Abstract 2562: Ultra sensitive immuno-array based multiplexed assay capable of profiling RTKs and signaling proteins, heterodimers, and growth factors for routine analysis of clinical samples with limited availability

• Published in: Cancer research (Chicago, Ill.) Ill.), 2012-04, Vol.72 (8_Supplement), p.2562-2562
• Main Authors: Hoe, Nicholas, Zhou, Jinyao, Kirkland, Richard, Nguyen, Anthony, Roxas, Gio, Kuy, Crystal, Mateling, Michael, Lee, Tani, Magonova, Katya, Kim, Phillip, Liu, Xinjun, Singh, Sharat
• Format: Article
• Language: English
• ISSN: 0008-5472; 1538-7445
• DOI: 10.1158/1538-7445.AM2012-2562

Patient stratification based on target protein expressions or mutations alone has limitations in selecting efficacious therapeutic regimens for solid tumors. Information on the activation status of signaling pathway proteins as determined by their phosphorylation states and the level of expression, profiling of growth factors responsible for its activation, and their dimerization partners, can significantly influence clinical decisions. Herein we report a comprehensive analysis of key receptor tyrosine kinases (HER1, HER2, p95HER2, HER3, cMET, IGF1R, FGFRs, and others) and their downstream signal proteins (PI3K, Shc, AKT, MEK, ERK, PRAS40, RPS6, P70S6K, RSK), and dimerization partners (HER1:HER2, HER2:HER3, HER1:HER3, and others), and secreted growth factors (AREG, EREG, NRG1, HB-EGF, HGF, BTC, EGF, TGF, VEGF, and IGF-1) based on a multiplexed immunoarray CEER (Collaborative Enzyme Enhanced Reactive-immunoassay) platform. CEER platform utilizes the formation of an unique “triple-antibody-enzyme-channeling” immuno-complex. This complex requires co-localization of two detector-antibodies conjugated with corresponding channeling-enzymes once target proteins are bound by the capture antibodies. The immuno-collaboration between two detector enzymes, glucose oxidase (GO, conjugated to anti-target protein antibodies) and horseradish peroxidase (HRP, conjugated to anti-phosphorylated sites within the target) via target-specific, tyramide-mediated signal amplification enables target protein expression /phosphorylation profiling with validated analytical sensitivity between 1 to 10 cells. Using the CEER platform, quantitative modulations of target protein(s) in response to targeted agent(s) and treatment effect on associated downstream and upstream pathway proteins were measured. Due to the extreme sensitivity and specificity, CEER can be utilized in clinical samples with limited availability (e.g., FNA or CTCs). A comprehensive signaling pathway analysis can be a critical guide for selecting effective clinical strategies for targeted drug combinations or sequential therapeutic regimens. In addition, continued signaling pathway profiling after drug administration can monitor drug response and provide valuable information on potential drug resistance mechanisms. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; C