Triage by PAX1 and ZNF582 methylation in women with cervical intraepithelial neoplasia grade 3: a multicenter case-control study

Background The colposcopy-conization inconsistency is common in women with cervical intraepithelial neoplasia grade 3 (CIN3). No adequate method has been reported to identify the final pathology of conization. In this study, we explored the ability of PAX1 and ZNF582 methylation to predict the pathological outcome of conization in advance. Methods This was a multicenter study and included 277 histologically confirmed CIN3 women who underwent cold knife conization (CKC) from January 2019 to December 2020. The methylation levels of PAX1 (PAX1m) and ZNF582 (ZNF582 m) were determined by quantitative methylation specific PCR (qMSP) and expressed in ΔCp. Receiver-operating characteristic (ROC) curves were used to evaluate predictive accuracy. Results The final pathological results in 48 (17.33%) patients were inflammation or low-grade squamous intraepithelial lesion (LSIL), 190 (68.59%) were high grade squamous intraepithelial lesion (HSIL) and 39 (14.08%) were squamous cervical cancer (SCC). PAX1 m and ZNF582 m increased as lesions progressed from inflammation/LSIL, HSIL to SCC. PAX1 and ZNF582 methylation yielded better prediction performance compared to common screening strategies, whether individually or combined. ΔCpZNF582 ≥19.18). A 6.53-fold increase in SCC risk was observed in patients with elevated ZNF582 methylation (ΔCpZNF582 < 7.09). Conclusion DNA Methylation would be an alternative screening method to triage and predict the final outcome of conization of the CIN3 cases.

Proliferative activity of the epithelium in squamous epithelial lesions of the cervix

As a criterion for precancerous changes in the stratified squamous epithelium of the cervix, its proliferative activity, studied using monoclonal antibodies PC-10 to the antigen of proliferating cell nuclei (PCNA), is considered. The results of the studies showed that patients with a low degree of squamous intraepithelial lesion are characterized by weak proliferative activity, and for patients with a high degree of lesion it is moderate and pronounced. An increase in proliferative activity is a prognostic factor that determines long-term persistence and the likely progression of the lesion.

A multicentre, prospective, randomised controlled trial to evaluate hexaminolevulinate photodynamic therapy (Cevira®) as a novel treatment in patients with high grade squamous intraepithelial lesion: APRICITY Phase 3 study protocol

IntroductionHigh-risk human papilloma virus (HPV)-associated cervical cancer is the fourth most common cancer in women worldwide. Current treatments of high grade squamous intraepithelial lesion (HSIL) of the cervix are based on invasive surgical interventions, compromising cervical competence and functionality.ObjectiveAPRICITY is a multicentre, prospective, double-blind, randomised controlled Phase 3 study further evaluating the efficacy and safety of Cevira®, an integrated drug- and light-delivery device for hexaminolevulinate photodynamic therapy, which shows promise as a novel, non-invasive therapy for women with HSIL.Methods and analysisPatients with biopsy-confirmed HSIL histology are invited to participate in the study currently being conducted at 47 sites in China and 25 sites in Ukraine, Russia and European Union. The aim is to include at least 384 patients which will be randomised to either Cevira® or placebo group (2:1). All patients will be assessed 3 months after first treatment and a second treatment will be administered in patients who are HPV positive or have at least low grade squamous intraepithelial lesion (LSIL). Primary endpoint is the proportion of the responders at 6 months after first treatment. Secondary efficacy endpoints and safety endpoints will be assessed at 6 months, and data for secondary performance endpoints for Cevira® device will be collected at 3 months and 6 months, in case second treatment was administered. All patients in the Cevira® group will be enrolled in an open, long-term extension study following patients for further 6 months to collect additional efficacy and safety data (study extension endpoints).ConclusionDue to its non-invasiveness and convenient application, Cevira® may be a favourable alternative to surgical methods in treatment of patients with HSIL.Ethics and disseminationThe study was approved by the ethics committee of the Peking Union Medical College Hospital and Hannover Medical University, Germany. Findings will be disseminated through peer review publications and conference presentations.Trial registration numberclinicaltrials.govNCT04484415

Biomarker Expression in Multifocal Vulvar High-Grade Squamous Intraepithelial Lesions

In patients with high-grade squamous intraepithelial lesion (HSIL) of the vulva, the presence of multiple lesions, called multifocal HSIL, is common. The aim of this exploratory study was to investigate biomarker expression profiles in multifocal HSIL. In total, 27 lesions from 12 patients with high-risk human papillomavirus (HPV)-positive multifocal HSIL were tested for HPV genotype, expression of p16INK4a and Ki-67, and DNA methylation of six genes. HPV16 was found most commonly in 21 (77.8%) HSILs. In two (16.4%) patients, HPV genotype differed between the lesions. All lesions demonstrated diffuse p16INK4a staining, of which three (11.1%) were combined with patchy staining. One patient (8.3%) demonstrated markedly different DNA methylation levels between lesions. Generally, heterogeneity in methylation profiles was observed between different patients, even when other biomarkers showed similar expression. In conclusion, this study is the first to demonstrate heterogeneity of individual lesions in patients with multifocal HSIL. The studied biomarkers have the potential to refine prognostic and predictive diagnostics. Future prospective, longitudinal studies are needed to further explore the potential of a biomarker profile for management of patients with multifocal HSIL.

The Expression of Oct3/4A mRNA and Not Its Isoforms is Upregulated by the HPV16 E7 Oncoprotein

Oct3/4 a transcription factor is involved in maintaining the characteristics of cancer stem cells. Oct3/4 can be expressed differentially with respect to the progression of CC. In addition, Oct3/4 can give rise to three isoforms by alternative splicing of the mRNA Oct3/4A, Oct3/4B and Oct3/4B1. The aim of this study was to evaluate the mRNA expression from Oct3/4A, Oct3/4B and Oct3/4B1 in low-grade squamous intraepithelial lesion (LSIL), high-grade squamous intraepithelial lesion (HSIL), cervical cancer (CC) samples, and measure the effect of the HPV16 E7 oncoprotein on the mRNA expression from Oct3/4 isoforms in the C-33 A cell line. The expression levels of Oct3/4A, Oct3/4B and Oct3/4B1 mRNA were analyzed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) in patients with LSILs, HSILs and CC. Additionally, C-33 A cells that expressed the HPV16 E7 oncoprotein were established to evaluate the effect of E7 on the expression of Oct3/4 mRNA isoforms. Oct3/4A (p=0.02), Oct3/4B (p=0. 001) and Oct3/4B1 (p<0. 0001) expression is significantly higher in patients with LSIL, HSIL and CC than in woman with non-IL. In the C-33 A cell line, the expression of Oct3/4A mRNA in the presence of the E7 oncoprotein increased compared to that in nontransfected C-33 A cells. Oct3/4B and Oct3/4B1 mRNA were expressed at similar levels among the different groups. These data indicate that only the mRNA of Oct3/4A is upregulated by the HPV16 E7 oncoprotein.