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

•Ambiguous p16 patterns refer to certain p16 IHC results that meet some but not all requirements for the Lower Anogenital Squamous Terminology “block-positive” pattern.•Seventy percent of p16 ambiguous cervical intraepithelial neoplasia (CIN) 2 lesions are negative for HR-HPV.•Sixteen percent of p16 ambiguous CIN 2 lesions reveal high-grade squamous intraepithelial lesion outcomes during 12-month surveillance.•“p16 ambiguous CIN 2” is a distinct category of cervical lesions that appear to pose an intermediate risk of persistence and progression.

Summaryp16INK4a 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.