Dual Stain Immunohistochemical Localization of p16INK4A and ki-67: A Synergistic Approach to Identify Clinically Significant Cervical Mucosal Lesions

Background: The primary goal of this study was to assess the clinical utility of a multiplexed immunohistochemical method using colocalization of p16(INK4a) and Ki-67 in identifying high-grade cervical mucosal lesions. Design: The study included formalin-fixed cervical biopsy specimens, representative of 297 diagnostic regions. They were subjected to 2 colors immunohistochemical staining for p16(INK4a) and Ki-67 using an EnVision polymer-based method. The chromogens used were of DAB brown for the detection of p16(INK4a) and alkaline phosphatase blue for Ki-67. Histologic regions were scored positive for either marker based on the detection of p16(INK4a) or Ki-67 in > 10% of the cells of interest. Result: Positive test results with colocalization of p16(INK4a)/Ki-67 were found in 20 of 40 cases of cervical intraepithelial neoplasia 1 (n = 40) and in all cases of cervical intraepithelial neoplasia 2/3 (n = 32), squamous cell carcinoma (n = 11), adenocarcinoma in situ (n = 10), and invasive adenocarcinoma (n = 8). Colocalization of p16(INK4a)/Ki-67 was also detected in few cells in 1 of 19 sections with tuboendometrial metaplasia but was not detected in normal squamous mucosa (n = 78), normal endocervical mucosa (n = 76), immature squamous metaplasia (n = 13), or in microglandular hyperplasia (n = 9). Conclusions: The p16(INK4a) and Ki-67 are coexpressed in virtually 100% of cases of high-grade squamous and glandular lesions, but they are rarely coexpressed in normal tissues or in benign lesions of the squamous and glandular mucosa. Thus, multiplexed colocalization of p16(INK4a) and Ki-67 is a practical and potentially powerful diagnostic approach to enhance the accuracy of cervical histopathologic diagnosis.