Profiling of receptor tyrosine kinases (RTK) activation in circulating tumor cells (CTCs) in metastatic tumors using proximity mediated microarray immunoassay

Abstract

11024 Background: The abnormal activation of HER1 and HER2 has been linked to various types of cancer progression, and the changes in their expression status between primary tumor and CTCs have been reported to occur at a significant frequency. Methods for detecting HER1/HER2 phosphorylation in serially collected CTCs may provide valuable insight into the overall disease profile shift, and therefore lead to better selection of therapy combination for individual patient. Methods: A triple-antibody-enzyme-channeling multiplexed protein microarray platform has been developed to detect the phosphorylation on target molecules. It utilizes a unique immuno-complex formation via co-localization of two detector enzyme-conjugated-antibodies once target proteins are captured on the microarray-surface. The channeling events between two detector enzymes in proximity enabled profiling of the RTKs with a single-cell level sensitivity. The simultaneous binding of three different types of target specific antibodies for signal amplification results in extremely high assay specificity. In order to validate the method on clinical samples, CTCs from 75 cancer patients (stage 3/4) on various therapy regimens were analyzed. Results: We identified 6 patients (8%) with activated HER1, 6 patients (8%) with activated HER2 and 14 (18.5%) patients with dual RTK activation in their CTCs from various types of metastatic cancer. 25 samples from normal subjects showed no detectable HER1/HER2 activation. We also observed discrepancies between HER2 activation status in CTCs and their corresponding primary HER2-IHC status among breast cancer patients. CTCs with activated-HER2 were found in 6 out of 16 (38%) patients with HER2-negative primary breast cancer. In addition, 2 out of 5 HER2-positive breast cancer patients had CTCs with no apparent HER2-activation. Conclusions: The multiplexed-proximity mediated immunoassay successfully detected the activation of RTKs in CTCs isolated from various cancer patients. As CTCs found in metastatic stage represent the most aggressive invading cell population, serial CTC-profiling can lead to better therapy selection/adjustment and disease/treatment monitoring as there are available options to choose appropriate kinase inhibitors for RTK-targeted therapies. [Table: see text]