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

Abstract

Grading cervical intraepithelial neoplasia (CIN) determines clinical management of women after abnormal cytology with potential for overdiagnosis and overtreatment. We studied a novel biomarker of human papillomavirus (HPV) life-cycle completion (panHPVE4), in combination with the minichromosome maintenance (MCM) protein cell-cycle marker and the p16INK4a transformation marker, to improve CIN diagnosis and categorization. Scoring these biomarkers alongside CIN grading by 3 pathologists was performed on 114 cervical specimens with high-risk (HR) HPV. Interobserver agreement for histopathology was moderate (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% confidence interval [CI]: 0.763-0.969; p16INK4a : κ=0.798; 95% CI: 0.712-0.884; MCM: κ=0.894; 95% CI: NC (this quantity cannot be calculated). Biomarker expression was studied by immunofluorescence and immunohistochemistry and was correlated with 104 final CIN diagnoses after histologic 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.

Figures

Figure 1

Comparison of detection of expression of panHPVE4 by immunofluorescence (IF; in green; nuclei counterstained using DAPI, in blue) (B, E) and immunohistochemistry (in brown) (C, F) in a productive CIN1 lesion. Images A, B, and C were captured at higher magnification than those shown in D, E, and Fto illustrate the wide distribution of HPVE4 that can sometimes be seen in low-grade CIN1 lesions.

Figure 2

Cervical squamous epithelium, negative for HPV DNA and by consensus histology (A): there is no panHPVE4 detected by IF and only parabasal MCM staining (in red; nuclei counterstained using DAPI, in blue) (B), and absent p16INK4a by IHC (C).

Figure 3

CIN1 lesion by consensus histology (A): strongly positive for panHPVE4 by IF (in green), widespread MCM staining (in red; nuclei counterstained using DAPI, in blue) (B), and p16INK4a staining of the lower third of the epithelium by IHC (C).

Figure 4

CIN2 lesion by consensus histology (A): panHPVE4 staining of upper quarter of the epithelium by IF (green; MCM red; DAPI blue) (B) and extensive p16INK4a staining by IHC (brown) (C).

Figure 5

CIN3 lesion by consensus histology (A): nopanHPVE4 expression by IF (green) with full thickness MCM (red; DAPI blue) (B) and p16INK4a expression by IHC (brown) (C).

Figure 6

Schematic diagram summarizing the panHPVE4, p16INK4a and MCM biomarker expression patterns in relation to histological diagnoses by CIN classification. Cells expressing E4 are shown in green, while those expressing p16 are shown in brown. Cells expressing MCM are indicated by the presence of red nuclei. Normal squamous epithelium does not express p16INK4a or E4 and has only (para)basal MCM staining. The productive pattern is extensively positive for E4 with widespread MCM staining, and p16INK4a staining typically restricted to the lower third of the epithelium. The intermediate pattern shows E4 staining of upper quarter or less of the epithelium and p16INK4a in lower two-thirds of epithelium. The transforming pattern shows limited or no E4 expression, with p16INK4a expression in two-thirds or more of the epithelium or full thickness.