Abstract 4578: Quantification of protein complexes and post-translational modifications in colorectal cancer utilizing combinted MultiOmyxTMand PLA assay from a single FFPE slide
Authors: Qingyan Au, Flora Sahafi, Kathy Nguyen, Raghav Padmanabhan, Edward J. Moler, Nicholas Hoe
Journal: Cancer Research (2016)

Abstract A comprehensive signaling pathway analysis is critical to characterize cancer pathogenesis, in which malignant cells evolve from nonmalignant cells, in heterogeneous tissues comprised of both healthy and pathological cells. Although significant advances in technologies have led to improved understanding of cancer biology, comprehensive profiling utilizing multiple biomarkers remains a technical challenge due to limited sample availability. GE Healthcare MultiOmyx multiplexed immunofluorescence (IF) assay overcomes this sample limitation and staining up to 60 protein biomarkers has been demonstrated in a single formalin-fixed, paraffin-embedded (FFPE) slide. In this study, proximity ligation assay (PLA) technology is adapted to expand MultiOmyx assay capabilities by enabling detection of protein-protein interactions (PPIs) and post-translational modification (PTMs) from a single FFPE slide. The MultiOmyx assay utilizes a pair of directly conjugated Cyanine dye-labeled (Cy3, Cy5) antibodies per round of staining. Each round of staining is imaged and followed by novel dye inactivation chemistry, enabling repeated rounds of staining. The PLA technology utilizes a pair of directly conjugated proximity probes to detect proteins of interest. Proximal binding of these probes lead to ligation and DNA amplification using rolling circle amplification (RCA). Amplified DNA is detected by hybridizing Cyanine dye-labeled oligonucleotides. Herein we report a comprehensive analysis of key receptor tyrosine kinases (RTKs) (HER1, HER2, HER3, cMET, others) along with their downstream signaling proteins (PI3K, phospho AKT, and phospho ERK1/2) in 10 colorectal cancer (CRC) samples using the standard MultiOmyx assay. A PLA-adapted MultiOmyx assay is utilized to detect dimerization partners (HER1:HER2, HER2:HER3, HER1:HER3) and RTK phosphorylation using separate antibodies against the RTK and the phosphorylation site. Protein IF staining revealed heterogeneous expression and activation across different samples. High EGFR and HER3 expression correlated with positive staining for AKT, through EGFR:HER3 dimer. High expression of EGFR correlated with positive staining for phospho Erk1/2, through EGFR:HER2 dimer. Additionally, intra-tumor heterogeneity was observed, with varied expression and activation of EGFR, HER2, HER3, and cMET. Current IHC and multiplexed IF assays measures the expression levels of individual proteins but overlook the measurement of PPIs and PTMs, which are crucial to understanding the biology of pathway signaling. The PLA-adapted MultiOmyx assay enables true comprehensive pathway signaling analysis at a single cell level by providing spatial context and quantitative analysis of protein expression, protein-protein interactions, and protein activations (phosphorylation). Citation Format: Qingyan Au, Flora Sahafi, Kathy Nguyen, Raghav Padmanabhan, Edward J. Moler, Nicholas Hoe. Quantification of protein complexes and post-translational modifications in colorectal cancer utilizing combinted MultiOmyxTM and PLA assay from a single FFPE slide. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4578.