Dual stain immunohistochemical localization of p16^INK4A 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 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 and Ki-67 using an EnVision polymer-based method. The chromogens used were of DAB brown for the detection of p16 and alkaline phosphatase blue for Ki-67. Histologic regions were scored positive for either marker based on the detection of p16 or Ki-67 in >10% of the cells of interest. Result: Positive test results with colocalization of p16/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/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 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 and Ki-67 is a practical and potentially powerful diagnostic approach to enhance the accuracy of cervical histopathologic diagnosis.