Highly sensitive proximity mediated immunoassay reveals HER2 status conversion in the circulating tumor cells of metastatic breast cancer patients

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.

Minimal HER1 and HER2 expressions in CHO and HEK-293 cells cause them appropriate negative cells for HERs-related studies

“…5 copy/µg) in MDA-MB-468 and HER2 (6.5622×10 6 copy/µg) in SKBR3 in our study are concordance to previous studies which reported that MDA-MB-468 and SKBR3 highly expressed HER1 and HER2, respectively (23)(24)(25)(26)(27)(28). While HER2 expression has not been detected in different studies (29,30), our results showed that MDA-MB-468 expressed HER2 as much as 1.5×10 4 copy/µg.…”

Discordance rate of HER2 status in primary breast carcinomas versus synchronous axillary lymph node metastases: a multicenter retrospective investigation

“…The conversion in HER2 status is consistent with the evidence that HER2 gene amplification can be acquired during disease progression or recurrence in BC patients 31,32. Indeed, breast tumor cells may have different pathophysiological states, divide continuously, and harbor the potential for further mutations.…”

The association of tissue anti-TNF drug levels with serological and endoscopic disease activity in inflammatory bowel disease: the ATLAS study

“…TNF and HER2 were measured using a proprietary, modified ELISA assay platform. The lowest limit of quantitation for TNF in serum and tissue were 0.056 and 0.064 pg/mL, respectively 18 19…”