A new standardization platform for Ki67 immunohistochemical staining

Researchers from the Department of Pathology, Yale University School of Medicine (New Haven, USA) and Department of Oncology and Pathology, Karolinska Institutet (Stockholm, Sweden), have developed a new tool for the technical standardization of the Ki67 immunohistochemical assay. The use of the tool to adjust Ki67 staining cutpoints, significantly improved the prognostic potential of Ki67 in breast cancer.

This work paves the way for the establishment of Ki67 as a standard-of-care biomarker in clinical pathology. The study was published in the journal Modern Pathology on February 3rd, 2021.

Ki67 as a marker of tumor cell proliferation in clinical specimens

Ki67 is a nuclear protein expressed in all mammalian cells throughout the cell cycle, but its expression is decreased at the G0 phase. This expression pattern has made Ki67 one of the most important markers of proliferation in mammalian cells and in human clinical specimens.1

In fact, the Ki67 proliferative index, defined as the percentage of Ki67+ cells in tumor tissues, has emerged as a promising predictive and prognostic biomarker in various types of cancer, including breast cancer. Specifically, high Ki67 levels indicating a greater proliferative index have been associated with aggressive tumor phenotypes and poor treatment outcomes.2

Ki67 has also been used to risk-stratify patients and identify patients likely to respond to standard chemotherapy and those who are unlikely to benefit from the treatment.3,4

Although Ki67 is widely used to evaluate cell proliferation in clinical pathology, there are no Ki67-based companion diagnostic assays. Despite progress in Ki67 staining interpretation by the International Ki67 Working Group in breast cancer, significant technical variability remains in anti-Ki67 antibody clones, staining protocols, antibody formats, and staining platforms used in different Ki67 immunohistochemical assays.5,6

According to the Tumor Marker Guidelines of the American Society of Clinical Oncology, there is not sufficient evidence to support the use of Ki67 as a biomarker in patients with breast cancer.5

“The International Working Group for Ki67 in Breast Cancer has spent 10 years examining Ki67 as a biomarker for breast cancer with a focus on methods for concordant assessment,” said David Rimm MD, PhD, Professor of Pathology at the Yale University School of Medicine and Director of Yale Pathology Tissue Services. “However, the group did not address the issue of pre-analytic variables, such as the difference between different stainers and assays at different sites.”

The lack of standardized Ki67 staining protocols and interpretation guidelines are the main factors hindering the establishment of Ki67 as a standard-of-care biomarker in clinical laboratories.

Establishment and validation of a new standardization method for Ki67 immunohistochemical staining

Working toward minimizing variabilities in Ki67 staining protocols and assessing the technical sensitivity and linearity of Ki67 assays, Aung et al.7 developed a new standardization tool for Ki67 staining.

Specifically, they established a cell line microarray system consisting of human Ki67+ cells (Karpas 299 or Jurkat cells) and Ki67 cells from the insect Spodoptera frugiperda (Sf9). In this cell line system, Ki67+ and Ki67 cells were mixed in incremental standardized ratios ranging between 0% and 100%.

To validate the standardization tool, they used six different ready-to-use anti-Ki67 antibodies from six vendors. Then, using these antibodies and IHC protocols for manual and automated platforms, they stained the cells to measure Ki67 proliferation indices.

Ki67 staining assays were conducted at three different laboratories at Yale University to confirm the reproducibility of the method. Ki67 staining was analyzed using two image analysis software packages: the open-source software QuPath and a commercially available image analysis package from Visiopharm.

No antibody cross-reactivity was observed between insect and human Ki67, with no Ki67 staining in Sf9 cells. In contrast, most Karpas 299 cells exhibited Ki67 immunoreactivity, confirming the specificity of the cell line-based standardization system.

Normalizing variability in Ki67 staining in clinical specimens

The researchers found that the different antibody clones provided statistically significant differences in Ki67 reactivity. The sensitivity of the antibody clones also differed considerably.

Furthermore, significant staining variability was observed among the three laboratories that conducted the assays using the same protocols. However, three of the antibody clones yielded similar staining results, regardless of the laboratory where the immunohistochemical assays were performed.

