Using quantitative image analysis of a putative immunohistochemistry assay against C-met to guide companion diagnostic decisions.

When developing an immunohistochemistry (IHC) based biomarker or companion diagnostic assay, establishing reagent specificity is very challenging. Because antibodies recognize three dimensional epitopes, epitope recognition may be based on a specific confirmation (activated, receptor occupied, etc) or biological state (glycosylation, cell surface vs cytoplasmic) of the target protein. Furthermore, recognition of the epitope in Formalin-Fixed, Paraffin Embedded (FFPE) specimens imposes additional challenges due to the fixation effects. For these reasons, different antibodies against the same target biomarker may demonstrate different prevalence, range, and staining patterns in the same specimens. Determining reagent specificity is a critical part of IHC assay development; but the typical approaches utilized (such as Western Blotting, etc) do not directly prove specificity in the FFPE setting. Additionally, the presence or absence of the protein in a particular tissue (tumor vs stroma) or cell (membrane/ cytosol/ nuclear) compartment may be critical to associate the drugs mechanism of action with efficacy. In the companion diagnostic (CDx) setting, the mechanism of the drug, epitope recognition, and staining features used to interpret and quantify the biomarker to predict patient response requires an evidence-based approach, where all features of an IHC assay are considered and tied empirically to patient response to the drug. In this study, we demonstrate these complexities in gastric cancer specimens, by comparing IHC assays using two antibodies against the intracellular vs extracellular domains of c-met (SP44/ C-term and EP154Y/ N-term) in the context of the ligand for c-met, Hepatocyte Growth Factor (HGF). Therapeutic antibodies targeting c-met [such as MetMab® (OA-5D5/ Roche)]; or HGF directly [such as Rilotumumab (AMG 102/ Amgen)], have linked c-met protein expression by IHC directly to patient response. Thus, we hypothesized that a link between c-met protein expression and HGF should be discernible. To test this hypothesis, we used image analysis approaches to determine the staining features of each IHC assay in relation to each other. Surprisingly, we found little concordance between the two c-met antibodies in evaluating c-met and HGF expression. We found distinct populations of c-met expressing vs HGF expressing specimens, whose HGF-c-met association differed with the c-met assay used. We examined the tissue and cell compartmentalization, and identified staining features of each reagent which would aid or impede in interpretation strategies for understanding the relationship between c-met and HGF. These results suggest that the epitope-specific features of each c-met antibody determines the relationship with HGF expression, and how quantitative image analysis endpoints can be used to make critical decisions during the development of an IHC companion diagnostic. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C34. Citation Format: Joseph S. Krueger, Brian Laffin, Mirza Peljto, Mahipal Suraneni, Holger Lange, David Young. Using quantitative image analysis of a putative immunohistochemistry assay against C-met to guide companion diagnostic decisions. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C34.