Detection of HPV DNA in Cervical Intraepithelial Neoplasia Using In Situ Hybridization

Background: Human papillomavirus (HPV)-related diseases remain a major global health problem, with cervical intraepithelial neoplasia (CIN) representing a key precursor to cervical cancer. Identification of high-risk HPV genotypes is essential for early diagnosis and appropriate management. This study aimed to evaluate the usefulness of in situ hybridization (ISH) for detecting HPV DNA in formalin-fixed, paraffin-embedded (FFPE) cervical tissue and to compare automated signal detection with manual histopathological assessment. Methods: This prospective, non-randomized study included 83 women undergoing diagnostic procedures for abnormal cytology or confirmed CIN between 2022 and 2023. Tissue specimens obtained during a loop electrosurgical excision procedure (LEEP) were examined using two ISH probes: ISH II for low-risk HPV types 6 and 11, and ISH III for high-risk HPV genotypes. Staining patterns and distributions were evaluated and correlated with molecular HPV testing and histopathological outcomes. Results: ISH II distribution was significantly associated with the presence of HPV type 6 or 11 (p < 0.001), although stain structure itself was not. ISH III stain structure was significantly associated with high-risk HPV genotypes (p = 0.020). A positive ISH II result predicted low-risk HPV infection with a sensitivity of 62.5% and specificity of 64.0%, while ISH III predicted high-risk HPV infection with a sensitivity of 86.36% but lower specificity (23.53%). Overall diagnostic accuracy was 63.86% for ISH II and 73.49% for ISH III. Conclusions: ISH proved to be a reproducible method for detecting HPV in archived cervical tissue, enabling assessment even years after specimen collection. Although PCR-based methods remain more widely used due to higher sensitivity and less invasive sampling, ISH provides valuable morphological context and may serve as a complementary diagnostic tool, particularly when only archival tissue is available.