Nondiffuse p16 Expression in HPV-Associated and HPV-Independent Cervical Squamous Cell Carcinomas

Negative or focal p16 expression may occur in cervical squamous cell carcinomas (SCCs) associated with high-risk or low-risk human papillomavirus (HPV), as well as in HPV-independent SCCs. However, this phenomenon is uncommon, and its pathologic and molecular characteristics remain poorly defined. In this single-institution study, we identified 11 cases (11/201 [5.5%]) with negative or focal p16 expression, including 4 (2.0%) associated with high-risk HPV, 6 (3.0%) HPV-independent, and 1 (0.5%) associated with low-risk HPV. Among the 4 high-risk HPV-associated SCCs with complete loss of p16 expression, 3 showed complete loss of the methylthioadenosine phosphorylase (MTAP). Notably, MTAP loss was also observed in 2 of 4 p16-negative high-grade squamous intraepithelial lesions (HSILs), suggesting that p16/MTAP codeletion may occur at both precursor and invasive stages. In contrast, all 6 HPV-independent SCCs retained MTAP expression. Two HPV-independent SCCs were associated with precursor lesions: 1 resembled verruciform acanthotic vulvar intraepithelial neoplasia (vaVIN), while the other was analogous to HSIL. Consistent with high-risk HPV infection, all 4 HPV-associated SCCs demonstrated partial or complete loss of RB1 expression. In comparison, only 2 of 6 HPV-independent SCCs showed partial RB1 loss, likely through HPV-independent mechanisms. A wild-type p53 immunostaining pattern was observed in all cases except 1 HPV-independent SCC (case 7), which exhibited a null pattern and harbored a TP53 nonsense mutation (c.430C>T; p.Gln144Ter). p16 promoter methylation was detected in case 9 (HPV-independent SCC). Somatic TERT promoter mutations were identified in 2 of 4 (50%) HPV-associated SCCs, including c.-131C>T, c.-148C>T, and c.-150C>T. Among HPV-independent SCCs, 4 of 6 cases (66.7%) harbored pathogenic TERT promoter mutations, including c.-138C>T (n=1) and c.-124C>T (n=3). No PIK3CA hotspot mutations were detected in p16-negative, high-risk HPV-associated SCCs. In contrast, 2 HPV-independent SCCs harbored PIK3CA hotspot mutations (p.E545K and p.H1047R), supporting a role for PI3K-AKT-mTOR signaling in this setting. Because nondiffuse p16 expression can occur in both HPV-associated (high-risk and low-risk HPV) and HPV-independent cervical SCCs, which may have different clinical outcomes, awareness of this phenomenon is important when interpreting negative p16 immunostaining. Although data are limited, routine assessment of both p16 immunohistochemistry and HPV status, or correlation with prior liquid-based HPV test results when available, is recommended.