Development of a novel, urine-based high-risk human papillomavirus polymerase chain reaction test to predict cervical intraepithelial neoplasia abnormalities associated with cervical cancer

This study evaluated and optimized a novel urine-based workflow using high-risk human papillomavirus (hrHPV) polymerase chain reaction (PCR) test to detect 14 hrHPV genotypes linked to cervical intraepithelial neoplasia (CIN) abnormalities for cervical cancer screening. In cohort 1 for clinical feasibility, 82 women with confirmed CIN2+ abnormalities provided paired urine and cervical brush samples before treatment. Urine was tested with the novel hrHPV workflow, and cervical brushes with the Cobas HPV Test (Roche). For Ct cut-off optimization in cohort 2, urine samples from 983 women in a primary screening, triage, and follow-up program were analyzed, optimizing CIN2+ and CIN3+ sensitivity and specificity by varying the 12-other hrHPV channel Ct cut-off from 28.0 to 38.0, while maintaining HPV16/18 Ct cut-offs at 38.0. In cohort 1, the urine hrHPV test showed 92.7% sensitivity for CIN2+ and 93.9% for CIN3+, exceeding cervical brush sensitivities of 78.1% and 90.9%, respectively. In cohort 2, the 12-other hrHPV Ct altered CIN2+ sensitivity from 76.6% to 93.6% and specificity from 83.1% to 75%. ROC analysis identified optimal cut-offs at 32.76 for CIN2+ and 30.47 for CIN3+. The proposed cut-offs are HPV16/18 at 38.0, and 12 other hrHPV at 33.0 for maximum sensitivity (93.62% CIN2+, 90.91% CIN3+) or at 30.5 for balanced performance (89.36% CIN2+ sensitivity, 80% specificity). The urine-based hrHPV test uses 40 mL of urine collected using a standard urine collection device, and demonstrated high sensitivity for CIN2+ prediction. Adjustable Ct thresholds allow tailored performance optimization, thereby enhancing accessibility, adherence, and early cervical cancer detection across diverse clinical settings.IMPORTANCEA novel urine-based high-risk human papillomavirus (hrHPV) polymerase chain reaction test was developed to detect 14 hrHPV genotypes, including individual HPV16 and HPV18 typing, associated with cervical cancer progression. By employing a liquid-liquid separation method to extract DNA from large-volume (40 mL) pot-collected urine samples, the test demonstrated sensitivities of 92.7% for cervical intraepithelial neoplasia grade 2 or higher (CIN2+) and 93.9% for CIN3+. Optimization of cycle threshold cut-offs yielded 93% sensitivity and 77% specificity for predicting CIN2+ in an enriched population. It can enhance accessibility, compliance, and early detection across diverse clinical settings.