Department of Pathology, Yale University School of Medicine, New Haven, CT, USA.
Department of Oncology and Pathology, CCK, Karolinska Institutet, Stockholm, Sweden.
Mod Pathol. 2021 Jul;34(7):1261-1270. doi: 10.1038/s41379-021-00745-6. Epub 2021 Feb 3.
Ki67, a nuclear proliferation-related protein, is heavily used in anatomic pathology but has not become a companion diagnostic or a standard-of-care biomarker due to analytic variability in both assay protocols and interpretation. The International Ki67 Working Group in breast cancer has published and has ongoing efforts in the standardization of the interpretation of Ki67, but they have not yet assessed technical issues of assay production representing multiple sources of variation, including antibody clones, antibody formats, staining platforms, and operators. The goal of this work is to address these issues with a new standardization tool. We have developed a cell line microarray system in which mixes of human Karpas 299 or Jurkat cells (Ki67) with Sf9 (Spodoptera frugiperda) (Ki67) cells are present in incremental standardized ratios. To validate the tool, six different antibodies, including both ready-to-use and concentrate formats from six vendors, were used to measure Ki67 proliferation indices using IHC protocols for manual (bench-top) and automated platforms. The assays were performed by three different laboratories at Yale and analyzed using two image analysis software packages, including QuPath and Visiopharm. Results showed statistically significant differences in Ki67 reactivity between each antibody clone. However, subsets of Ki67 assays using three clones performed in three different labs show no significant differences. This work shows the need for analytic standardization of the Ki67 assay and provides a new tool to do so. We show here how a cell line standardization system can be used to normalize the staining variability in proliferation indices between different antibody clones in a triple negative breast cancer cohort. We believe that this cell line standardization array has the potential to improve reproducibility among Ki67 assays and laboratories, which is critical for establishing Ki67 as a standard-of-care assay.
Ki67 是一种与核增殖相关的蛋白，在解剖病理学中被广泛应用，但由于检测方案和解读的分析变异性，它并未成为伴随诊断或标准护理生物标志物。国际乳腺癌 Ki67 工作组已经发表了关于 Ki67 解读标准化的努力，并正在进行中，但他们尚未评估代表多种变异来源的检测生产的技术问题，包括抗体克隆、抗体形式、染色平台和操作人员。这项工作的目标是使用新的标准化工具来解决这些问题。我们开发了一种细胞系微阵列系统，其中混合了人 Karpas 299 或 Jurkat 细胞（Ki67）与 Sf9（Spodoptera frugiperda）（Ki67）细胞，以递增的标准化比例存在。为了验证该工具，我们使用了六种不同的抗体，包括来自六个供应商的即用型和浓缩型，使用 IHC 方案在手动（台式）和自动化平台上测量 Ki67 增殖指数。这些检测由耶鲁大学的三个不同实验室进行，并使用两个图像分析软件包（包括 QuPath 和 Visiopharm）进行分析。结果表明，每个抗体克隆之间的 Ki67 反应性存在统计学上的显著差异。然而，使用三个克隆在三个不同实验室进行的 Ki67 检测亚组没有显示出显著差异。这项工作表明 Ki67 检测需要进行分析标准化，并提供了一种新的工具来实现这一目标。我们在这里展示了如何使用细胞系标准化系统来归一化不同抗体克隆之间三阴性乳腺癌队列中增殖指数的染色变异性。我们相信，这种细胞系标准化阵列有可能提高 Ki67 检测和实验室之间的重现性，这对于将 Ki67 确立为标准护理检测至关重要。
