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P9. Cell lines as the basis for quality assurance of immunohistochemical assay for Ki67 in tissue

Marina Sørensen, Line Sloth Hansen and Søren Nielsen

Institute of Pathology, Aalborg University Hospital, Aalborg, Denmark

BACKGROUND: Breast cancer (BC) is the most common female malignancy. Ki67 is a nuclear protein expressed by proliferating cells and can be identified by immunohistochemistry for the assessment of proliferation level in BC. Ki67 has a prognostic and predictive value. However, clinical use of this biomarker is impeded by a large intra- and interlaboratory variability and low comparability of Ki67 studies. One of the sources of those discrepancies is related to different immunohistochemical (IHC) staining procedures used by the individual laboratories. Control procedure is an important component of IHC. However, the current approach with use of tissue control material may be insufficient due to the low reproducibility and accuracy. A novel approach is to use cell lines with homogenous and well characterised antigenic expression.

MATERIALS AND METHODS: Ki67 staining was performed using the standard immunohistochemical method from Aalborg University Hospital. Mouse monoclonal antibody clone MIB-1 was utilised to detect Ki67. The slides were stained on BenchMark ULTRA, Ventana. The homogeneity of the Ki67 expression in tissue and cell line material was evaluated in three different levels of both specimen types. Capability to identify fluctuations of the IHC protocol with respect to quantitative and qualitative assessment of Ki67 was evaluated by simultaneous staining with standard and modified settings for Ki67. Digital image analysis (DIA) was performed at the digital image of cell line microarray using VIS-software (Visopharm integrator System, v. 4.6.3.1050, Denmark). The counting procedure for the tonsil control tissue was carried out with a use of eye-balling method of three pathologists. One way ANOVA, F-test, Welch ANOVA and Chi-square test was performed in R-studio (RStudio Desktop 0.98.1103) to test the hypotheses of the study.

RESULTS: The study shows superiority of homogeneity and counting ability of cell lines compared to the tissue control material. This conclusion is based on brief visual assessment of digital images and DIA slides. Cell line material also demonstrates superiority to the tissue material as a robust and more reliable control for quantitative and qualitative assessment of Ki67. This can be stated as cell lines demonstrate more close distribution of H-score means with respect to common mean of the group and no overlap in four different dilutions of Ki67 antibody compared to tonsil control tissue that demonstrates a large spread of overlapping values.

CONCLUSION: Cell lines are more robust and reliable control material for the technical evaluation of the analytical component of a Ki67 assay compared to tissue material.

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