Separation of complex protein mixtures that have a wide dynamic range of concentration, such as plasma or serum, is a challenge for proteomic analysis. Sample preparation to remove high-abundant proteins is essential for proteomics analysis. Immunoglobulin yolk (IgY) antibodies have unique and advantageous features that enable specific protein removal to aid in the detection of low-abundant proteins and biomarker discovery. This report describes the efficiency and effectiveness of IgY microbeads in separating 12 abundant proteins from plasma with an immunoaffinity spin column or LC column. The protein separation and sample preparation process was monitored via SDS-PAGE, 2-DE, LC-MS/MS, or clinical protein assays. The data demonstrate the high specificity of the protein separation, with removal of 95-99.5% of the abundant proteins. IgY microbeads against human proteins can also selectively remove orthologous proteins of other mammals such as mouse, rat, etc. Besides the specificity and reproducibility of the IgY microbeads, the report discusses the factors that may cause potential variations in protein separation such as protein-protein interactions (known as "Interactome"), binding and washing conditions of immunoaffinity reagents, etc. A novel concept of Seppromics is introduced to address methodologies and science of protein separation in a context of proteomics.

PurposeThe aim of this study was to utilize the proteomics-based Collaborative Enzyme Enhanced Reactive (CEER) immunoassay to investigate protein tyrosine phosphorylations as diagnostic markers in gastric cancers (GCs).Experimental DesignProtein lysates from fresh-frozen 434 advanced stage GCs were analyzed for phosphorylation of HER1, HER2, p95HER2, HER3, cMET, IGF1R and PI3K. The pathway activation patterns were segregated based on the tumor HER2 status. Hierarchical clustering was utilized to determine pathway coactivations in GCs. Prognostic value of pathway activation patterns was determined by correlating disease-free survival times of the various GC subgroups using Kaplan-Meier survival analysis. CEER was also used to determine the presence of tyrosine phosphorylated signaling cascades in circulating tumor cells (CTCs) and ascites tumor cells (ATCs).ResultsUtilizing a novel diagnostics immunoassay, CEER, we demonstrate the presence of p95HER2 and concomitantly activated signaling pathways in GC tumor tissues, CTCs and ATCs isolated from GC patients for the first time. p95HER2 is expressed in ∼77% of HER2(+) GCs. Approximately 54% of GCs have an activated HER1, HER2, HER3, cMET or IGF1R and demonstrate a poorer prognosis than those where these receptor tyrosine kinases (RTKs) are not activated. Hierarchical clustering of RTKs reveals co-clustering of phosphorylated HER1:cMET, HER2:HER3 and IGF1R-PI3K. Coactivation of HER1 with cMET renders GCs with a shorter disease-free survival as compared to only cMET activated GCs.ConclusionsOur study highlights the utility of a novel companion diagnostics technology, CEER that has strong implications for drug development and therapeutic monitoring. CEER is used to provide an increased understanding of activated signaling pathways in advanced GCs that can significantly improve their clinical management through accurate patient selection for targeted therapeutics.

