Digital image analysis of Ki67 proliferation index in breast cancer using virtual dual staining on whole tissue sections: clinical validation and inter-platform agreement

The Ki67 proliferation index is a prognostic and predictive marker in breast cancer. Manual scoring is prone to inter- and intra-observer variability. The aims of this study were to clinically validate digital image analysis (DIA) of Ki67 using virtual dual staining (VDS) on whole tissue sections and to assess inter-platform agreement between two independent DIA platforms.

Serial whole tissue sections of 154 consecutive invasive breast carcinomas were stained for Ki67 and cytokeratin 8/18 with immunohistochemistry in a clinical setting. Ki67 proliferation index was determined using two independent DIA platforms, implementing VDS to identify tumor tissue. Manual Ki67 score was determined using a standardized manual counting protocol. Inter-observer agreement between manual and DIA scores and inter-platform agreement between both DIA platforms were determined and calculated using Spearman's correlation coefficients. Correlations and agreement were assessed with scatterplots and Bland-Altman plots.

Spearman's correlation coefficients were 0.94 (p <0.001) for inter-observer agreement between manual counting and platform A, 0.93 (p <0.001) between manual counting and platform B, and 0.96 (p <0.001) for inter-platform agreement. Scatterplots and Bland-Altman plots revealed no skewness within specific data ranges. In the few cases with > 10% difference between manual counting and DIA, results by both platforms were similar.

DIA using VDS is an accurate method to determine the Ki67 proliferation index in breast cancer, as an alternative to manual scoring of whole sections in clinical practice. Inter-platform agreement between two different DIA platforms was excellent, suggesting vendor-independent clinical implementability.