What is Ki-67 (Kiel-67, proliferation marker)?

What is Ki-67?

Ki-67 is a nuclear protein encoded by the MKI67 gene that serves as a well-established proliferation marker, present during all active phases of the cell cycle (G1, S, G2, and mitosis) but absent from resting cells in the G0 phase. 1

Molecular Characteristics

Ki-67 is a large nuclear protein whose expression is strictly associated with cell proliferation 2. The protein exhibits distinct cellular localization patterns:

• During interphase: Exclusively detected within the nucleus 2
• During mitosis: Most of the protein relocates to the surface of chromosomes, forming part of the perichromosomal layer 1, 3
• In resting cells (G0): Completely absent, making it an excellent marker for determining the growth fraction of cell populations 2

Functional Roles

While Ki-67's function was historically unclear, recent research has revealed multiple molecular functions 3:

• Interphase functions: Required for normal cellular distribution of heterochromatin antigens and nucleolar association of heterochromatin 3
• Mitotic functions: Essential for formation of the perichromosomal layer and prevents aggregation of mitotic chromosomes 3
• Cell cycle regulation: Phosphorylation and dephosphorylation of Ki-67 are controlled by the cyclin B/cdc2 complex and occur at two major checkpoints during mitosis 4

Clinical Applications

Ki-67 is extensively used in tumor diagnostics to estimate the growth fraction and proliferative activity of malignant cells 1, 2:

Detection Methods

The most commonly used antibody is the MIB1 monoclonal antibody clone, which is considered the "gold standard" for Ki-67 immunohistochemistry in formalin-fixed paraffin-embedded tissue 1. The MIB1 antibody has favorable properties including:

• Detection of an epitope motif repeated 16 times in the protein (enhancing sensitivity) 1
• Consistent performance across a wide range of antibody dilutions 1
• Robust results with neutral buffered formalin fixation for 4-48 hours 1

An alternative antibody, SP6 (rabbit anti-human Ki-67 monoclonal), may provide additional improvements in sensitivity and quantitative image analysis 1.

Scoring and Interpretation

Ki-67 is a nuclear protein, and only nuclear staining (plus mitotic figures) should be incorporated into the Ki-67 score 1. The score is defined as the percentage of positively stained cells among the total number of malignant cells scored 1.

Important caveats:

• Cytoplasmic or membrane staining can occur (especially in breast cancer with squamous metaplastic changes) and should be ignored 1
• Weak counterstaining can result in overestimation of the Ki-67 index 1
• At least three randomly selected high-power fields should be counted if staining is homogeneous 1

Diagnostic Applications

Dual-color Ki-67/MART-1 immunohistochemistry specifically detects cell proliferation activity in melanocytes/melanoma cells by eliminating actively proliferating non-melanocytes such as reactive lymphocytes, endothelial cells, or keratinocytes 1. This technique is particularly useful for:

• Distinguishing melanomas from benign nevi 1
• Differentiating capsular nevi from metastatic melanomas in lymph nodes 1
• Identifying lack of maturation in deep dermal melanocytes, which correlates with melanoma 1

Prognostic Value

The Ki-67 labeling index is often correlated with clinical course of disease 2. For breast and prostate carcinomas, the prognostic value for survival and tumor recurrence has been proven in univariate and multivariate analyses 2.

However, current guidelines note that data are insufficient to recommend routine use of Ki-67 to assign breast cancer patients to prognostic groupings for clinical decision-making 1. For neuroendocrine tumors, when accurate mitotic rate is precluded by inadequate tissue (such as small biopsies), Ki-67 index is the preferred method of establishing proliferative rate 1.

Technical Considerations

• Tissue storage: Loss of Ki-67 immunoreactivity occurs if sections are stored on glass slides exposed to room air for 3 months or longer 1
• Fixation: Neutral buffered formalin fixation for 4-48 hours is adequate, with antigenicity well preserved in paraffin blocks for decades 1
• Epitope retrieval: Heat-induced methods are preferred; protease and low pH methods should be avoided 1