Ki‑67 Hot Spot Assessment in Breast Cancer

Definition and Biological Significance

Ki‑67 hot spots are focal regions within a breast tumor where immunohistochemical staining for Ki‑67 is markedly higher than in the surrounding tissue, reflecting substantially elevated proliferative activity. 1
These hot spots represent areas of biological heterogeneity, with proliferating‑cell percentages that can be dramatically higher than the rest of the specimen (e.g., ~90 % versus ~30 % Ki‑67 positivity in the same tumor). 3
Hot spots constitute one of two main patterns of Ki‑67 heterogeneity; the other pattern is a gradient of increasing staining toward the invasive tumor edge. 4
The invasive edge is generally regarded as the most biologically active tumor region and is most likely to influence disease outcome. 5

Investigators differ in how they handle hot spots: some focus analysis exclusively on hot spots, others incorporate them into a global assessment, and some recommend avoiding hot spots altogether. 6
Relying solely on hot‑spot evaluation can overestimate overall tumor proliferative activity, potentially leading to misclassification of tumor aggressiveness. 1
Inconsistent selection of hot spots contributes to inter‑observer variability, underscoring the need for standardized approaches. 8

When hot spots are present, pathologists should evaluate the entire tissue section and record the overall average Ki‑67 score to ensure reproducibility across laboratories. 7
This recommendation emphasizes consistency over the potential benefit of capturing the most aggressive tumor biology, a question that remains under investigation. 8

Count a minimum of 500 tumor cells, aiming for at least 1 000 cells, across representative fields to obtain a reliable Ki‑67 estimate. 9
If Ki‑67 staining appears homogeneous, assess at least three randomly selected high‑power fields (×40 objective) to calculate the score. 10
For heterogeneous specimens containing hot spots, average the Ki‑67 score across the whole section rather than focusing exclusively on the high‑proliferation areas. 1