Abstract 826: Expression levels of circulating soluble-EphA2 receptor in cancer patients
Authors: Bradley Stephen P. , Hamer Peter J. , Carney Walter P. 
Published: February 2011

Abstract
EphA2 is a receptor tyrosine kinase that critically controls many aspects of cell behavior. It is expressed in a manner that is consistent with the regulation of cell growth, migration and invasion. Analysis by IHC has shown that elevated expression of EphA2 exhibits predictive value in lung cancer survival and recurrence. Importantly, current data suggests that targeting EphA2 could be beneficial and is currently being tested in an ovarian cancer setting. We developed a prototype ELISA for circulating EphA2 receptor in order to investigate expression levels of the soluble form of EphA2 in certain cancer types. We have developed a prototype ELISA which measures circulating human EphA2 receptor. 96 well Nunc plates were coated with an affinity-purified goat polyclonal antibody specific for the extracellular domain (ECD) of human EphA2. Human serum and plasma samples were diluted 5-fold in a sample diluent with blocking reagents. The standard was a mammalian cell-derived form of the ECD for the human EphA2 receptor. Standards and samples were incubated on the plates for 2 Hours, 37C. The detector antibody was a biotinylated affinity-purified goat polyclonal antibody and was added to the plates for 1 Hour, 37C. Plates were washed and a streptavidin-HRP conjugate was added to the plates for 30 minutes, 22C. A final wash was performed and a TMB substrate was added to the plates for color development. The reaction was stopped with sulfuric acid and plates were read at an absorbance of 450nm. Data generated to date using this prototype ELISA indicates that over-expression of soluble-EphA2 occurs in many cancers versus normal samples tested. The 95% CI of total normal plasma and serum was 672_1306 and 894_1429pg/mL, respectively. Capture Inhibition studies revealed specificity of the assay in human samples (100% inhibition). Parallelism studies revealed acceptable performance in serially diluted samples. Significant over-expression of soluble-EphA2 was observed in benign lung cancers versus normals (100%, 10/10) while staged lung cancers exhibited significantly lower levels of circulating EphA2 (p&lt;0.001) compared to benign (96%, 148/154). Elevations of circulating EphA2 were found in glioblastoma (73%, 22/30), ovarian (48%, 28/59), pancreatic (46%, 19/41), and late stage breast (44%, 15/34) cancer patients. Immunoprecipitation experiments are ongoing to visualize soluble EphA2 by western blot, with initial data showing a specific band at ∼60kD. This study describes the discovery of circulating EphA2 receptor in human samples using a prototype ELISA. Levels of soluble-EphA2 are over-expressed in glioblastoma, ovarian, pancreatic, and late stage breast cancer samples. Decreased expression of the circulating form of EphA2 is observed in staged lung cancer versus benign. Measuring the levels of the soluble-EphA2 in circulation may have potential utility for cancer patients who may benefit from EphA2 targeted therapies.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 826.