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]. For melanocytic lesions, the number and distribution of Ki-67-positive cells can be used to differentiate melanomas from nevi, because the number of positive cells is significantly increased in melanomas compared to nevi, see [3]. However, prominent lymphatic infiltrate is often present in melanomas and nevi, which may make it difficult to distinguish the two from each other. To make sure that the Ki-67 staining patterns are not misinterpreted, an IHC cocktail detecting both Ki-67 and the melanocytic marker Melanoma Antigen Recognized by T-cells (MART1, sometimes also called MelanA) can be used to distinguish Ki-67-positive melanocytes from Ki-67-positive non-melanocytes, see [3].
This protocol automatically detects and quantifies Ki-67 positive cells within tumor regions. No manual outlining of the tumor is needed, since the tumor region is automatically identified based on the Ki-67/MART1-IHC double staining. Thus, only an overall region of interest (ROI) has to be defined. 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.