Background: Targeted therapeutic strategies are currently limited to patients with hormone receptors and/or HER2 positive disease in breast cancer (BCA) treatment. However, patients often develop resistance to these therapies. The ability to functionally profile a whole spectrum of pathway proteins (and their variants) in tumor may provide valuable information about the potential mechanism for drug resistance and evidence for rational selection of suitable targeted therapies. Here we report a comprehensive profile of HER1, HER2, p95HER2, HER3, cMET, IGF1R, PI3K, Shc, AKT and other signal transduction pathway proteins in BCA tissues and their matched adjacent normal tissues (ANTs). Methods: A multiplexed Collaborative Proximity ImmunoAssay (COPIA), antibody-microarray platform requiring co-localization of 2 detector antibodies on captured biomarker proteins has been used for comprehensive pathway analysis. Channeling events between 2 detector enzymes (glucose oxidase & horse radish peroxidase) in proximity enabled the profiling of the target biomarkers with extreme sensitivity and specificity, and a direct comparison to electrochemiluminescence based immunoassay platform (MSD) was performed for pathway proteins in tumor vs. ANTs for their expression and activation in samples collected from 20 BCA patients. Results: Three dilutions of lysate (10ug, 1ug, 0.1ug) were analyzed for quantitative differential pathway modulation for COPIA. - Substantially higher cytokeratin (CK) levels were found in 16/20 tumor samples when compared to paired-ANT; 3/20 samples showed high levels of CK in ANTs. Substantial levels of HER3 and IGF1R expression was detected in 9 and 5 tumor samples respectively. - Over-expression of HER2 with high degree of activation was found in 2 patients. In one of the HER2-overexpressing patients, HER3 was also highly expressed and moderately phosphorylated. Co-expression of cMET and IGF1R was evident as well. - A significant degree of HER2 phosphorylation was found in many patients with low level HER2 expression; this may be due to co-expression of high level of HER3 and other RTKs with trans-activational potential. Evidence of activated PI3K complex will be reported. - In direct comparison to MSD, COPIA detected activated pathway proteins in samples that were not detectable with MSD. MSD was sensitive enough to detect the very extreme cases. COPIA appeared to be a more desirable method for detection of protein expression and activation for samples with limited availability. The distinct pathway modulation in each patient (detected by COPIA) will be reported. Discussion: COPIA was used to detect the differential expression and phosphorylation of HER2, other RTKs and pathway proteins in 20 paired tumor and matched ANTs. As this platform requires magnitudes lower amounts of specimen, it can be used to profile tumors at different metastatic sites and could provide comprehensive metastatic profiles. The comprehensive functional pathway profiling of tumor specimen may provide insightful information for potential drug-resistant mechanisms and may guide appropriate selection of targeted drug-combinations or drug-sequencing. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-06-13.

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.

Although gastrectomy is the only curative treatment in gastric cancer (GCA) patients, a high recurrence rate ranging from 40 ∼ 60% following curative surgery still accounts for poor overall survival. In order to further improve survival, there is an urgent need to identify reliable molecular prognostic markers for survival or recurrence following adjuvant chemoradiation therapy, which will evolve to the development of patient-tailored treatment strategies. The improvement of gastric cancer therapy will eventually depend on novel therapeutic approaches targeting molecules critical for cancer proliferation. As the transduction pathway proteins function in cell signaling and transmit signals regulating growth, differentiation, adhesion, migration, and apoptosis, they have become targets of various therapeutic agents. Hence, we have developed a multiplexed immunoassay platform for functional profiling of the transduction pathway proteins. The COllaborative Proximity ImmunoAssay (COPIA) is a multiplexed protein microarray platform that utilizes the formation of a unique immuno-complex requiring co-localization of two detector-antibodies. The detector-antibodies are conjugated with corresponding channeling-enzymes, glucose oxidase (GO) and horseradish peroxidase (HRP). Once target proteins are bound by the capture antibodies, the channeling events between GO and HRP in proximity enables the profiling of the target proteins with extreme sensitivity. COPIA delivers extremely high analytical specificity as it requires multiple entities within target specific proximity for the signal generation/amplification. COPIA can also be configured for each specific target protein to allow differential detection of truncated targets (i.e., p95HER2) from their normal counter parts (i.e., HER2). We applied COPIA to investigate the levels of expression and activation of signaling proteins in frozen tissues collected from GCA patients. Here we report the prevalence of HER1, HER2, p95HER2, HER3, IGF1-R, c-MET, PI3K, Shc, VEGFR, panCK, and other signal transduction pathway protein expression and their levels of activation in GCA patients. The improvement of gastric cancer therapy will eventually depend on novel therapeutic approaches targeting specific markers identified by functional profiling. As the disease profile often shifts in recurrent cancers and under different therapeutic pressure, clinical information obtained by analyzing samples (often with limited availability) obtained from ‘evolving / heterogeneous disease’ can help clinicians adjust their disease treatment options for each patient according to ‘personal’ cancer profile-shift. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4019.