Abstract PD06-03: Development of a highly reproducible clinical test for Ki67 using AQUA technology
Authors: Murphy DA , Pacia E , Beruti S , Lamoureux J , Dabbas B , Diver J , Christiansen J 
Published: August 2014

Abstract
Background: The measurement of Ki67 is showing promise in the management of breast cancer patients. It has shown potential for prognosis and as a chemotherapeutic response predictor, and also has a role in multivariate assessment of residual risk. However, the advancement in clinical testing has been arduous due to a lack of clinically validated scoring and/or cutpoints to guide clinicians. Standardization of Ki67 scoring is hindered by inter-laboratory variability in analysis of Ki67 in clinically relevant whole tissue sections, which are complicated not only by the subjective measurement required for IHC but also by heterogeneous expression due to biological variation and inconsistent fixation. In our diagnostic laboratory, we observed a significant amount of variability in the assessment of Ki67 using manual IHC scoring for percentage of positively stained nuclei. To attempt to reduce the variations in Ki67 assessment in clinically relevant specimens, our laboratory investigated the use of image analysis methods. Fluorescence immunohistochemistry (FIHC) coupled with the AQUA® technology has been established as a diagnostic test using image analysis and has been shown to eliminate much of the variation introduced in typical visual assessment.
Materials and methods: FIHC, based on the SP6 antibody clone, was used with AQUA technology to assess Ki67 expression as measured by percentage of positively stained nuclei. Results were compared to manually scored IHC results generated using the commonly used anti-Ki67 MIB1 antibody. IHC interpretation was performed by three pathologists on the same slides.
Results: The Ki67 assay using FIHC and AQUA technology was validated for use in a CLIA environment. Inter-reader variability by IHC was first demonstrated on whole tissues (Average difference in percent positive nuclei was 18% and ranged from 0–50%). This was further assessed on tissue microarrays, which mitigate the heterogeneity component of the tissue and resulted in the same high inter-reader variability (average percent difference was 18%). A significant correlation was observed for Ki67 percent positivity between AQUA and IHC in TMAs (Spearman: r = 0.88, p &lt; 0.0001). FIHC staining and scoring for Ki67 positivity by AQUA resulted in a 3-fold reduced variation compared to IHC and manual reads (6% variation). Using clinically relevant whole tissue sections, the accuracy of Ki67 measurement by AQUA was compared to IHC performed by an outside laboratory and resulted in a significant correlation between the two methods (Spearman: r=0.78, p &lt; 0.0001). The inter-instrument scoring variability by FIHC of a subset of the whole tissue specimens was substantially reduced compared to manual reads (Average difference in percentage of positive nuclei was 2%).
Discussion: The use of image analysis has been shown to be of significant value in expression assessment due to increased objectivity and the potential for broader standardization. We have demonstrated that the use of advanced image analysis when coupled with fluorescence immunohistochemistry can produce a system with greatly improved precision over traditional IHC methods in clinically relevant samples. These results indicate that image analysis of Ki67 is advantageous over manual IHC in clinical diagnostic assessment.
Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD06-03.