Performance of ASLC1/LHX8 DNA methylation and extended HPV genotyping in first-void urine for high-grade cervical intraepithelial neoplasia detection in an HPV-positive referral population - a cross-sectional study

Background: First-void urine (FVU) collection for high-risk human papillomavirus (HPV) testing is a promising tool to reach un(der)-screened women in cervical cancer screening programs. This cross-sectional study investigated the clinical performance of host-cell DNA methylation markers ASCL1 and LHX8 in HPV-positive FVU to detect high-grade cervical intraepithelial neoplasia and cancer (CIN2+ and CIN3+). Secondly, comparative analysis to paired HPV-positive clinician-collected cervical samples (CS) and HPV genotyping was examined. Methods: Paired FVU and CS samples were collected from 286 women aged 23-64 years referred for colposcopy or cervical excision. Histological endpoints included 123 ≤CIN1 (no dysplasia and CIN1), 38 CIN2, and 123 CIN3/AIS and 2 cancers. FVU and CS were tested for HPV DNA and ASCL1 / LHX8 methylation. Methylation test performance was evaluated by area under the curve (AUC) and logistic regression analysis. Accuracy differences between paired samples and across methylation, HPV16/18, and extended 16/18/31/33/52 genotyping testing in FVU were tested using McNemar’s test. Results: ASCL1 and LHX8 methylation levels in HPV-positive FVU increased significantly with disease severity. Methylation testing yielded an AUC of 0.76 (95% CI: 0.70-0.82) for CIN3+ and 0.73 (95% CI: 0.67-0.79) for CIN2+, with corresponding sensitivities of 79.2% (95% CI: 71.0-85.9%) and 75.5% (95% CI: 68.1-81.9%) and a specificity of 57.0% (95% CI: 47.8-65.8%) for ≤CIN1. In CS, methylation testing yielded an AUC of 0.84 (95% CI: 0.79-0.89) for CIN3+ and 0.80 (95% CI: 0.75-0.85) for CIN2+, corresponding to significantly higher sensitivities (p≤0.02) but lower specificity (p=0.04) compared to FVU. In women aged ≥30 years, CIN3+ sensitivity and specificity for ≤CIN1 were similar between FVU and CS (both p=0.09). Methylation testing in HPV-positive FVU had similar diagnostic accuracy as extended genotyping (p≥0.35), but had a higher sensitivity (p=0.01) and a lower specificity (p=0.01) than HPV16/18 genotyping. Conclusions: ASCL1/LHX8 methylation testing in HPV-positive FVU showed promise for detecting high-grade cervical disease. ASCL1/LHX8 methylation performance in FVU was similar to extended HPV genotyping and, in women aged ≥30 years, similar to performance in CS. This supports the potential of methylation analysis as a direct and single triage test in urine-based cervical cancer screening, removing the need for follow-up cervical sampling. Trial registration: Clinicaltrials.gov: NCT05065853