Abstract 2014: MultiOmyxTM: A multiplexed immunofluorescent assay capable of profiling protein expression and phosphorylation, in combination with next-generation sequencing from a single FFPE tissue section
Authors: Au Qingyan , Fu Maoyong , Bordwell Alexander , Pinky Tripathi , Lazare Michael , Tran Nam , Hoe Nicholas 
Published: August 2015

Abstract
A comprehensive signaling pathway profile in combination with mutational analysis may be a critical guide for selecting effective clinical strategies for targeted drugs in combinations or in sequential regimens. In the United States, colorectal cancer (CRC) is the third most common cancer and the third leading cause of tumor associated death in men and women. CRC is a heterogeneous disease defined by different receptor tyrosine kinase (RTK) activation, signaling through phosphatidylinositol 3-kinase (PI3K)/AKT and RAS/MAP2K pathways. In addition, activating mutations in RTK and/or activating or loss-of-function mutations in downstream intracellular signaling proteins, can alter the efficacy of targeted drugs resulting in an ineffective treatment.
GE Healthcare, through its affiliate Clarient Diagnostic Services Inc., has developed a novel hyperplexed multi-omic technology, MultiOmyx, to enable visualization and characterization of multiple biomarkers across multiple assays on a single 4μm tissue section. MultiOmyx protein immunofluorescence (IF) assays utilize 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 and deactivation for up to 60 protein biomarkers. The same protein IF processed slide is then used to perform DNA FISH assay, followed by DNA extraction using laser capture microdissection (LCM) from region(s) of interest for next generation sequencing.
Herein, we report an analysis of the key receptor tyrosine kinases (EGFR, HER2, HER3, and cMET) and their downstream signaling proteins (PI3K, phospho AKT, and phospho ERK1/2) in 10 colon tumor samples using the MultiOmyx technology. Mutational analysis was performed on LCM extracted tumor and tumor adjacent regions, and sequenced using the Ion AmpliSeq cancer panel, consisting of 50 targeted genes. Protein IF staining revealed heterogeneous expression and activation across different samples. With high EGFR and HER3 expression correlating with activation of AKT, possibly through EGFR:HER3 dimer, to high expression of EGFR correlating with positive staining for phospho ERK1/2. Additionally, intra-tumor heterogeneity was observed within the same tumor tissue, with varied expression of EGFR, HER2, and HER3. Preliminary mutational analysis also detected BRAF G469A known to increase kinase activity of BRAF and downstream activation of pERK1/2 in tumor but not in tumor-adjacent region.
The MultiOmyx assay can be utilized for clinical samples testing with limited size and availability. A comprehensive mutational and signaling protein analysis can guide oncologists tailored either a single or combinational targeted therapy to patient's individual tumor profile.
Citation Format: Qingyan Au, Maoyong Fu, Alexander Bordwell, Tripathi Pinky, Michael Lazare, Nam Tran, Nicholas Hoe. MultiOmyxTM: A multiplexed immunofluorescent assay capable of profiling protein expression and phosphorylation, in combination with next-generation sequencing from a single FFPE tissue section. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2014. doi:10.1158/1538-7445.AM2015-2014