Selected reaction monitoring (SRM) analysis of epidermal growth factor receptor (EGFR) in formalin fixed tumor tissue

被引：45

作者：

Todd Hembrough

Sheeno Thyparambil

Wei-Li Liao

Marlene M Darfler

Joseph Abdo

Kathleen M Bengali

Paul Taylor

Jiefei Tong

Humberto Lara-Guerra

Thomas K Waddell

Michael F Moran

Ming-Sound Tsao

David B Krizman

Jon Burrows

机构：

[1] Onco Plex Diagnostics Inc., Rockville,MD 20850

[2] Program in Molecular Structure and Function, Hospital for Sick Children, Toronto, ON M5G 1X8

[3] Toronto General Hospital Research Institute, Division of Thoracic Surgery, University Health Network, Toronto

[4] Ontario Cancer Institute, University Health Network, Princess Margaret Hospital, Toronto

[5] Molecular Genetics,Banting and Best Department of Medical Research, Institute of Medical Science, Toronto

[6] Department of Surgery, University of Toronto, Toronto, ON M5S 1A8

[7] Laboratory Medicine and Pathology, Medical Biophysics Institute of Medical Science, Toronto

[8] Onco Plex Diagnostics Inc., Rockville20850 MD

来源：

Clinical Proteomics | 2012年 / 9卷 / 1期

基金：

加拿大健康研究院;

关键词：

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

D O I：

10.1186/1559-0275-9-5

中图分类号：

学科分类号：

摘要：

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. © 2012 Hembrough et al.; licensee BioMed Central Ltd.

引用

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