The Expression of HPV E6/E7 mRNA in Situ Hybridization in HPV Typing-negative Cervical Cancer

Background: High-risk human papilloma virus (HR-HPV) persistent infection is the major tumorigenesis factor for cervical cancer (CC) and cervical intraepithelial neoplasia (CIN). However, the incidence of HPV-negative CC is approximately 5%-30% with different HPV detection methods. HR-HPV E6/E7 mRNA in situ hybridization (RISH) can detect HPV-driven tumours. Our study aimed to explore whether HPV typing-negative CC was caused by HPV infection.Methods: The records of CC patients with HPV typing results who had undergone cervical biopsies, cervical conization or hysterectomies were collected from April 2018 to September 2021 at the Affiliated Hospital of Weifang Medical University. RISH was detected using RNAscope chromogenicin in the tissues of patients with CC. Immunohistochemistry (IHC) was performed to evaluate the expression of p16INK4a and Ki67. CC patients with positive HPV typing in the same period were collected as controls.Results: A total of 308 women with HPV typing results were enrolled, and 30 (9.74%) cases of HPV typing were negative, including 22/30 (73.3%) cases of squamous cell carcinoma (SCC) and 8/30 (26.7%) cases of adenocarcinoma. In the CC patients who were HPV typing-negative, 28/30 (93.3%) were positive for RISH and p16INK4a block+ staining, which contained 22/22 (100%) SCCs and 6/8 (75%) adenocarcinomas. RISH was positive in 278/278 (100%) HPV typing-positive CCs, which included 232/232 (100%) SCCs and 46/46 (100%) adenocarcinomas. While 273/278 (98.2%) showed p16INK4a block+ staining, the five negative cases were SCC. Positive RISH in HPV typing-negative CC was significantly lower than in the HPV typing-positive group (P=0.002, 95% CI: 0.848-1.027). However, this significant difference only existed in adenocarcinoma, and positive RISH in HPV typing-negative SCC was the same as in the HPV typing-positive group. No significant differences were seen in the expression of p16INK4a and Ki67 (P=0.291, 95% CI: 0.863-1.047 and P=0.174, 95% CI: 0.905-1.033).Conclusions: HPV typing may cause misdiagnosis in 9.74% of CC patients, and HPV E6/E7 mRNA can detect the majority of CC with HPV-related status even in HPV typing-negative patients. This approach could provide a novel option to accurately detect HR-HPVs in cervical tumours and help to eliminate the percentage of misdiagnosed HPV-related cases.