Abstract C25: Enabling in situ quantitative biomarker analysis in the context of tissue morphology
Authors: Holden Elena , Henriksen Melvin , Luther Ed 
Published: June 2012

Abstract
Modern personalized medicine makes use of individual molecular biomarkers to subdivide tumors into subsets that behave differently from each other. A large number of clinical trials are evaluating several novel therapeutic targets and relevant biomarkers. Tissue biomarkers are gaining prominent position in drug discovery and development programs and are commonly included in clinical trial testing to identify patients that would profit from particular treatments, measure drug efficacy and/or monitor the success of therapeutic interventions.
The demands on accuracy and reliability of tests are very high. The quality of tissue biomarker testing can be affected by various pre-analytic factors (time to fixation, time of fixation, type of fixation, method of tissue processing), analytic factors (assay validation, equipment validation, test reagents, standardized control materials) and post-analytic factors (interpretation criteria, use of image analysis, quality assurance procedures). In this poster we will focus on analytic and post-analytic aspects, and more specifically - imaging cytometry as a method of choice in automated analysis of tissue and tissue microarray specimens.
Materials and Methods: 1. Standard cytometric QC materials - for within-run precision of fluorescence measurements, Flow Check beads (Beckman-Coulter), for run-to-run precision of fluorescence measurements, CaliBrite Beads (B.D. Biosciences), for run-to-run precision of chromatic staining, negative and positive punch cores of breast tissue labeled for HER2/Neu with CB11 antibody developed with DAB and counterstained with Hematoxylin.
2. Tissue microarrays - whole mouse, rat and major human organ TMAs (Invitrogen).Arrays stained with dual IHC protocols for CD31/CD34, cytokeratin 7/CD31, S100/GFAP, kappa/lambda (in situ hybridization) and Ki67/CD68.
3. Specimens were analyzed on an iCys® Imaging Cytometer (CompuCyte Corporation, Westwood, MA).
Quality control measures and Results: a) within-run precision of fluorescence measurement - a Coefficient of Variation (CV) of fluorescence intensity of less than 3% measured over a large bead population; b) run-torun precision of fluorescence measurements, the mean of fluorescence intensity measurements within +/− 2 Standard Deviations (SDs) as established by baseline runs; c) run-to-run precision of chromatic staining - the mean DAB laser light loss measurements within +/− 2 Standard Deviations (SDs) as established by baseline runs.
TMA analysis: instrument automatically identified core elements, performed spectral deconvolution to isolate markers of interest. Chromatic measurements by laser-light-loss and fluorescence measurements were obtained simultaneously and combined to quantify the expression of the markers of interest. High resolution images ofthe cores were obtained simultaneously with the quantitative analytical data. Quantitative end-points on a per-core basis included: percent positive marker expression, mean expression level, total marker expression level and co-localization of markers. The technique of “image masking” effectively reduced the background levels in tissue elements.
Summary: LSC technology provides accurate and reproducible measurements of tissue biomarkers in unbiased, highly quantitative manner. It is well suited for tissue-based biomarker discovery and clinical trials.
Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C25.