Ki-67 is a nuclear protein accidentally discovered during the screening of monoclonal antibodies in the early 1980s. Although Ki-67 has been used in the field of breast cancer for decades, its potential as a breast cancer biomarker has not yet been fully realized. Understanding the capabilities and limitations of Ki-67 assessment methods is crucial for realizing the potential of this biomarker. Therefore, this series of tweets compiles and interprets the latest literature by Professor Allen M. Gowntitled ‘The Biomarker Ki-67: Promise, Potential, and Problems in Breast Cancer,’ introducing the history and issues of Ki-67, as well as the research by the International Ki-67 in Breast Cancer Working Group, to share with all teachers!
Ki-67 was discovered in the 1980s in the laboratory of German hematopathologist Dr. Harold Stein. While working at Kiel University in Germany, Dr. Stein used the then-novel monoclonal antibody technology to discover a series of tumor-associated proteins with biomarker potential, including Ki-67, named because it was cloned from the 67th well of a microtiter plate. Dr. Harold Stein collaborated with pathologist/molecular biologist Johannes Gerdes and used crude nuclear extracts from the Hodgkin’s lymphoma cell line L428 to generate the monoclonal antibody named Ki-67, finding that it reacted with nuclei of Hodgkin’s lymphoma patients and non-tumor germinal center nuclei in frozen sections. In fact, in this influential paper, the Ki-67 antibody was also found to react with cells in proliferative zones of other tissue types, including basal epithelial cells.
The discussion section of this paper includes a prescient comment: ‘Ki-67 may become an important tool for reliably and rapidly determining the proliferation rate of malignant tumors.’ Shortly thereafter, the rapid development of IHC technology created conditions for innovations such as frozen tissue section techniques, the use of deparaffinized, formalin-fixed tissues, and antibody use and antigen or epitope retrieval. About 10 years later, using recombinant Ki-67 protein, a series of monoclonal antibodies (from MIB-1 to MIB-3) were generated against the Ki-67 antigen, allowing Ki-67 to be visualized in deparaffinized, formalin-fixed tissues through epitope retrieval.
Since Ki-67 was only accidentally discovered as a monoclonal antibody, it took many years to reveal its protein nature, and only part of it has been revealed so far. The gene has about 30,000 bp, and sequencing found little similarity to other proteins. Its half-life is about 90 minutes, and the original Ki-67 antibody has been shown to decorate nuclear nucleoli during the G1, S, and G2 phases of the cell cycle but is absent in the G0 phase. Ki-67 levels gradually increase as the cell cycle progresses, reaching a maximum in the M phase. In prophase, the Ki-67 antigen moves to the surface of newly formed chromosomes and remains until telophase. In mitotic cells, Ki-67 covers condensed chromosomes and binds to reassembled nucleoli when cells enter the G1 phase.
The function of Ki-67 is related to regulating cell proliferation, but in some experimental mice with Ki-67 knocked out, despite the absence of Ki-67 expression, the mice developed normally and cells proliferated normally. It has been proposed that Ki-67 appears to play a major role in the constitution of heterochromatin, thereby controlling gene expression.
Gerdes et al., using the original Ki-67 monoclonal antibody on frozen sections, designed a ‘Ki-67 index,’ first applied to malignant lymphomas, determined by calculating the percentage of Ki-67-positive tumor cells among 200-500 tumor cells. Subsequently, Gerdes et al. applied the concept of the Ki-67 index to breast cancer and compared the results with ER, demonstrating a negative correlation between the Ki-67 index and ER status. At this early stage, Gerdes et al. speculated that this relationship might explain why some ER-positive breast cancers respond poorly to endocrine therapy (ET), and the suggestion that the current Ki-67 index might predict response to cell cycle-targeted therapies was very prescient.
Ki-67 as a Prognostic Biomarker
Numerous published studies have explored the correlation between Ki-67 analysis and various cancer prognoses, such as disease-free survival and overall survival. However, it is practically impossible to compare the results of these different studies with each other, as shown in a meta-analysis of 46 published studies (involving 12,155 patients): dividing the Ki-67 index into ‘high’ and ‘low,’ a large portion of studies did find that a ‘high’ Ki-67 index (ranging from 5% to 32%) was associated with poorer outcomes, but since researchers all chose their own cutoff values to distinguish ‘high’ and ‘low’ Ki-67, the 46 studies could not be compared.
In another meta-analysis based on 25 studies involving 64,196 patients, Petrelli et al. again demonstrated the modest prognostic value of ‘high’ versus ‘low’ Ki-67 (again, using the definitions of the original study authors), with a hazard ratio of about 1.5 for patients with ‘high’ Ki-67. In another large study involving 3,658 patients, after adjusting for age, menopausal status, tumor size, lymph node status, histology, grade, lymphatic invasion, vascular invasion, hormone receptor status, and HER-2 status, the Ki-67 index could predict clinical outcomes; however, this relationship was found to be nonlinear. Figure 1 shows examples of breast tumors with ‘low’ and ‘high’ Ki-67 indices.
Figure 1. Examples of breast tumors with (A) low and (B) high Ki-67 indices
Ki-67 Index, Oncotype Dx, and Prediction of Response to Chemotherapy
The Oncotype Dx (21-gene assay for breast cancer) recurrence score has been shown to be an excellent prognostic tool, especially in patients with ER-positive, node-negative breast cancer. Oncotype Dx is a reverse transcription polymerase chain reaction assay that combines the expression results of 16 genes, heavily weighting genes related to cell proliferation, including the Ki-67 gene. Recently, the Oncotype Dx recurrence score has also been shown to be a predictive marker for chemotherapy response. Given the demonstrated strong correlation between the Ki-67 index and the Oncotype Dx recurrence score, it becomes a reasonable question whether the Ki-67 index alone can predict response to chemotherapy.
Viale et al., in a study of 1,924 patients from two different randomized trials of adjuvant chemotherapy for node-negative breast cancer patients, found that for all patients, regardless of treatment modality, ‘high’ Ki-67 was associated with poorer prognosis, but a ‘high’ Ki-67 index did not predict a better response to adjuvant chemotherapy. In a recently published study on neoadjuvant chemotherapy for breast cancer patients, although the Ki-67 index was associated with pathological response, the authors concluded that Ki-67 itself is not a suitable marker for deciding whether breast cancer patients should receive neoadjuvant chemotherapy. Therefore, the substantial evidence to date indicates that while the Ki-67 index can be used as a prognostic marker, unlike the Oncotype Dx assay, Ki-67 is not used as a biomarker for predicting chemotherapy response.
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Antibody Name
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Product Number
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Clone Number
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Cellular Localization
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Ki-67
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RMA-0731
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MXR002
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Nuclear
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MAB-0672
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MX006
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Nuclear
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RMA-0542
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SP6
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Nuclear
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References:
Gown Allen M.The Biomarker Ki-67: Promise, Potential, and Problems in Breast Cancer[J]. Applied Immunohistochemistry & Molecular Morphology, DOI:10.1097/PAI.0000000000001087.
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