Using Histogram Image Analysis To Improve the Interpretation of Ac-Histone H3 Biomarker Expression in HDAC Inhibitor Treated Xenograft
Y Gao, R Mosher, J Deeds. Novartis Institutes for BioMedical Research, Inc., Cambridge, MA
Background: Acetylated-histone H3 (Ac-H3) increases with HDAC inhibition. Ac-H3 was examined in treated xenograft models. In initial studies, thresholds to determine levels of Ac-H3 were set empirically with binned values. The purpose of this study was to determine if additional information could be obtained by looking at this IHC data as a continuous series, rather than discrete values.
Design: The Ac-H3 antibody was evaluated on tissue sections from three animals treated with 20 mg/kg of an HDAC inhibitor over a series of dilutions using the Ventana Immunostainer. Whole tissue images were analyzed using the Aperio Scanscope and the Aperio Imagescope IHC Nuclear Algorithm Version 8.0 software. First, the slides were examined by setting manual thresholds for positive signal. The slides were re-examined using a histogram representation of all the data points, to see if additional information could be revealed.
Results: By eye, a greater difference could be seen between groups as the antibody was titrated. At a titration of 1:1000, we could easily set an empirical threshold for staining that allowed us to bin samples into treated vs untreated. When frequency data was plotted for all optical density values, and ROC curves generated, we could see that the by eye threshold favored specificity over sensitivity. Furthermore, while the higher concentration assays were difficult to interpret by eye, the higher antibody concentration caused the ROC curve to shift to the upper left hand corner.
Conclusions: 1) Empirical interpretation can be improved by examining a series of titrations. 2) Empirical threshold can be examined against ROC curves to understand the relative value of sensitivity and specificity. 3) Image analysis methods may perform best at titrations that are not optimal for empirical reads.
Wednesday, March 11, 2009 1:00 PM
Poster Session VI # 250, Wednesday Afternoon