Abstract B27: Proximity ligation assays as biomarkers for receptor tyrosine kinase activity in lung tumors.
Authors: Smith Matthew A. , Hall Richard , Fisher Kate , Chen Ann , Haura Eric B. 
Published: August 2014

Abstract
In the era of precision medicine, matching patients to targeted therapies is a critical goal and novel biomarker strategies must be implemented as new therapeutic options are introduced into the clinic. However, most traditional biomarkers fail to capture the actual signaling activity occurring within a patient's tumor. Proximity ligation assays (PLA) represent a novel approach to quantify receptor tyrosine kinase activity in clinical specimens, yielding a fluorescent readout of in situ signaling complexes. We have developed PLA assays for multiple receptor tyrosine kinase-adaptor pairs, including EGFR in complex with its major adaptor protein, GRB2. Here, we demonstrate robust performance of these assays in both cell lines and FFPE tissue specimens and provide evidence of predictive biomarker capacity for tyrosine kinase inhibitor response in lung cancer patients.
For EGFR-GRB2, we demonstrate PLA is highly sensitive and specific, reflecting kinase activity and not merely expression levels of EGFR or GRB2. In non-small cell lung cancer (NSCLC) cell lines, high PLA signal is associated with EGFR mutation status and levels of phospho-EGFR. PLA also correlates with results from co-immunoprecipitation experiments and is abrogated by erlotinib in a dose-dependent manner. In multiple cell line xenograft models, PLA signal is observed in cytokeratin-staining regions and reveals dissociation of EGFR-GRB2 signaling complexes in response to erlotinib. In patient-derived lung adenocarcinoma xenografts, significantly higher PLA signal is observed in EGFR mutant xenografts relative to wildtype xenografts and is strongly reduced with erlotinib treatment. We have analyzed three cohorts of mixed histology NSCLC tumor specimens (N =354 patients) and scored these on a 0-3+ scale, finding good agreement of incidence of ≥ 2 PLA score across the three cohorts. EGFR protein expression levels as determined by Automated Quantitative Analysis (AQUA) correlate with EGFR-GRB2 PLA signal, but clear instances are defined where PLA signals are discordant, indicating “value-added” over EGFR expression analysis. High PLA score was not limited to adenocarcinomas and was observed in multiple histologies, with significant enrichment in metastatic brain lesions (P&lt;.0001). PLA signal was enriched in tumors with known EGFR mutation status (P&lt;0.01 by x2), but we also identified high signal in 24.5% (13 of 53) of KRAS mutant lung cancers and in 34% (26 of 77) of lung cancers lacking either EGFR or KRAS mutations (WT/WT). Among erlotinib-treated patients with follow-up data (N=91), we identified a strong trend of higher EGFR-GRB2 PLA signal associated with improved overall survival (P = 0.127) and a near doubling in median survival (10 months vs. 20 months) which was not observed when EGFR expression was evaluated by AQUA. Furthermore, PLA assays can be used on archival specimens in which phospho-IHC fails and can also be conducted using brightfield microscopy detection.
Collectively, our data indicate that PLA assays measuring EGFR:GRB2 signaling complexes can serve as biomarkers for druggable kinase activity. We believe this novel proteome-level approach has the potential to be applied in a predictive capacity in multiple malignancies.
Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B27.
Citation Format: Matthew A. Smith, Richard Hall, Kate Fisher, Ann Chen, Eric B. Haura. Proximity ligation assays as biomarkers for receptor tyrosine kinase activity in lung tumors. [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 B27.