Abstract 4640: Highly sensitive proximity mediated immunoassay can determine functional VEGFR2 status in clinical samples for routine clinical uses.
Authors: Hester Kelly D. , Hoe Nicholas , Zhou Jinyao , Kuy Crystal , Zhang Liching , Kim Phillip , Liu Xinjun , Singh Sharat 
Published: August 2014

Abstract
VEGFR2 is the primary signaling receptor for VEGF-A, and is considered to be the major initiator of angiogenesis. Binding of the growth factor activates VEGFR2 via tyrosine phosphorylation and results in induction of angiogenic and lymphangiogenic signals. An understanding of the VEGFR2 status of human tumors may help to identify patients who would benefit most from treatment with VEGFR2 inhibitors. Currently there are several drugs targeting VEGFR2 either approved for clinical use or in various stages of clinical trials. Bevacizumab, a monoclonal antibody to VEGF-A is used to treat patients with lung, colon and breast cancer. Sunitinib targeting VEGFR2, PDGFRβ and c-Kit was approved for treating carcinoid tumors in the gastro-intestinal stroma. VEGFR2 profiling could be used to monitor the efficacy of anti-angiogenic treatment in cancer patients. Here we report the development of immunoarray assays for functional characterization of VEGFR2 and their application in profiling VEGFR2 status in preclinical and clinical specimens.
The CEERTM (Collaborative Enzyme Enhanced Reactive-immunoassay) assays use an antibody-microarray based platform which utilizes the formation of a unique “triple-antibody-enzyme-channeling” immuno-complex capable of measuring expression (total) and activation (phospho) of VEGFR2 in tumor tissues or surrogate tissues with limited availability. CEERTM platform can detect levels of expression and activation of VEGFR2 in as little as 10 cells due to its unique proximity channeling assay configuration. Specimens are analyzed along with standards so quantitative read out can be compared longitudinally. Assay specificity was established by comparing levels of phosphorylated VEGFR2, between the non-treated and treated with VEGF. Phosphorylation levels were increased in VEGFR2 positive cell lines HUVEC and H441 when treated, while VEGFR2 negative cell lines MDA-MB-134 IV, BT474, SNU16, Ramos lymphoma, and K562 did not. In clinical setting, robust quantifiable signals were detected in FNAs or core biopsy tissues obtained from various cancer patients. Ongoing analysis of clinical samples from colorectal, lung and renal cancer patients indicates an increase of VEGFR2 phosphorylation in tumor samples compared to healthy tissues and a reduction of VEGFR2 phosphorylation post VEGFR2-targeted therapy in some patients. 47% (18/38) of NSCLC patients have shown evidence of activated VEGFR2, and it would be interesting to determine if patients with activated VEGFR2 respond to Bevacizumab.
The CEER-based VEGFR2 assay can provide quantitative analysis of clinical samples with limited availability providing critical information for developing effective strategies for treatment selection, monitoring drug response and overcoming potential resistance in cancer patients.
Citation Format: Kelly D. Hester, Nicholas Hoe, Jinyao Zhou, Crystal Kuy, Liching Zhang, Phillip Kim, Xinjun Liu, Sharat Singh. Highly sensitive proximity mediated immunoassay can determine functional VEGFR2 status in clinical samples for routine clinical uses. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4640. doi:10.1158/1538-7445.AM2013-4640