The p16 gene (also called CDKN2A and INK4) is located on chromosome 9 in the region 9p21. This region is either deleted or mutated in a wide variety of tumors, such as pancreatic cancer. Two alternatively spliced gene products are encoded by p16: Protein P16 and p16ARF Protein. Both gene products have tumor suppressor functions.

The P16 Protein is one of the inhibitors for cyclin-dependent kinases (CDKs), e.g. CDK4. CDKs are a group of serine/threonine kinases that form active heterodimeric complexes by binding to their regulatory subunits, cyclins. Several CDKs, mainly CDK2, CDK4, and CDK6, work cooperatively to drive cells from G₁ phase into S phase.

CDK4 and CDK6 are involved in early G₁ phase, whereas CDK2 is required to complete G₁ phase and initiate S phase. Both CDK4 and CDK6 form active complexes with the D type of cyclins (cyclins D1, D2, and D3). CDK2 is sequentially activated by the E type of cyclins, cyclins E1 and E2, during G₁/S transition stage.

A type cyclins, cyclin A1 and A2, play a role during the S phase. CDK2/cyclin A complex appears during the late S phase and plays a role in progression of DNA replication. The cyclins that are involved in regulating the passage of the cell from the G₂ checkpoint into M phase are known as mitotic cyclins and they associate with mitotic CDKs.

Similarly, cyclins that are involved in the passage of cells from the G₁ checkpoint into S phase are called G₁ cyclins. Once the CDKs have completed their role, they undergo a rapid programmed proteolysis via ubiquitin-mediated delivery to the proteasome complex.

The enzymatic activity of a CDK is regulated at three levels: cyclin association, subunit phosphorylation, and association with CDK inhibitors. When cyclins initially bind to CDKs, the resulting complex is inactive. The phosphorylation of CDKs by CDK activating kinases leads to their activation.

The p16 gene is inactivated by promoter hypermethylation (5-30%) in a number of different cancers, such as lung cancer (NSCLC, non-small cell lung cancer), colon cancer, lymphomas (depending on stage) and bladder cancer.

PyroMark p16 assay detects the level of methylation in a region +148 to +161 in exon 1 of the gene (Ensembl gene: ENSG00000147889).