Prevalence of Activated &amp; Total p95HER2 and Other Receptor Tyrosine Kinases in Breast Cancer.
Authors: X. Liu, P. Kim, R. Kirkland, T. Lee, F. Lin, L. Liu, L. Zhang, K. Magonova, B. Ybarrondo, S. Singh
Journal: Cancer Research (2009)

Abstract Background: HER2-overexpressing breast cancer has poor prognosis and is often resistant to HER2 targeted monoclonal antibody therapy. One of the mechanisms of de novo or acquired resistance is expression of p95HER2, various forms of truncated HER2 receptors with missing amino-terminal extra cellular domains. Methods for profiling various forms of HER2 receptors and other receptor tyrosine kinases (RTKs) with transactivation potential in primary and metastatic tumors may provide valuable insight into the shifting disease pathogenesis.Methods: A novel technology capable of specifically detecting phosphorylation events in ErbB family RTKs has been developed. This multiplexed protein microarray platform utilizes the formation of a unique immuno-complex requiring the co-localization of two detector enzyme-conjugated-antibodies once target proteins are captured on the microarray-surface. The channeling events between two detector enzymes (glucose oxidase &amp; horse radish peroxidase) in proximity enabled the profiling of the RTK with extreme sensitivity. The analytical specificity is greatly enhanced given the requirement for simultaneous binding of three different antibodies. Different configuration of detector antibodies allowed differential detection of truncated targets (i.e., p95HER2) from their normal counter parts (i.e., HER2). Here we report successful profiling of signal transduction pathway molecules for their expression and activation using 240 FNA samples collected from breast cancer patients (stage II to IV) with various ER/PR/HER2 status.Results: The FNA samples collected using G23 gauge needles were analyzed for expression and activation status for various RTKs and downstream signal transduction molecules including p95HER2, HER2, HER1, HER3, IGF-1R, PI3K and Shc. We found:Ø 100% concordance between primary HER2-IHC status and HER2 expression analysis based on proximity mediated immuno-microarray system Ø presence of varying degree of p95HER2 in over 40% of HER2-positive (HER2: 3+ and 2+ with FISH+) patients Ø ∼50% of p95HER2 expressors had activated p95HER2 Ø 25% of HER2-positive samples also had some levels of HER1 and HER3 activation Ø HER2-negative (HER2: 2+ with FISH-, 1+ and 0) samples also had 100% concordant HER2 expression, but number of them showed low but significant levels of HER2, HER3 and HER 1 activation.These findings may have implication on selection of appropriate targeted therapeutics.Discussion: The multiplexed-immuno microarray was utilized to detect the expression and phosphorylation of p95HER2, HER2, other RTKs (including HER3, HER1, IGF-1R, Shc, and PI3K) in 240 FNA samples collected from unique breast cancer patients with various ER/PR/HER2 status. Having the ability to profile tumors at different metastatic sites with an expanded pathway panel could provide information on their differential metastatic potentials; hence minimally invasive single-passage-mFNA samples may be utilized to tailor therapy options as disease-profile changes. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3053.