p16 (p16ink4a) is one of multiple independent proteins transcribed from the gene CDKN2A. p16 can prevent phosphorylation of Rb protein (RB1) by inhibiting CDK4 and CDK6, and it manages the G1 to S transition in the cell cycle via this p16-Rb pathway. Furthermore, expression of p16 is involved in regulating cellular senescence. Cancers see frequent mutation, deletion and hypermethylation of CDKN2A, or alternatively may show overexpression of transcribed p16 protein alongside knockout of its repressor RB1. Thus, both up- and down-regulation of p16 as part of the dysregulation of the p16-Rb pathway can be indicative of loss of senescent properties and a breakdown of the negative regulation of proliferation in the cell. In immunohistochemistry, p16 antibodies are frequently used when examining anogenital lesions and in the diagnosis of HPV-associated precancerous lesions. Overexpression correlates with oncogenic HPV infection, particularly alongside loss of RB expression. Typical positive staining of p16 is described as a “block” of ten to twenty cells that together show strong nuclear and cytoplasmic expression. p16 is also positive in squamous cell carcinoma, Müllerian serous carcinoma, and head and neck carcinoma, and high expression is correlated with high grade cancers. Lastly, it should be noted that p16-specific antibodies do not cross-react with p14ARF (also from CDKN2A), as these two proteins are transcribed from alternative open reading frames. References: Park, Ben Ho et al. Holland-Frei Cancer Medicine. 6th edition. Hamilton (ON): BC Decker; 2003, URL: https://www.ncbi.nlm.nih.gov/books/NBK13507/; Int J Cancer. 2012 Apr 15;130(8):1715-25, PMID: 22025288; Cancer Res. 1994 Dec 1;54(23):6078-82, PMID: 7954450; Hum Pathol. 2017 Aug;66:144-151, PMID: 28705710