positive women to reduce unnecessary over-referrals. TRIAL REGISTRATION: ChiCTR2100048972. Registered on 19 July 2021.

Association of HLA class I and II genes with cervical cancer susceptibility in a Han Chinese population.

Cervical cancer (CC) is one of the leading causes of cancer-related death in females worldwide. Genome-wide association studies (GWASs) have identified CC-related susceptibility loci in HLA regions. To investigate the associations between HLA genes and cervical intraepithelial neoplasia (CIN) or cervical cancer (CC), six loci of HLA class I (HLA-A, -B, and -C) and II (HLA-DRB1, -DPB1, and -DQB1) were selected for genotyping, and the associations between these alleles or their haplotypes with CIN or CC risk or protection from disease were evaluated. In total, 2193 participants, including 909 healthy individuals in the control group, 769 patients with CC, and 515 patients with CIN2+ (CIN II and III), were enrolled in the current study. HLA genes were genotyped using the NGSgo Illumina MiSeq workflow, and the associations between these loci and CIN2+ or CC at the allele and haplotype levels were analyzed. The allele frequencies of HLA-A*33:03, B*58:01, C*03:02, DPB1*05:01, and DRB1*12:01 were lower in both the CC and CIN2+ groups than in the control group, whereas those of B*55:02, C*04:03, and DPB1*03:01 were higher in the CC group than in the control group. In the histologic CC type analysis, the differences in the frequencies of these alleles in squamous cell carcinoma (SCC) of the cervix and stage I CC showed a consistent trend. In the haplotype analysis, the frequency of A*33:03-C*03:02-B*58:01 was lower in the CC and CIN2+ groups than in the control group, and that of A*24:02-C*04:03-B*15:25 was higher in the CC group than in both the control and CIN2+ groups. These three different haplotype frequencies were also identified in the FIGO CC stage analysis. In addition, in human papilloma virus (HPV) genotype analyses, the frequencies of HLA-C*03:02 and DPB1*05:01 were significantly lower in the CC and CIN2+ groups than in the control group, and in SCC subgroup, the frequencies of HLA-DQB1*04:01 and DRB1*04:05 were higher in the HPV other genotype infection group than in the HPV16 infection group. In both HPV16 single infection and coinfection with other HPVs, the frequency of haplotype A*33:03-C*03:02-B*58:01 was lower in both CC and CIN2+ than in the control group, while the frequencies of A*11:01-C*14:02-B*51:01 and A*24:02-C*03:04-B*13:01 were higher in the CIN2+ than in CC and the control group. In the HPV16 and other HPV infection comparisons, the frequencies of DRB1*04:05-DQB1*04:01-DPB1*02:01 and DRB1*11:01-DQB1*03:01-DPB1*05:01 were lower in the HPV16 infection group than in the other HPV infection group. Our results suggest that the HLA class I and II genes may affect the risk of CIN and CC as well as the histologic CC types and FIGO stages of CC in the Han Chinese population. In addition, HLA genes were associated with HPV16 infection at both the allelic and haplotype levels.

Folate deficiency modifies the risk of CIN3+ associated with DNA methylation levels: a nested case-control study from the ASCUS-COL trial.

PURPOSE: To our knowledge, there are very few studies evaluating if the levels of folate modify the risk of cervical intraepithelial neoplasia grade 2 and higher (CIN2+ and CIN3+) associated with the levels of HPV genome methylation, two cofactors related to single carbon metabolism and independently associated with cervical cancer in previous studies. We conducted a case-control study nested in a three-arm randomized clinical pragmatic trial (ASCUS-COL trial) to evaluate the risk of CIN3+ associated with methylation levels according to serum folate concentrations. METHODS: Cases (n = 155) were women with histologically confirmed CIN2+ (113 CIN2, 38 CIN3, and 4 SCC) and controls were age and follow-up time at diagnosis-matched women with histologically confirmed ≤ CIN1 (n = 155), selected from the 1122 hrHPV + women of this trial. The concentrations of serum folate were determined by the radioimmunoassay SimulTRAC-SNB-VitaminB12/Folate-RIAKit and the methylation levels by the S5 classifier. Stepwise logistic regression models were used to estimate the association between folate or methylation levels and CIN2+ or CIN3+. The joint effect of folate levels and methylation on the risk of CIN3+ was estimated using combinations of categorical stratifications. RESULTS: Folate levels were significantly lower in women with CIN3+ than in other diagnostic groups (p = 0.019). The risk of CIN3+ was eight times higher (OR 8.9, 95% CI 3.4-24.9) in women with folate deficiency and high methylation levels than in women with normal folate and high methylation levels (OR 1.4, 95% CI 0.4-4.6). CONCLUSION: High methylation and deficient folate independently increased the risk of CIN3+ while deficient folate combined with high methylation was associated with a substantially elevated risk of CIN3+.

