Investigating Diagnostic Problems of CIN1 and CIN2 Associated With High-risk HPV by Combining the Novel Molecular Biomarker PanHPVE4 With P16INK4a

Grading cervical intraepithelial neoplasia (CIN) determines clinical management of women after abnormal cytology with potential for over-diagnosis and overtreatment. We studied a novel biomarker of HPV life-cycle completion (panHPVE4), in combination with the MCM cell-cycle marker and the p16INK4a transformation marker to improve CIN diagnosis and categorization. Scoring these biomarkers alongside CIN grading by three pathologists was performed on 114 cervical specimens with high-risk (HR-) HPV. Inter-observer agreement for histopathology was moderate (kappa (κ): 0.43 for CIN1/negative, 0.54 for CIN2/≤CIN1, and 0.36 for CIN3). Agreement was good or excellent for biomarker scoring (E4: κ=0.896; 95%CI: 0.763-0.969, p16INK4a: κ=0.798; 95%CI: 0.712-0.884, MCM: κ=0.894; 95%CI: n.c.). Biomarker expression was studied by immunofluorescence and immunohistochemistry and correlated with 104 final CIN diagnoses following histological review. All 25 histologically negative specimens were p16INK4a and panHPVE4 negative although 9 were MCM positive. There were variable extents of p16INK4a positivity in 11/11 CIN1, and extensive panHPVE4 staining in 9/11. Ten CIN2 lesions expressed panHPVE4 and p16INK4a and 13 CIN2 expressed only p16INK4a. CIN3 showed extensive p16INK4a positivity with no/minimal panHPVE4 staining. PanHPVE4, unlike MCM, distinguished CIN1 from negative. PanHPVE4 with p16INK4a separated CIN2/3 showing only expression of p16INK4a indicating transforming HR-HPV E7 expression, from CIN1/2 showing completion of HR-HPV life-cycle by E4 expression and variable p16INK4a expression. PanHPVE4 and p16INK4a staining are complementary markers that could provide simple, reliable support for diagnosing CIN. Their value in distinguishing CIN1/2 that supports HR-HPV life cycle completion (and which might ultimately regress), from purely transforming CIN2/3 needing treatment warrants further research.