Selected Reaction Monitoring (SRM) Analysis of Epidermal Growth Factor Receptor (EGFR) in Formalin Fixed Tumor Tissue
Open Access
- 3 May 2012
- journal article
- Published by Springer Science and Business Media LLC in Clinical Proteomics
- Vol. 9 (1), 5
- https://doi.org/10.1186/1559-0275-9-5
Abstract
Background: Analysis of key therapeutic targets such as epidermal growth factor receptor (EGFR) in clinical tissue samples is typically done by immunohistochemistry (IHC) and is only subjectively quantitative through a narrow dynamic range. The development of a standardized, highly-sensitive, linear, and quantitative assay for EGFR for use in patient tumor tissue carries high potential for identifying those patients most likely to benefit from EGFR-targeted therapies. Methods: A mass spectrometry-based Selected Reaction Monitoring (SRM) assay for the EGFR protein (EGFR-SRM) was developed utilizing the Liquid Tissue®-SRM technology platform. Tissue culture cells (n = 4) were analyzed by enzyme-linked immunosorbent assay (ELISA) to establish quantitative EGFR levels. Matching formalin fixed cultures were analyzed by the EGFR-SRM assay and benchmarked against immunoassay of the non-fixed cultured cells. Xenograft human tumor tissue (n = 10) of non-small cell lung cancer (NSCLC) origin and NSCLC patient tumor tissue samples (n = 23) were microdissected and the EGFR-SRM assay performed on Liquid Tissue lysates prepared from microdissected tissue. Quantitative curves and linear regression curves for correlation between immunoassay and SRM methodology were developed in Excel. Results: The assay was developed for quantitation of a single EGFR tryptic peptide for use in FFPE patient tissue with absolute specificity to uniquely distinguish EGFR from all other proteins including the receptor tyrosine kinases, IGF-1R, cMet, Her2, Her3, and Her4. The assay was analytically validated against a collection of tissue culture cell lines where SRM analysis of the formalin fixed cells accurately reflects EGFR protein levels in matching non-formalin fixed cultures as established by ELISA sandwich immunoassay (R2 = 0.9991). The SRM assay was applied to a collection of FFPE NSCLC xenograft tumors where SRM data range from 305amol/μg to 12,860amol/μg and are consistent with EGFR protein levels in these tumors as previously-reported by western blot and SRM analysis of the matched frozen tissue. In addition, the SRM assay was applied to a collection of histologically-characterized FFPE NSCLC patient tumor tissue where EGFR levels were quantitated from not detected (ND) to 670amol/μg. Conclusions: This report describes and evaluates the performance of a robust and reproducible SRM assay designed for measuring EGFR directly in FFPE patient tumor tissue with accuracy at extremely low (attomolar) levels. This assay can be used as part of a complementary or companion diagnostic strategy to support novel therapies currently under development and demonstrates the potential to identify candidates for EGFR-inhibitor therapy, predict treatment outcome, and reveal mechanisms of therapeutic resistance.Keywords
This publication has 41 references indexed in Scilit:
- EGFR Protein Expression in Non–Small Cell Lung Cancer Predicts Response to an EGFR Tyrosine Kinase Inhibitor—A Novel Antibody for Immunohistochemistry or AQUA TechnologyClinical Cancer Research, 2011
- Utility of formalin‐fixed, paraffin‐embedded liver biopsy specimens for global proteomic analysis in nonalcoholic steatohepatitisProteomics – Clinical Applications, 2011
- Initial Development and Validation of a Novel Extraction Method for Quantitative Mining of the Formalin-Fixed, Paraffin-Embedded Tissue Proteome for Biomarker InvestigationsJournal of Proteome Research, 2010
- Performance Metrics for Liquid Chromatography-Tandem Mass Spectrometry Systems in Proteomics AnalysesMolecular & Cellular Proteomics, 2010
- Equivalence of Protein Inventories Obtained from Formalin-fixed Paraffin-embedded and Frozen Tissue in Multidimensional Liquid Chromatography-Tandem Mass Spectrometry Shotgun Proteomic AnalysisMolecular & Cellular Proteomics, 2009
- Multi-site assessment of the precision and reproducibility of multiple reaction monitoring–based measurements of proteins in plasmaNature Biotechnology, 2009
- LC/MS-Based Quantitative Proteomic Analysis of Paraffin-Embedded Archival Melanomas Reveals Potential Proteomic Biomarkers Associated with MetastasisPLOS ONE, 2009
- Application of a Global Proteomic Approach to Archival Precursor Lesions: Deleted in Malignant Brain Tumors 1 and Tissue Transglutaminase 2 Are Upregulated in Pancreatic Cancer PrecursorsPancreatology, 2008
- Mass spectrometry-based proteomicsNature, 2003
- The EGFR family and its ligands in human cancerEuropean Journal Of Cancer, 2001