Therapeutic Implications of Detection of Amplification and Activation of HER2 and Other Receptor Tyrosine Kinases (RTKs) in Circulating Tumor Cells (CTCs) in Recurrent Breast Cancer.

Abstract

Background: HER2 is one of four trans-membrane RTKs in epidermal growth factor receptor family, and HER2-positive phenotype has been associated with aggressive subtype of breast cancer with HER2 gene amplification. Approximately 15-20% of breast cancers are considered HER2-positive by IHC or FISH analysis. Recently, changes in HER2 expression status between primary tumor and CTCs found in recurrent metastatic disease have been reported to occur at a significant frequency. Methods for detecting HER2 expression and phosphorylation in serially collected CTCs may provide valuable insight into the overall disease profile shift, and therefore lead to better selection of therapy for each patient.Methods: A triple-antibody-enzyme-channeling multiplexed protein microarray platform has been developed to detect the phosphorylation on target molecules. Extremely high assay specificity was achieved by 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. In order to validate the method on clinical samples, CTCs from 77 breast cancer patients on different therapy regimens were analyzed at various time points along their course of therapy.Results: Whole blood of 77 metastatic cancer patients and 60 healthy volunteers were analyzed for CTC-HER2 expression and activation. We observed significant HER2 status conversion with recurrent disease. 29% of patients with negative HER2 expression in the primary tumor showed HER2-amplification in isolated CTCs. Phosphorylated HER2 receptors were found in 52% of patients with primary HER2 negative disease. The enhancement of assay sensitivity and specificity using proximity mediated immuno-assay made the detection of HER2 activation (even without amplification) possible when isolated CTCs were stimulated with ligands to other RTKs with transactivation potential.Discussion: The multiplexed-proximity mediated immunoassay successfully detected the expression of HER2 RTKs and their degree of activation in CTCs isolated from recurrent breast cancer patients. As we hypothesize that CTCs found in metastatic stage represent the most aggressive and invasive 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. While significant number of patients acquired HER2-amplification in their CTCs, substantially higher rate of CTC-HER2 activation was found in relapsed metastatic disease. The unique triple-antibody mediated immuno-microarray analysis allowed a single cell level profiling of the CTC-HER2. The ability to profile serially collected CTCs will provide valuable information on changes occurring in tumor cells as a function of time and treatment. This method can provide guidance, not only for initial selection of targeted therapeutics, but also in subsequent monitoring for rapidly 'evolving' disease in individual patient. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3010.