Clinical Implication of p16, Ki-67, and Proliferating Cell Nuclear Antigen Expression in Cervical Neoplasia: Improvement of Diagnostic Accuracy for High-grade Squamous Intraepithelial Lesion and Prediction of Resection Margin Involvement on Conization Specimen

BACKGROUND Cervical intraepithelial neoplasia (CIN) grading is subjective and affected by substantial rates of discordance among pathologists. Although the use of p16INK4a (p16) staining has been proven to improve diagnostic accuracy for high-grade squamous intraepithelial lesion (HSIL), the clinical evidence for use of Ki-67 and proliferating cell nuclear antigen (PCNA) is insufficient to make an independent recommendation for use, alone or in combination. The primary objective was to evaluate clinical utility of Ki-67 and PCNA in combination with p16 in diagnosing HSIL. Also, we assessed the correlation between expressions of three biomarkers and resection margin status of conization specimen. METHODS The expressions of p16, Ki-67, and PCNA were evaluated by immunohistochemical methods in 149 cervical tissues encompassing 17 negative lesion, 31 CIN 1, 25 CIN 2, 41 CIN 3, and 35 invasive squamous cell carcinoma. The immunohistochemical staining results were classified into four grades: 0, 1+, 2+ and 3+. RESULTS The expression of three biomarkers was positively associated with CIN grade. Ki-67 immunostaining did not increase the accuracy of HSIL diagnosis when combined with p16 immunostaining compared with p16 immunostaining alone. In contrast, combining the staining results for p16 and PCNA (p16 = 3+ and PCNA ≥2+) increased its specificity (66.7% vs. 75.0%, P = 0.031) without decrease of its sensitivity (98.7% vs. 98.7%) for diagnosis of CIN 3 and more sever lesion. Subgroup analysis for conization specimen with CIN 2 and CIN 3 showed that positive Ki-67 immunostaining was an independent risk factor for predicting resection margin positivity (odds ratio = 6.52, 95% confidence interval 1.07-39.64). CONCLUSIONS We found that the combined use of p16 and PCNA immunostaining enhanced diagnostic accuracy for HSIL. Positive Ki-67 immunostaining was associated with incomplete excision.