Cervical Cytology and HPV16/18/45 mRNA Co-Testing Improve Risk Stratification in Routine Clinical Practice

<b>Background/Objectives:</b> Co-testing may improve cervical cancer prevention by stratifying women into groups with different absolute risks of CIN2+, CIN3+, and cervical cancer. We evaluated real-world co-testing with cervical cytology and a genotype-specific HPV <i>E6/E7</i> mRNA assay targeting HPV16, HPV18, and HPV45 (PreTect SEE) in routine clinical practice. <b>Methods:</b> We conducted a retrospective, registry-based cohort study at the Department of Clinical Pathology, University Hospital of North Norway. Eligible co-test samples (liquid-based cytology with concurrent HPV mRNA testing, both with valid results) from routine screening, follow-up, and clinically indicated testing were identified from the laboratory information system and passively followed for worst histological outcome through December 2025. Outcomes were no biopsy/<CIN2, CIN2+, CIN3+, and cervical cancer. <b>Results:</b> Among 116,217 eligible co-test samples (mean age 43.9 years), cumulative risks were 4.4% for CIN2+, 1.5% for CIN3+, and 0.1% for cervical cancer. Baseline HPV mRNA positivity was 3.9%, and cytology was ASC-US+ in 12.2% of samples. Co-testing produced a clear stepwise risk gradient. Double-negative results (NILM/mRNA-; 86.7%) had very low risks (CIN3+ 0.2%; cervical cancer 0.02%). ASC-US+/mRNA- results (9.3%) showed intermediate risks (CIN3+ 4.1%; cervical cancer 0.2%). NILM/mRNA+ results (1.1%) showed substantially higher risks despite normal cytology (CIN3+ 13.0%; cervical cancer 0.5%). Double-positive results (ASC-US+/mRNA+; 2.8%) had the highest risks (CIN3+ 28.5%; cervical cancer 2.3%). Within NILM, mRNA positivity captured 42.7% of CIN3+ cases and 25.0% of cancers. Genotype-specific analyses showed highest risks for HPV16, followed by HPV18 and HPV45. <b>Conclusions:</b> Co-testing with cervical cytology and a 3-type HPV mRNA assay provided strong, clinically interpretable risk stratification and identified a small but high-risk subgroup among women with normal cytology. These findings support genotype-specific HPV mRNA testing as an adjunct to cytology in routine care.