The image analysis platforms also contributed to staining variabilities, although to a lower extent than the different antibody clones used and the laboratories conducting the staining.

The researchers also used the new standardization system to normalize staining variability in proliferation indices in clinical specimens from patients with triple-negative breast cancer stained with different anti-Ki67 antibody clones.

Specifically, they stained clinical specimens using two different anti-Ki67 antibodies. Then, using the cell line-based standardization tool, they normalized Ki67 staining intensities between the two assays to minimize staining variabilities and determine an optimal cutpoint for Ki67. Notably, this normalization increased the prognostic value of the Ki67 proliferative index in patients with breast cancer.

“In Aung et al., we use synthetic cell mixtures of cells to show that different sites can get different results depending on the stainer or assay they use,” said Dr David Rimm, the corresponding author of the study. “Then, we show how to use this tool (the synthetic cell mixture calibration slide) to be sure each individual site is getting the same result, thereby standardizing and unifying the assay, so it is not susceptible to inaccuracy due to pre-analytic variables.”

This calibration array is already commercially available from Array Science LLC (https://www.arrayscience.com/).

Future perspectives

Improving the reproducibility among Ki67 staining methods is essential for establishing Ki67 as a standard-of-care assay in clinical laboratories. This work provides a robust cell line-based system for technical standardization of Ki67 immunohistochemical assays.

Although many studies show that the reading of Ki67 assays is a significant source of variability, this standardization tool does not address variability in the interpretation of the Ki67 assay.

Minimizing technical variation in Ki67 assays represents significant progress toward the clinical implementation of the Ki67 proliferative index; nevertheless, standardized interpretation guidelines for Ki67 immunohistochemical staining are needed to establish Ki67 as a predictive and prognostic biomarker.

In addition, validation of this new standardization system by multiple laboratories across different centers is required, as all three laboratories evaluating the performance and reproducibility of the system were within the same institution.

Moreover, the tool does not take into account the impact of variability in nuclear counterstain. For example, weak nuclear counterstaining can lead to a higher Ki67 proliferative index and affect Ki67 assay interpretation; thus, a standardization tool taking into account counterstaining intensity would be ideal.

To learn more about the new platform for analytic standardization of Ki67 immunohistochemical assays, read the article by Aung et al., “A new tool for technical standardization of the Ki67 immunohistochemical assay,” Modern Pathology 34, 1261–1270 (2021).


  1. Sun X, Kaufman PD. Ki-67: more than a proliferation marker. Chromosoma. 2018;127(2):175-186. doi:10.1007/s00412-018-0659-8
  2. Inwald EC, Klinkhammer-Schalke M, Hofstädter F, et al. Ki-67 is a prognostic parameter in breast cancer patients: results of a large population-based cohort of a cancer registry. Breast Cancer Res Treat. 2013;139(2):539-552. doi:10.1007/s10549-013-2560-8
  3. Assersohn L, Salter J, Powles TJ, et al. Studies of the potential utility of Ki67 as a predictive molecular marker of clinical response in primary breast cancer. Breast Cancer Res Treat. 2003;82(2):113-123. doi:10.1023/B:BREA.0000003968.45511.3f
  4. Jones RL, Salter J, A’Hern R, et al. The prognostic significance of Ki67 before and after neoadjuvant chemotherapy in breast cancer. Breast Cancer Res Treat. 2009;116(1):53-68. doi:10.1007/s10549-008-0081-7
  5. Nielsen TO, Leung SCY, Rimm DL, et al. Assessment of Ki67 in Breast Cancer: Updated Recommendations from the International Ki67 in Breast Cancer Working Group. J Natl Cancer Inst. December 2020. doi:10.1093/jnci/djaa201
  6. Rimm DL, Leung SCY, McShane LM, et al. An international multicenter study to evaluate reproducibility of automated scoring for assessment of Ki67 in breast cancer. Mod Pathol an Off J United States Can Acad Pathol Inc. 2019;32(1):59-69. doi:10.1038/s41379-018-0109-4
  7. Aung TN, Acs B, Warrell J, et al. A new tool for technical standardization of the Ki67 immunohistochemical assay. Mod Pathol. 2021;34(7):1261-1270. doi:10.1038/s41379-021-00745-6

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