Ki67

The Ki-67 protein, encoded by the MKI67 gene on chromosome 10, is crucial for cell proliferation. It coats the surface of chromosomes during mitosis and prevents them from collapsing. By acting as a kind of “biological surfactant”, Ki-67 ensures that each chromosome remains physically separate from its neighbors, enabling the proper distribution of chromosomes into daughter cells. Ki67 is expressed in all active phases of the cell cycle, starting in mid-G1, with levels escalating through the S and G2 phases and peaking during the M phase before being rapidly degraded at the end of mitosis. The Ki67 proliferation index (PI), which represents the proportion of Ki67-positive cells in a tissue, reflects the growth fraction.

For many tumors, cell proliferation rates determined by Ki67 immunoreactivity correlate with tumor grades and clinical outcomes. Importantly, Ki67 plays a central role in diagnosis, prognosis, and predicting the progression of various neuroendocrine neoplasms (NENs) and lymphomas.

The Ki67 proliferation index (PI) is essential for classifying gastrointestinal and pancreatic neuroendocrine neoplasms (NENs): neuroendocrine tumor (NET) grade 1 with a Ki67 PI <3%; grade 2 with a Ki67 PI of 3–20%; and grade 3, along with neuroendocrine carcinoma (NEC), including both small cell and large cell types, with a Ki67 PI >20%. Mixed neuroendocrine/non-neuroendocrine neoplasms exhibit a variable Ki67 PI.

In lung NENs, while Ki67 PI is not essential for diagnosis, the 2021 WHO classification uses it to distinguish entities: up to 5% for typical carcinoid, up to 30% for atypical carcinoid, and over 30% for NECs. Ki67 has gained importance for diagnosing lung NENs in biopsies, especially when crush artifacts affect morphology, ensuring accurate pre-operative diagnosis and preventing misdiagnosis of NETs and NECs.

The 2022 WHO classification for head and neck tumors incorporates the Ki67 index as one of the three diagnostic criteria for classifying NENs, using a cutoff value of 20%.

Ki67 is used in lymphoma diagnostics, where the distribution and number of Ki67-positive cells in a lymph node is used to differentiate reactive lymphocytic changes and neoplasia. The Ki67 PI is also used as a prognostic indicator but no general specific cut-off values for Ki67 PI have been identified for this purpose.

In the diagnosis of pheochromocytoma and paraganglioma, the Ki67 PI serves primarily as a prognostic marker but may also have a negative diagnostic role. Typically, the Ki67 PI in pheochromocytomas and paragangliomas is less than 10%; values exceeding this threshold should suggest consideration of a differential diagnosis.

Currently, the Ki67 PI holds limited significance in the diagnostic and prognostic evaluation of urogenital, thyroid, parathyroid, or pituitary neuroendocrine neoplasms (NENs).

Ki67, in combination with p16 immunohistochemistry, plays an important role in distinguishing HPV-related neoplastic lesions from reactive conditions especially within gynecological cervical lesions. Markedly increased Ki67 expression extending into the middle and upper epithelial layers of squamous epithelium, along with strong, diffuse (block-like) p16 staining and co-expression of Ki67 and p16 in the same cells, favors the diagnosis of HPV-related neoplastic lesions.

The Ki67 PI has been used alongside with gene expression testing to subclassify breast carcinoma with emphasis on diagnostic, predictive and prognostic information.

• Correlation of Ki67 PI with tumor grades and clinical outcomes in various tumor types.
• Classification and diagnosis of NENs in gastrointestinal, pancreatic, lung, and head and neck tissues, where the Ki67 PI is used to differentiate tumor subtypes and guide clinical decisions.
• Distinguishing between reactive lymphocytic changes and lymphoma. 
• Distinguishing HPV-related neoplastic lesions from other reactive changes when used together with p16.
• Use of Ki67 PI as a prognostic marker or a negative diagnostic tool in pheochromocytomas and paragangliomas.

At present tonsil is recommended as a positive and negative tissue control. Virtually all B-cells in the dark zones of the germinal centers should exhibit moderate to strong nuclear staining reaction, while at least a weak to moderate staining reaction should be observed in most B-cells in the light zones. No staining should be seen in the vast majority of mantle zone B-cells. Liver or pancreas can be used as supplementary negative tissue controls, where fewer than 1% of hepatocytes and epithelial cells of the exocrine pancreatic glands should show positivity. However, inflammatory and reactive conditions may lead to an elevated Ki67 score. For unexpected reasons dormant neutrophils can display a weak nuclear Ki67 reaction.