# Optimization of RNAscope for HPV mRNA detection in liquid-based cervical cytology samples

## TL;DR

This study adapts RNAscope technology for liquid-based cervical cytology to detect HPV mRNA, improving diagnostic accuracy for cervical cancer and precancerous lesions.

## Contribution

The study adapts RNAscope for liquid-based cytology, enabling simultaneous cytological and HPV mRNA analysis.

## Key findings

- RNAscope in LBC showed high positive rates for HPV E6/E7 mRNA in cervical lesions.
- Diagnostic concordance with histopathology reached 95.65% compared to HPV PCR testing.
- Expression patterns were clearly visualized in LSIL, HSIL, SCC, and adenocarcinoma cases.

## Abstract

To establish an RNAscope-based joint detection scheme compatible with liquid-based cytology (LBC) slides and staining systems, enabling simultaneous observation of cytological phenotype and HPV E6/E7 mRNA expression in cervical cancer and precancerous lesions.

From March 2023 to February 2024, 115 LBC samples were collected at the Affiliated Obstetrics and Gynecology Hospital of Fudan University. Histopathological diagnosis included 30 LSIL, 33 HSIL, 29 HPV-related SCC, 8 HPV-related adenocarcinoma and 15 negative for cervical intraepithelial lesions cases. RNAscope technology, originally applied to paraffin samples, was adapted for LBC by modifying slide type selection, extending target retrieval, and enhancing permeabilization. The improved RNAscope in situ hybridization was then employed to detect high-risk HPV E6/E7 mRNA. Positive rates, diagnostic concordance with histopathology and, expression pattern were statistically analyzed.

RNAscope combined with LBC showed high positive rates, 93.33% for LSIL, 96.97% for HSIL, 100.00% for SCC, and 87.50% for HPV-related adenocarcinoma. Compared with HPV PCR testing, the diagnostic concordance rate reached 95.65% (P<0.001). Figures illustrate RNAscope positive expression patterns in LSIL, HSIL, SCC and, HPV-related adenocarcinoma.

The adaptation of RNAscope technology, originally designed for paraffin samples, enables accurate diagnosis in cervical LBC, enhances consistency with histopathology, and provides a novel diagnostic approach for cervical lesions.