Abstract
PurposeThe 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.MethodsSerial 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.ResultsSpearman’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.ConclusionsDIA 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.
Highlights
Breast cancer is the most common type of cancer among women worldwide, and one of the leading causes of cancer-related death [1]
Resection specimens of 154 consecutive primary invasive breast carcinomas treated in the University Medical Center Groningen (The Netherlands) between August 2015 and February 2017 were prospectively included
Of these 37 cases, 32 were misaligned in both digital image analysis (DIA) platforms, 3 cases were misaligned in only one platform, and 2 cases could not be aligned by one platform as the stains were mirrored
Summary
Breast cancer is the most common type of cancer among women worldwide, and one of the leading causes of cancer-related death [1]. Immunohistochemistry for Ki67 (MKI67), a nuclear antigen which is present in all but the G0 phase of the cell cycle and expressed in proliferating cells, can be used to determine tumor proliferation index [3]. Ki67 is a prognostic and predictive marker in breast cancer. Breast Cancer Research and Treatment (2018) 169:33–42 patients used in both clinical practice and clinical trials [4, 5]. Ki67 staining is subject to intra-tumoral heterogeneity and Ki67 scoring is prone to inter- and intraobserver variability, especially with ‘eyeballing’ [6,7,8,9,10]. Manual counting is time-consuming as at least 500–1000 cells have to be counted to achieve acceptable error rates and to correct for heterogeneity [4, 5]
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