Abstract 1173: Development of calibrators and controls as quality control tools for clinical implementation of quantitative immunofluorescence assays for pharmacodynamic biomarkers of molecular targeted agents in tumor biopsies.
Authors: Ferry-Galow Katherine V. , Navas Tony , Lawrence Scott M. , Mutreja Karun , Butcher Donna , Hollingshead Melinda , Parchment Ralph , Tomaszewski Joseph E. , Doroshow James H. , Kinders Robert J. 
Published: August 2014

Abstract
The application of robust pharmacodynamic (PD) assays as part of clinical trial design is valuable for confirming putative drug mechanisms in patients. Our group supports the development, validation and clinical implementation of these PD assays including quantitative immunofluorescence (qIFA) measurements in formalin-fixed, paraffin-embedded (FFPE) human tissue biopsies. One such assay is a multiplex qIFA method for evaluating histone H2AX phosphorylated at serine 139 (γH2AX), activated caspase 3 (cCasp3), Ki67, and DAPI (referred to as Rx1-IFA4) in response to DNA-damaging agents. Integral PD quality control tools were developed as part of the Rx1-IFA4 and included xenograft tissue calibrators, which are used as visual and quantitative reference standards representing a range of biomarker expression levels for drug effect on tumor, and mouse testis and jejunum tissues, which are used as positive or negative controls for endogenous tissue expression for each biomarker. Rx1-IFA4 tissue calibrators were prepared with tumor quadrants derived from human breast MDA-MB-231 xenografts treated with vehicle, low, or high doses of a cIAP inhibitor. Xenografts were prescreened for levels of each biomarker, and tissues containing defined ranges of biomarker expression were re-embedded in a block to represent each of the treatment groups. Use of tumor quadrants as opposed to biopsies allows for a large number of calibrator slides to be produced from a single qualified block. Qualification of each lot of calibrator slides includes qIFA analysis of representative slides from which specifications for acceptable ranges of biomarker expression for that lot are determined. Whole fluorescent slide imaging using Aperio® was performed to document distribution patterns of the biomarkers within each tumor quadrant. Staining analysis of control tissues also enabled performance assessment of all assay antibodies applied on each clinical slide without the need for duplicate slides as controls. Application of tissue calibrators and controls to the qIFA assay, as opposed to the more typical use of cell pellet-derived controls provides many advantages. Biomarker expression levels with drug treated cells rarely correlate with expression levels in vivo. Additionally, quantitative image analysis for this assay utilizes image analysis software and custom macros that require the appropriate selection of a representative area of background for each analyzed image and tissue context is critical for accurate quantitation. These quality control tools are effective for monitoring the performance of the qIFA assay across laboratories and clinical assay runs. The first three authors listed contributed to this work equally. Funded by NCI Contract No HHSN261200800001E.
Citation Format: Katherine V. Ferry-Galow, Tony Navas, Scott M. Lawrence, Karun Mutreja, Donna Butcher, Melinda Hollingshead, Ralph Parchment, Joseph E. Tomaszewski, James H. Doroshow, Robert J. Kinders. Development of calibrators and controls as quality control tools for clinical implementation of quantitative immunofluorescence assays for pharmacodynamic biomarkers of molecular targeted agents in tumor biopsies. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1173. doi:10.1158/1538-7445.AM2013-1173