Selected Reaction Monitoring (SRM) Analysis of Epidermal Growth Factor Receptor (EGFR) in Formalin Fixed Tumor Tissue

Clinical Proteomics

Joseph Abdo⁵  Ming-Sound Tsao⁴  Paul Taylor³  David B Krizman⁶  Kathleen M Bengali⁵  Humberto Lara-Guerra¹  Jiefei Tong³  Marlene M Darfler⁵  Todd Hembrough⁵  Michael F Moran²  Wei-Li Liao⁵  Sheeno Thyparambil⁵  Jon Burrows⁵  Thomas K Waddell¹ 

[1] Division of Thoracic Surgery, Toronto General Hospital Research Institute, University Health Network, Toronto, CanadaDivision of Thoracic Surgery, Toronto General Hospital Research Institute, University Health Network, Toronto, CanadaDivision of Thoracic Surgery, Toronto General Hospital Research Institute, University Health Network, Toronto, Canada;Program in Molecular Structure and Function, Hospital For Sick Children, Toronto, CanadaOntario Cancer Institute, Princess Margaret Hospital, University Health Network, Toronto, CanadaMolecular Genetics, Banting and Best Department of Medical Research, Institute of Medical Science, Toronto, CanadaDepartment of Surgery, University of Toronto, Toronto, CanadaProgram in Molecular Structure and Function, Hospital For Sick Children, Toronto, CanadaProgram in Molecular Structure and Function, Hospital For Sick Children, Toronto, CanadaOntario Cancer Institute, Princess Margaret Hospital, University Health Network, Toronto, CanadaMolecular Genetics, Banting and Best Department of Medical Research, Institute of Medical Science, Toronto, CanadaDepartment of Surgery, University of Toronto, Toronto, CanadaOntario Cancer Institute, Princess Margaret Hospital, University Health Network, Toronto, CanadaProgram in Molecular Structure and Function, Hospital For Sick Children, Toronto, CanadaOntario Cancer Institute, Princess Margaret Hospital, University Health Network, Toronto, CanadaMolecular Genetics, Banting and Best Department of Medical Research, Institute of Medical Science, Toronto, CanadaDepartment of Surgery, University of Toronto, Toronto, CanadaMolecular Genetics, Banting and Best Department of Medical Research, Institute of Medical Science, Toronto, CanadaProgram in Molecular Structure and Function, Hospital For Sick Children, Toronto, CanadaOntario Cancer Institute, Princess Margaret Hospital, University Health Network, Toronto, CanadaMolecular Genetics, Banting and Best Department of Medical Research, Institute of Medical Science, Toronto, CanadaDepartment of Surgery, University of Toronto, Toronto, CanadaDepartment of Surgery, University of Toronto, Toronto, CanadaProgram in Molecular Structure and Function, Hospital For Sick Children, Toronto, CanadaOntario Cancer Institute, Princess Margaret Hospital, University Health Network, Toronto, CanadaMolecular Genetics, Banting and Best Department of Medical Research, Institute of Medical Science, Toronto, CanadaDepartment of Surgery, University of Toronto, Toronto, Canada;Program in Molecular Structure and Function, Hospital For Sick Children, Toronto, CanadaProgram in Molecular Structure and Function, Hospital For Sick Children, Toronto, CanadaProgram in Molecular Structure and Function, Hospital For Sick Children, Toronto, Canada;Ontario Cancer Institute, Princess Margaret Hospital, University Health Network, Toronto, CanadaLaboratory Medicine and Pathology, Medical Biophysics Institute of Medical Science, Toronto, CanadaDepartment of Surgery, University of Toronto, Toronto, CanadaOntario Cancer Institute, Princess Margaret Hospital, University Health Network, Toronto, CanadaOntario Cancer Institute, Princess Margaret Hospital, University Health Network, Toronto, CanadaLaboratory Medicine and Pathology, Medical Biophysics Institute of Medical Science, Toronto, CanadaDepartment of Surgery, University of Toronto, Toronto, CanadaLaboratory Medicine and Pathology, Medical Biophysics Institute of Medical Science, Toronto, CanadaOntario Cancer Institute, Princess Margaret Hospital, University Health Network, Toronto, CanadaLaboratory Medicine and Pathology, Medical Biophysics Institute of Medical Science, Toronto, CanadaDepartment of Surgery, University of Toronto, Toronto, CanadaDepartment of Surgery, University of Toronto, Toronto, CanadaOntario Cancer Institute, Princess Margaret Hospital, University Health Network, Toronto, CanadaLaboratory Medicine and Pathology, Medical Biophysics Institute of Medical Science, Toronto, CanadaDepartment of Surgery, University of Toronto, Toronto, Canada;Onco Plex Diagnostics Inc., 9620 Medical Center Drive, Rockville, USAOnco Plex Diagnostics Inc., 9620 Medical Center Drive, Rockville, USAOnco Plex Diagnostics Inc., 9620 Medical Center Drive, Rockville, USA;Onco Plex Diagnostics Inc., 9620 Medical Center Drive, Rockville, USAOnco Plex Diagnostics Inc., 9620 Medical Center Drive, Rockville, USAOnco Plex Diagnostics Inc., 9620 Medical Center Drive, Rockville, USAOnco Plex Diagnostics Inc., 9620 Medical Center Drive, Rockville, USAOnco Plex Diagnostics Inc., 9620 Medical Center Drive, Rockville, USAOnco Plex Diagnostics Inc., 9620 Medical Center Drive, Rockville, USAOnco Plex Diagnostics Inc., 9620 Medical Center Drive, Rockville, USAOnco Plex Diagnostics Inc., 9620 Medical Center Drive, Rockville, USA

关键词: Formalin fixed;    FFPE;    EGFR;    Gefitinib;    Targeted therapy;    Patient tissue;    Quantitative;    Personalized medicine;    Molecular diagnostics;    Non-small cell lung cancer;   

DOI  :  10.1186/1559-0275-9-5

来源: Humana Press Inc

【 摘 要 】

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.

【 授权许可】

Unknown