Abstract 5129: Mapping the activity of oncogenic signaling networks with phospho-specific Liquid Tissue® mass spectrometry

Abstract

Abstract Many of the current targeted therapies in oncology target the activity of either receptor tyrosine kinases (Her2, EGFR, IGF1R, cMet, FGFR, PGDFR) or cytoplasmic tyrosine kinases (cSrc, AurA, PI3K, MEK, Raf, Akt). The current slate of clinically useful diagnostic tests measure target expression either directly by immunohistochemistry or indirectly by extrapolation from the level of gene or mRNA expression/amplification. These assays are limited by lack of quantitation, and no assay can directly assess the activation state of the signaling pathway components. The lack of information regarding target activation and downstream signal transduction can be overcome using the Liquid Tissue®-SRM technology platform. This approach enables relative and absolute quantification of multiple proteins and their phosphorylation status directly in formalin fixed tissue. In order to fill this diagnostic ‘gap’ we have used this platform to develop a quantitative multiplexed phospho-target assay format which measures the specific phosphorylation state of many clinically relevant oncogenic kinases (EGFR, cMet, Her3, Erk, cSrc, Mek, Akt, p70S6K) directly in FFPE tumor tissue. This phosphopeptide multiplex assay was initially preclinically validated on the A431 tumor cell line which harbors an amplification of the EGFR gene. These cells were stimulated with a dose range of EGF (50-200ng/ml) or in a time course study (EGF 50ng/ml for 5-30min). Confluent, EGF stimulated cells were then formalin fixed, subjected to Liquid Tissue® processing, and then phospho-enriched using TiO2 magnetic beads. The resulting enriched phosphopeptides were then analyzed by mass spectrometry. This method demonstrated the feasibility, and reproducibility of this method for quantitating EGFR pY1197, EGFRpT693, AKT pS473, p-p70S6K pS447, ERK pT202/pY204. We extended this study by performing phosphoenrichment and mass spectrometric analysis of human tumor xenografts, primary human tumor NSCLC explants and clinical trial tissue from EGFR inhibitor treated NSCLC and colorectal cancer patients. In each case we were able to enrich and measure the phosphorylation of a large set of important oncogenic kinases. Our intention is to develop this diagnostic tool to provide a multiplex assay format in formalin fixed tissue that can be applied from preclinical to clinical studies that will impact both targeted drug development and patient stratification needs in this era of personalized healthcare. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5129. doi:10.1158/1538-7445.AM2011-5129