Cervicovaginal microbiota disorder combined with the change of cytosine phosphate guanine motif- toll like receptor 9 axis was associated with cervical cancerization.

BACKGROUND: Convincing studies demonstrated that cervicovaginal microbiota disorder and toll-like receptor 9 (TLR9) high expression were related to cervical carcinogenesis. However, the effects of cervicovaginal microbiota integration TLR9 in cervical cancerization are unclear. Based on the biological basis that unmethylated cytosine-phosphate-guanine (CpG) motifs of bacteria could activate TLR9, we explored the effects of cervicovaginal microbiota disorder and CpG motif-TLR9 axis change in cervical carcinogenesis. METHODS: A total of 341 participants, including 124 normal cervical (NC), 90 low-grade cervical intraepithelial neoplasia (CIN1), 78 high-grade cervical intraepithelial neoplasia (CIN2/3) and 49 squamous cervical cancer (SCC), diagnosed by pathology were enrolled in the study. Here, metagenomic shotgun sequencing was used to reveal cervicovaginal microbiota characteristics, and TLR9 protein was detected by western blotting. RESULTS: Our results showed that the diversity of cervicovaginal microbiota gradually increased along with the poor development of cervical lesions, showing the abundance of Lactobacillus crispatus and Lactobacillus iners decreased, while the abundance of pathogenic bacteria gradually increased. The level of TLR9 expression was gradually increased with cervicovaginal microbiota diversity increasing, the abundance of Lactobacillus decreasing, and we found a positive correlation dependency relationship (r = 0.384, P = 0.002) between TLR9 and GTCGTT motif content. Stratified analysis based on HPV16 infection, we found that the characteristics of cervicovaginal microbiota and increased TLR9 expression were also closely related to HPV16 infection. CONCLUSIONS: Cervicovaginal microbiota dysbiosis might lead to the CpG motif increased, which was closely associated with TLR9 high expression, and ultimately might promote the progression of cervical lesions.

¿Está la virtud en el punto medio? La incertidumbre del CIN2 y el papel del VPH / Is virtue at the midpoint? The uncertainty of CIN2 and the role of HPV

Objetivo: La biopsia guiada por colposcopia (BGC) marca el manejo de la neoplasia intraepitelial cervical. El objetivo de este estudio fue evaluar la concordancia de los resultados entre la BGC y la escisión amplia de la zona de transformación (LLETZ, large loop excision of the transformation zone), y la utilidad del genotipado del virus del papiloma humano (VPH) para seleccionar a las pacientes con riesgo de lesión intraepitelial escamosa de alto grado/neoplasia intraepitelial cervical 3 (HSIL/CIN3). Método: Se compararon los resultados de la BGC y de la LLETZ, siendo esta última el método de referencia. Se evaluó la relación del genotipo del VPH con el diagnóstico final de HSIL/CIN3. Resultados: La precisión de la biopsia comparada con LLETZ fue del 61,4%. La tasa de concordancia fue del 64,4% para CIN1, del 31,4% para CIN2 y del 77,4% para CIN3. La tasa global de sobrediagnóstico fue del 18,68% y la de subdiagnóstico del 19,89%. En mujeres menores de 30 años, la concordancia fue del 62,79% (CIN1 65%, CIN2 39,58% y CIN3 73,08%), la tasa de sobrediagnóstico del 22,67% y la tasa de subdiagnóstico del 15,11%. La infección por VPH16 tuvo una odds ratio de 3,86 para el diagnóstico final de HSIL/CIN3+. Conclusiones: El diagnóstico de CIN2 por BGC parece insuficiente para seleccionar a las pacientes para tratamiento escisional, principalmente en mujeres jóvenes. El hallazgo de VPH16 es un factor de riesgo de HSIL/CIN3+ independientemente del resultado de la biopsia.

Objective: Colposcopy-guided biopsy (CGB) is a basic tool for the management of cervical intraepithelial neoplasia. The aim of this study is to evaluate the concordance of results between CGB and large loop excision of the transformation zone (LLETZ), and the usefulness of human papillomavirus (HPV) genotyping to select patients at risk of H-SIL/CIN3. Method: The results of colposcopy-guided biopsy and LLETZ were compared, with LLETZ being the gold standard. The relationship of HPV genotype to the final diagnosis of CIN3 was assessed. Results: The accuracy of CGB compared to LLETZ was 61.4%. The concordance rate was 64.4% for CIN1, 31.4% for CIN2 and 77.4% for CIN3. The overall overdiagnosis rate was 18.68% and underdiagnosis rate was 19.89%. In women under 30 years of age the concordance rate was 62.79% (CIN1 65%, CIN2 39.58% and CIN3 73.08%), and the rate of overdiagnosis and underdiagnosis was 22.67% and 15.11%, respectively. HPV16 infection had an odds ratio of 3.86 for the final diagnosis of CIN3+ and the result was significant regardless of the biopsy result. Conclusions: The CGB result as CIN2 is inaccurate and seems insufficient to select patients for excisional treatment, mainly in young women. HPV16 infection is a risk factor for CIN3+ regardless of the colposcopy-guided biopsy result.

