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

## TL;DR

Adding an HPV mRNA test to routine Pap tests helps better identify women at higher risk of cervical cancer in everyday clinical practice.

## Contribution

This study demonstrates the clinical utility of HPV16/18/45 mRNA co-testing with Pap tests for risk stratification in routine care.

## Key findings

- Double-negative co-test results (normal Pap and negative HPV mRNA) were associated with very low risks of cervical cancer.
- Women with normal Pap but positive HPV mRNA had significantly higher risks of cervical cancer.
- HPV16 posed the highest risk, followed by HPV18 and HPV45.

## Abstract

We evaluated whether adding a targeted HPV mRNA test to routine Pap testing could better identify women at increased risk of serious cervical precancer or cervical cancer in everyday clinical practice. The test detects activity from three HPV types (16, 18, and 45) strongly linked to cervical cancer. Using routine data from Northern Norway, we analyzed more than 116,000 combined Pap and HPV mRNA tests and linked them to later biopsy outcomes. Combined testing separated women into clear risk groups: risk was very low when both tests were negative and increased when one or both tests were positive. Women with a normal Pap test but a positive HPV mRNA test had a clearly higher later risk of cervical cancer than women with both tests negative, identifying a small but clinically important risk group. These findings support HPV mRNA testing as an adjunct to cytology for risk-based follow-up.

Background/Objectives: 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 E6/E7 mRNA assay targeting HPV16, HPV18, and HPV45 (PreTect SEE) in routine clinical practice. Methods: 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. Results: 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. Conclusions: 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.