Abstract
BackgroundThe clinical benefits associated with targeted oncology agents are generally limited to subsets of patients. Even with favorable biomarker profiles, many patients do not respond or acquire resistance. Existing technologies are ineffective for treatment monitoring as they provide only static and limited information and require substantial amounts of tissue. Therefore, there is an urgent need to develop methods that can profile potential therapeutic targets with limited clinical specimens during the course of treatment.MethodsWe have developed a novel proteomics-based assay, Collaborative Enzyme Enhanced Reactive-immunoassay (CEER) that can be used for analyzing clinical samples. CEER utilizes the formation of unique immuno-complex between capture-antibodies and two additional detector-Abs on a microarray surface. One of the detector-Abs is conjugated to glucose oxidase (GO), and the other is conjugated to Horse Radish Peroxidase (HRP). Target detection requires the presence of both detector-Abs because the enzyme channeling event between GO and HRP will not occur unless both Abs are in close proximity.ResultsCEER was able to detect single-cell level expression and phosphorylation of human epidermal growth factor receptor 2 (HER2) and human epidermal growth factor receptor 1 (HER1) in breast cancer (BCa) systems. The shift in phosphorylation profiles of receptor tyrosine kinases (RTKs) and other signal transduction proteins upon differential ligand stimulation further demonstrated extreme assay specificity in a multiplexed array format. HER2 analysis by CEER in 227 BCa tissues showed superior accuracy when compared to the outcome from immunohistochemistry (IHC) (83% vs. 96%). A significant incidence of HER2 status alteration with recurrent disease was observed via circulating tumor cell (CTC) analysis, suggesting an evolving and dynamic disease progression. HER2-positive CTCs were found in 41% (7/17) while CTCs with significant HER2-activation without apparent over-expression were found in 18% (3/17) of relapsed BCa patients with HER2-negative primary tumors. The apparent 'HER2 status conversion' observed in recurrent BCa may have significant implications on understanding breast cancer metastasis and associated therapeutic development.ConclusionCEER can be multiplexed to analyze pathway proteins in a comprehensive manner with extreme specificity and sensitivity. This format is ideal for analyzing clinical samples with limited availability.
Highlights
The clinical benefits associated with targeted oncology agents are generally limited to subsets of patients
Collaborative Enzyme Enhanced Reactiveimmunoassay (CEER) can detect ErbB-receptor tyrosine kinases (RTKs) activation status at the single cell level in breast cancer cell lines CEER was used to detect the expression and activation of human epidermal growth factor receptor 1 (HER1) and human epidermal growth factor receptor 2 (HER2), receptor members of the ErbB-RTK family, at a sensitivity level of a single cell in breast cancer cell lines, MDA-MB-468 and SKBr3, respectively (Figure 2a). These cell lines have been well characterized for their ErbB-RTK expression status [17,18,19,20,21]
RTK expression is approximately 1 to 2 × 106 HER1 or HER2 receptors per cell in MDA-MB-468 and SKBr3, respectively, only subsets of these receptors are phosphorylated at any given instance
Summary
The clinical benefits associated with targeted oncology agents are generally limited to subsets of patients. There is an urgent need to develop methods that can profile potential therapeutic targets with limited clinical specimens during the course of treatment. With the advent of HER2-targeted therapies, most notably trastuzumab, the natural progression of HER2-positive breast cancers can be dramatically blunted [3,4]. Only approximately 50% of HER2-positive patients initially respond to trastuzumab-complemented treatments while the rest show inherent resistance and can metastasize to distant sites. Even the patients who demonstrate a dramatic initial response to trastuzumab eventually develop resistance [6]. If there were a way to prospectively predict the course of breast cancer progression and strategically segregate the responders from the non-responders, it would eliminate uncertainty in treatment and save valuable time providing most effective evidence-based therapeutic outcome
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
