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Optimization of RNAscope for HPV mRNA detection in liquid-based cervical cytology samples
Tianqi Liu1, Xianrong Zhou1, Xiang Tao1
1Department of Pathology, Obstetrics & Gynecology Hospital of Fudan University, Shanghai Key Lab of Reproduction and Development, Shanghai Key Lab of Female Reproductive Endocrine Related Diseases, Shanghai, China.
Frontiers in Oncology
|March 27, 2026
Summary
This study adapted RNAscope technology for liquid-based cytology (LBC) slides, enabling simultaneous detection of cervical cell phenotype and HPV E6/E7 mRNA. The novel method shows high accuracy for diagnosing cervical lesions.
Area of Science:
- Gynecologic Oncology
- Molecular Pathology
- Cytopathology
Background:
- Cervical cancer diagnosis relies on cytological and molecular methods.
- Simultaneous detection of cytological phenotype and human papillomavirus (HPV) E6/E7 mRNA in liquid-based cytology (LBC) slides is challenging.
- Existing RNAscope technology is primarily optimized for paraffin-embedded tissues.
Purpose of the Study:
- To develop and validate an RNAscope-based assay for simultaneous detection of cytological phenotype and HPV E6/E7 mRNA expression in cervical LBC samples.
- To establish a joint detection scheme compatible with LBC slides and automated staining systems.
- To enable precise diagnosis of cervical precancerous lesions and cancer.
Main Methods:
- Adaptation of RNAscope in situ hybridization for LBC slides, including modifications to slide type, target retrieval, and permeabilization.
- Collection and histopathological diagnosis of 115 LBC samples, categorized into LSIL, HSIL, HPV-related SCC, HPV-related adenocarcinoma, and negative cases.
- Statistical analysis of positive rates, diagnostic concordance with histopathology, and HPV PCR testing, alongside visualization of expression patterns.
Main Results:
- The adapted RNAscope assay demonstrated high positive rates across various cervical lesions: 93.33% for LSIL, 96.97% for HSIL, 100.00% for SCC, and 87.50% for adenocarcinoma.
- The diagnostic concordance rate between RNAscope and HPV PCR testing reached 95.65% (P<0.001).
- RNAscope successfully visualized distinct HPV E6/E7 mRNA expression patterns in different cervical lesions.
Conclusions:
- The adapted RNAscope technology provides an accurate and reliable method for diagnosing cervical lesions directly from LBC samples.
- This novel approach enhances diagnostic consistency with histopathology and offers a valuable tool for cervical cancer screening and management.
- Simultaneous detection of cytological and molecular markers improves the diagnostic capability for cervical intraepithelial neoplasia and cancer.
