Using p16 immunohistochemistry to classify morphologic cervical intraepithelial neoplasia 2: correlation of ambiguous staining patterns with HPV subtypes and clinical outcome.

p16INK4a immunohistochemistry (IHC) is widely used to facilitate the diagnosis of human papillomavirus (HPV)-associated cervical precancerous lesions. Although most p16 results are distinctly positive or negative, certain ones are ambiguous: they meet some but not all requirements for the "block-positive" pattern. It is unclear whether ambiguous p16 immunoreactivity indicates oncogenic HPV infection or risk of progression. Herein, we compared HPV genotypes and subsequent high-grade squamous intraepithelial lesion (HSIL) outcomes among 220 cervical biopsies with a differential diagnosis of cervical intraepithelial neoplasia 2 based on hematoxylin and eosin morphology and varying degrees of p16 immunoreactivity. p16 results were classified as block positive (n=40, 18%), negative (n=130, 59%), or ambiguous (n=50, 23%), a category we further grouped into 3 patterns: strong/basal (n=18), strong/focal (n=15), and weak/diffuse (n=17). Seventy percent of ambiguous p16 lesions were negative for the most common low- and high-risk HPV types; the remaining 30% were positive for HPV 16, 18, 45, 58, 59, or 66. Three patterns revealed comparably low HPV detection rates (28%, 27%, and 35%). During 12-month surveillance, HSILs were detected in 35% of the p16 block-positive group, 1.5% of negative group, and 16% of the ambiguous group. The accuracy of ambiguous p16 immunoreactivity in predicting oncogenic HPV and HSIL outcome is significantly lower than that of the block-positive pattern but greater than negative staining. Specific guidelines for this intermediate category should prevent diagnostic errors and help implement p16 IHC in general practice.