Abstract 41: Development of a quantitative colorectal cancer SRM assay for use in FFPE tumor tissues.
Authors: Hembrough Todd , Liao Wei-Li , Henderson Les , Xu Peng , Thyparambil Sheeno , Burrows Jon , Catenacci Daniel V. 
Published: August 2014

Introduction: Aberrant over-expression of receptor tyrosine kinases, including the MET, HER, FGFR, and IGFR families along with other critical downstream oncogenic mediators including KRAS, BRAF, PI3 Kinase and SRC are known drivers of colorectal cancer (CRC), subdividing the disease into distinct molecular subsets. Inter-patient tumor heterogeneity suggests that an expedient, reliable, medium throughput oncogene protein expression profiling will provide vital information to better personalize cancer care. Moreover, intra-patient tumor heterogeneity from primary tumor to metastatic disease is likely to influence biomarker prediction of response to specific targeted agents. 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. Other protein quantification methods (ELISA, ECL) would require non-standard tissue processing for analysis. We present a quantitative mass spectrometric (MS) assay for CRC utilizing Liquid Tissue - Selected Reaction Monitoring (SRM), with subsequent multiplex quantification of relevant oncoproteins in a cohort of CRC paired primary and metastatic tumor tissues.

Methods: 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 ‘CRC-plex’ MS assay with multiplexed SRM quantification of Met, RON, EGFR, HER2, HER3, IGF1R, FGFR2, KRAS and cSRC on 42 primary human CRC cancer tissues, with paired metastases when available obtained from core biopsy or metastatectomy, using laser capture microdissection of the target material from a single unstained 10μm thick section per sample.

Results: Validation of the CRC-plex SRM assay on cell lines and FFPE tissues revealed very high concordance when compared to IB and IHC. Multiplex oncogene quantification of all tissues, to the attomole/microgram level, will be presented, highlighting inter-patient and intra-patient (from primary to metastasis) heterogeneity of samples.

Conclusions:

Taken together, these data demonstrate a sensitive, accurate, and quantitative assay to measure relevant actionable oncoproteins in FFPE clinical samples. The CRC-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/or post-treatment assessment.

Citation Format: Todd Hembrough, Wei-Li Liao, Les Henderson, Peng Xu, Sheeno Thyparambil, Jon Burrows, Daniel V. Catenacci. Development of a quantitative colorectal cancer SRM 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 41. doi:10.1158/1538-7445.AM2013-41