Abstract 1616: Novel caged hapten proximity assay platform enables detection of oncogenic signaling-associated complexes in the clinical pathology laboratory setting
Authors: Kelly Brian D. , Polaske Nathan W. , Belosludtsev Yuri , Smith Matthew A. , Boyle Theresa , Chen Y. Ann , Haura Eric B. 
Published: August 2018

Assays measuring protein complexes represent a novel class of protein-based biomarkers. Using proximity-based readouts, we and others have previously shown utility in monitoring signaling-associated complexes to predict response to targeted therapeutics in lung cancer. However, fluorescence-based proximity assay readouts are difficult to implement in a clinical setting and are not conducive to typical pathology laboratory workflow. Here, we provide evidence of a novel proximity-based technology platform that utilizes a “caged hapten” proximity readout followed by chromogenic immunohistochemistry (IHC) detection. We have synthesized haptens modified with a phosphorylated quinone methide precursor. This enzyme- labile caging group abrogates antibody binding; however, upon exposure to alkaline phosphatase (AP) the native hapten is regenerated. These caged haptens may be applied in a proximity assay format by the use of a first antibody labeled with caged haptens that can be uncaged by AP conjugated to the second antibody. Only when the two epitopes of interest are in close proximity to one another will the AP interact with the caged hapten and uncage it. The native hapten, which represents the protein complex population, can then be visualized by an anti-hapten antibody conjugated to horseradish peroxidase (HRP) followed by 3,3'-diaminobenzidine (DAB) detection. These reagents and assays are fully automated on the VENTANA DISCOVERY ULTRA platform. We demonstrate detection of β-Catenin:E-Cadherin, EGFR:GRB2 and MET:GRB2 complexes in formalin-fixed paraffin embedded (FFPE) lung cancer cell lines, patient-derived xenografts (PDX) and non-small cell lung cancer (NSCLC) patient specimens. Assay specificity was confirmed through technical controls involving reagent omission experiments, and biologically by treatment with small molecule kinase inhibitors that disrupt kinase:adaptor interactions. We revealed heterogeneous EGFR and MET signaling in MET-amplified PDX specimens, replicating previously-published findings. For EGFR:GRB2, we developed a pathologist-guided ordinal scoring system and evaluated inter-rater concordance using a tissue microarray containing 120 NSCLC cores. Kappa statistics and inter-rater correlations reveal excellent correlation among multiple raters.Collectively, these data support the adaptation of proximity-based approaches in clinical pathology settings. The use of caged hapten proximity assays on VENTANA automated slide stainers provides a novel and robust platform to evaluate protein complexes as potential biomarkers that may be incorporated as molecular correlates in ongoing early phase clinical trials. This approach also facilitates analysis of a large number of slides in a reproducible assay format enabling retrospective evaluation of tumor specimens from completed trials.

Citation Format: Brian D. Kelly, Nathan W. Polaske, Yuri Belosludtsev, Matthew A. Smith, Theresa Boyle, Y. Ann Chen, Eric B. Haura. Novel caged hapten proximity assay platform enables detection of oncogenic signaling-associated complexes in the clinical pathology laboratory setting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1616.