Abstract 1207: Development of a quantitative gastroesophageal cancer selected reaction monitoring mass Spectrometric Multiplex Assay for use in FFPE tumor tissues.
Authors: Catenacci Daniel V. , Xu Peng , Henderson Les , Liao Wei-Li , Thyparambil Sheeno , Burrows Jon , Hembrough Todd 
Published: August 2014

Abstract
Aberrant over-expression of receptor tyrosine kinases, (e.g. MET, HER, FGFR, and IGFR) as well as other oncogenic mediators (e.g. KRAS, PI3 Kinase and SRC) are known drivers of gastroesophageal adenocarcinoma (GEC), subdividing the disease into distinct molecular subsets. Inter/intrapatient tumor heterogeneity suggests that an expedient, reliable, medium throughput oncogene protein expression profiling will provide vital information to better personalize cancer care. To date, clinical quantification of protein in formalin fixed paraffin embedded (FFPE) tissues is limited to immunohistochemistry (IHC), which is semi-quantitative at best. Moreover, IHC of multiple proteins of interest is laborious, time consuming, wasteful of scarce tissue, and costly. We present a quantitative mass spectrometric (MS) assay for FFPE GEC utilizing Liquid Tissue - Selected Reaction Monitoring (SRM), with subsequent multiplex quantification of relevant oncoproteins in a panel of gastroesophageal cancer (GEC) cell lines and tissues.
Using trypsin digestion mapping of recombinant oncoproteins, we identified unique peptide sequences, and built quantitative MS assays which could be multiplexed into a single SRM analysis of 1μg of tumor protein. Assays were preclinically validated on 10 different formalin fixed (FF) cell lines.
We then tested the GEC-plex assay on a panel of FFPE GEC cell lines characterized by immunoblot (IB), IHC, and gene copy number by FISH. In addition to RON, we multiplexed SRM quantification of Met, EGFR, HER2, HER3, IGF1R, FGFR2, KRAS and cSRC. We evaluated 17 GEC lines including AGS wild type, scrambled shRNA (AGS-SC) and RON shRNA knockdown (AGS-KD) to assess ‘post-treatment’ changes in oncogene expression. We then evaluated 100 GEC human cancer tissues with paired peritoneal metastases when available and select paraneoplastic normal tissues using laser microdissection of tumor tissue from a single unstained 10μm thick section.
Validation of the GEC-plex SRM assay on GEC cell lines revealed very high concordance when compared to IB and IHC measurement. The AGS-WT/SC cells showed comparable levels of RON (284/323 amol/μg cell protein), while RON was not detected in AGS-KD cells, as expected. Measurement of oncoproteins in GEC cell lines and tissues correlated well with IHC and FISH data. Multiplex oncogene quantification of all cell lines and tissues, along with expression profile changes in the AGS RON KD line compared to AGS-WT/SC will be presented.
Taken together, these data demonstrate a sensitive, accurate, and quantitative assay to measure relevant actionable oncoproteins in FF cells. The GEC-plex multiplexed oncogene expression of these tumors was feasible and expedient using limited tissue from clinical samples, and is a novel clinically applicable approach for tumor characterization for baseline and post-treatment assessment.
Citation Format: Daniel V. Catenacci, Peng Xu, Les Henderson, Wei-Li Liao, Sheeno Thyparambil, Jon Burrows, Todd Hembrough. Development of a quantitative gastroesophageal cancer selected reaction monitoring mass Spectrometric Multiplex Assay for use in FFPE tumor tissues. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1207. doi:10.1158/1538-7445.AM2013-1207