Abstract 2499: Application of a multiplexed fluorescence microscopy method (MultiOmyxTM) to dissect proteomic biomarkers of (18)F-fluorodeoxy-glucose ((18)FDG) uptake in breast cancer
Authors: Sood Anup , Miller Alexandra M. , Ginty Fiona , McDonough Elizabeth , Sui Yunxia , Bordwell Alexander , Li Qing , Kaanumalle Sireesha , Pang Zhengyu , Torres Franklin , Brogi Edi , Larson Steven , Mellinghoff Ingo 
Published: May 2015

Abstract
Background: While routinely collected human tumor specimen can now readily be examined for genetic alterations on a genome wide scale, multi-parameter measurements of protein expression remain challenging. We developed a multiplexed fluorescence microscopy method (MultiOmyxTM) for the quantitative characterization of multiple analytes in formalin-fixed paraffin-embedded tissue (PMID23818604). We now applied this platform to human breast cancer samples to determine the expression of 25 proteins at a single-cell level and examine their relationship to tumor uptake of the Positron Emission Tomography (PET) radiotracer (18)F-fluorodeoxy-glucose ((18)FDG). Methodology: We stained a single 5 µm section of breast carcinoma from each of 18 patients with antibodies against members of growth factor signaling pathways (HER2, IGF1R, PTEN, p-EGFR, p-PDK1, p-ERK1/2, p-S6 Ribosomal Protein, p-4EBP1, p-eIF4E), the glycolysis pathway (Glut-1, HK2, LDH-A), hormone receptors (ER, PR, AR), tumor cell proliferation markers (KI-67 and phospho-histone H3), and markers of hypoxia (HIF-1α, CA IX), and angiogenesis (CD31). 28-30 representative fields of view (FOVs) were randomly selected in each tumor, each comprising 300-500 cells. Images were automatically separated into subcellular and histopathological compartments based on the staining of tumor cells with a panel of segmentation markers (NaK-ATPase, pan-Cadherin, pan-cytokeratin, S6, and DAPI). A breast pathologist assessed and annotated the histologic composition of each FOV. Further analysis focused on FOVs (n=390) containing only invasive ductal carcinoma without admixed DCIS or normal breast tissue. Multivariate analysis between marker expression and FDG uptake was performed using logistic regression and Cox proportional hazard models. All patients had (18)-FDG PET within four weeks prior to collection of the tumor specimen. Results: Staining results with our MultiOmyxTM platform correlated closely with the results from CLIA-certified single marker biomarker assays (e.g., ER IHC and HER2 FISH assay) performed independently on the same set of samples (PMID: 21646475). Nuclear ER staining was associated with low FDG uptake (p=0.02). KI-67 was higher in tumors with high FDG uptake (p=0.04). K-median clustering identified molecular breast cancer subtypes. Conclusions: Our study illustrates the feasibility of quantitative proteomic measurements using a new in-situ multiplexed fluorescence microscopy platform on only a single routinely collected FFPE tissue section. Quantitative staining results from many thousand cells per tumor support the previously reported relationship between hormone receptor status, tumor cell proliferation, and FDG-uptake in breast cancer. Multiple protein markers showed a high degree of intra- and inter-tumoral heterogeneity.
Citation Format: Anup Sood, Alexandra M. Miller, Fiona Ginty, Elizabeth McDonough, Yunxia Sui, Alexander Bordwell, Qing Li, Sireesha Kaanumalle, Zhengyu Pang, Franklin Torres, Edi Brogi, Steven Larson, Ingo Mellinghoff. Application of a multiplexed fluorescence microscopy method (MultiOmyxTM) to dissect proteomic biomarkers of (18)F-fluorodeoxy-glucose ((18)FDG) uptake in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2499. doi:10.1158/1538-7445.AM2014-2499