EVX2 methylation assay enables risk assessment of cervical cancer in high-risk HPV-infected individuals

BACKGROUND: Stratifying cancer risk in high-risk human papillomavirus (hr-HPV) infected individuals can effectively reduce colposcopy referrals and minimize invasive procedures. Current assay methods need to be improved and perfected. METHODS: Four methylation markers (ZNF135, EVX2, MARCHF11, and UBD) were identified to discriminate cervical cancer (CC) from normal mucosal tissues through the analysis of TCGA/GEO cohorts. Subsequently, a specific methylation marker (EVX2) was further identified via immunohistochemical (IHC) assays and an extensive literature review. After validating the applicability of this technique for EVX2 methylation detection via MALDI-TOF mass spectrometry, a methylation model was constructed based on the selected markers in a retrospective cohort of cervical exfoliated cells (n = 104) and validated in an independent cohort (n = 22) using the identical technique and random forest algorithm. The diagnostic performance was systematically compared with LCT and high-oncogenicity HPV16/18 testing. RESULTS: Four methylation markers (ZNF135, EVX2, MARCHF11, and UBD) were identified that could discriminate CC from normal squamous epithelia. Among these markers, EVX2 was further pinpointed as the key candidate based on gene expression and literature review. A diagnostic model constructed via EVX2 methylation status exhibited robust diagnostic performance in differentiating patients with squamous intraepithelial lesion (SIL) or CC from healthy normal controls, and its diagnostic efficacy was significantly superior to that of LCT and HPV16/18 assays (AUC: 0.95 vs. 0.85 for LCT and 0.57 for HPV16/18 testing). This superiority was more pronounced in detecting HSIL and CC, with a detection rate of 96.97% versus 57.14% (LCT) and 37.5% (HPV16/18 testing). Subsequent analyses of TCGA databases further validated the diagnostic value of EVX2 methylation in CC. CONCLUSIONS: EVX2 methylation detection via MALDI-TOF mass spectrometry reliably discriminates normal squamous epithelium from HSIL and above cervical malignant lesions, and may serve as a rapid, non-invasive, and promising tool for cancer-risk screening and triage in hr-HPV-positive patients.