The Ki-67 protein is associated with cellular proliferation, and the protein is present in the nucleus of all cells that are in the active phase of the cell cycle, but absent in resting cells, see [1]. The cell proliferation rate can be assessed by Ki-67-immunohistochemical (IHC) staining, and this can be correlated to the tumor grade and the clinical course for the patient, see [2]. To identify the Ki-67 positive cells within the tumor regions on an image, these regions must be outlined manually, which can be a tedious and time-consuming task. However, tumor detection can be automated by performing a physical double staining (PDS) with cytokeratin 5 (CK5). CK5 is overexpressed in tumors located in the oral cavity, oropharyngeal, hypopharyngeal and laryngeal areas, see [3]. CK5 can be used as a tumor marker, and Ki-67 nuclei can thus be classified as belonging to a tumor based on the staining of the surrounding tissue. By using PDS with CK5, automated segmentation of relevant nuclei may be more precise and reproducible, see [4].
This protocol automatically detects and quantifies Ki-67 positive nuclei within neck tissue tumor regions. No manual outlining of the tumor is needed, since the tumor region is automatically identified based on the CK5+Ki-67 PDS. The protocol provides the number of positive nuclei within the tumor region as well as the ratio compared to the total number of nuclei within the tumor region.