Identification of a methylation panel as an alternative triage to detect CIN3+ in hrHPV-positive self-samples from the population-based cervical cancer screening programme.

BACKGROUND: The Dutch population-based cervical cancer screening programme (PBS) consists of primary high-risk human papilloma virus (hrHPV) testing with cytology as triage test. In addition to cervical scraping by a general practitioner (GP), women are offered self-sampling to increase participation. Because cytological examination on self-sampled material is not feasible, collection of cervical samples from hrHPV-positive women by a GP is required. This study aims to design a methylation marker panel to detect CIN3 or worse (CIN3+) in hrHPV-positive self-samples from the Dutch PBS as an alternative triage test for cytology. METHODS: Fifteen individual host DNA methylation markers with high sensitivity and specificity for CIN3+ were selected from literature and analysed using quantitative methylation-specific PCR (QMSP) on DNA from hrHPV-positive self-samples from 208 women with CIN2 or less (< CIN2) and 96 women with CIN3+. Diagnostic performance was determined by area under the curve (AUC) of receiver operating characteristic (ROC) analysis. Self-samples were divided into a train and test set. Hierarchical clustering analysis to identify input methylation markers, followed by model-based recursive partitioning and robustness analysis to construct a predictive model, was applied to design the best marker panel. RESULTS: QMSP analysis of the 15 individual methylation markers showed discriminative DNA methylation levels between < CIN2 and CIN3+ for all markers (p < 0.05). The diagnostic performance analysis for CIN3+ showed an AUC of ≥ 0.7 (p < 0.001) for nine markers. Hierarchical clustering analysis resulted in seven clusters with methylation markers with similar methylation patterns (Spearman correlation> 0.5). Decision tree modeling revealed the best and most robust panel to contain ANKRD18CP, LHX8 and EPB41L3 with an AUC of 0.83 in the training set and 0.84 in the test set. Sensitivity to detect CIN3+ was 82% in the training set and 84% in the test set, with a specificity of 74% and 71%, respectively. Furthermore, all cancer cases (n = 5) were identified. CONCLUSION: The combination of ANKRD18CP, LHX8 and EPB41L3 revealed good diagnostic performance in real-life self-sampled material. This panel shows clinical applicability to replace cytology in women using self-sampling in the Dutch PBS programme and avoids the extra GP visit after a hrHPV-positive self-sampling test.

NOVAprep-miR-Cervix: New Method for Evaluation of Cervical Dysplasia Severity Based on Analysis of Six miRNAs.

Cervical cancer is one of the most common gynecological malignancies and it is preventable through the yearly diagnosis and management of pre-cancerous cervical disease. The profile of miRNA expression in cervical epithelium cells is altered with cervical dysplasia development and further progression. The NOVAprep-miR-CERVIX is a new approach for the assessment of cervical dysplasia through the analysis of six marker miRNAs. This study aims to evaluate theperformance and diagnostic potency of the new method. Cytological smears from 226 women (NILM, n.114; HSIL, n.112) were included in the study. A VPH test was performed with RealBest DNAHPV HR screen Kit, six marker miRNAs (miR-21, -29b, -145, -451a, -1246, -1290) were assayed using NOVAprep-miR-CERVIX kit. Obtained data were analyzed using the Delta Ct method and random forest machine learning algorithm. The results of the quantitative analysis of six microRNAs were expressed as a miR-CERVIX parameter, which ranged from 0 to 1, where "0" corresponded to the healthy cervical epithelium, while "1" corresponded to high-grade squamous intraepithelial dysplasia. The average value of miR-CERVIX differed in groups of NILM and HSIL samples (0.34 vs. 0.72; p < 0.000005). An estimation of miR-CERVIX allowed for the differentiation between healthy and pre-cancerous samples with sensitivity of 0.79 and specificity of 0.79, as well as to confirm HSIL with specificity of 0.98. Interestingly, the HSIL group included HPV(+) and HPV(-) samples, which were statistically significantly different in terms of miR-CERVIX value. Analysis of CC-associated miRNAs in material of cervical smear might serve as an additional method for the evaluation of cervical dysplasia severity.