Comparative Validation of the SP6 and MIB1 Antibodies to Ki67 and Their Use in Tissue Microarray (TMA) and Image Analysis for Breast Cancer.

Abstract

Abstract Aim: To compare SP6 and MIB1 antibodies against Ki67 for use with visual scoring, image analysis and TMA (tissue microarray) analysis.Background: Immunohistochemical detection of Ki67 has been widely used in assessing the proliferation fraction in breast cancer. Neoadjuvant studies revealed that changes in Ki67 predict benefit from endocrine treatment, and that on-treatment Ki67 measurements are better predictors of long-term outcome than pretreatment levels (Dowsett at al, JNCI 2007; 99:167-170). As a result Ki67 is the primary end-point for some pre-surgical trials. MIB-1 has been the preferred Ki67 antibody for over 10 years. A rabbit monoclonal antibody, SP6, has become available with apparently improved performance, e.g. reduced background non-nuclear staining. The importance of Ki67 led us to conduct a systematic series of studies to validate SP6 in comparison to MIB1.Methods: We used TMAs constructed from (i) 177 archival FFPE blocks of primary or locally recurrent breast tumours from women treated with an aromatase inhibitor (AI) for advanced disease (AI TMAs) and (ii) one set of replicate blocks (200 tumours) from the TransATAC program. The TMAs were of 600µm cores. Three replicate TMAs (A, B, C) were used. Staining was performed using DAKO Autostainer and DAKO REAL Kit Detection System. MIB-1 antibody (DAKO) was used at a dilution of 1:40, whereas 1:100 dilution was used for SP6 antibody (Abcam). Stained sections were examined either visually or using the Ariol image analysis system (Genetix Ltd.).Results: There was a strong correlation between the antibodies in AI TMAs scored manually (n=161, r=0.93, p<0.0001). Coefficient of variation was calculated in all cases with ≥2 available cores. MIB1 showed higher median CV [%] compared with SP6 (43.7 vs. 36.7, respectively) in AI TMAs, but a lower CV in TransATAC TMAs (42.0 vs. 51.4, respectively). In a univariate analysis of manual scores from AI TMAs there was a similar association of Ki67 with time to treatment failure (TTF) with the two antibodies (HR: 1.15; 95% CI: 1.00-1.33; p=0.052 and HR: 1.19; 95% CI: 1.00-1.43; p=0.045 for MIB1 and SP6 respectively). Correlation between manual and image analysis scores was markedly better with the SP6 antibody (r=0.71 and r=0.88 for MIB1 and SP6, respectively).Conclusions: SP6 and MIB1 provide highly comparable measures of Ki67 that predict progression of advanced disease similarly. SP6 is substantially better suited than MIB1 to image analysis, and is now our preferred antibody for future studies.Funded by Breakthrough Breast Cancer and The Mary-Jean Mitchell Green Foundation. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3048.