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

Clinical Proteomics

Jon Burrows²  David B Krizman¹  Ming-Sound Tsao⁴  Michael F Moran⁴  Thomas K Waddell³  Humberto Lara-Guerra³  Jiefei Tong⁵  Paul Taylor⁵  Kathleen M Bengali²  Joseph Abdo²  Marlene M Darfler²  Wei-Li Liao²  Sheeno Thyparambil²  Todd Hembrough² 

[1] Onco Plex Diagnostics Inc., 9620 Medical Center Drive, Rockville, 20850, Maryland, USA;Onco Plex Diagnostics Inc. 9620 Medical Center Drive, Rockville, Maryland, 20850, USA;Division of Thoracic Surgery, Toronto General Hospital Research Institute, University Health Network, 610 University Avenue, Toronto, ON, M5G 2 M9, Canada;Department of Surgery, 2374 Medical Sciences Building, 1 King's College Circle, University of Toronto, Toronto, ON, M5S 1A8, Canada;Program in Molecular Structure and Function, Hospital For Sick Children, 555 University Avenue, Toronto, ON, M5G 1X8, Canada

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

Others  :  1026359
DOI  :  10.1186/1559-0275-9-5

 received in 2011-12-31, accepted in 2012-05-03,  发布年份 2012

【 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, predict treatment outcome, and reveal mechanisms of therapeutic resistance.

【 授权许可】

   
2012 Hembrough et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1 .

Figure 2 .

Figure 3 .

【 参考文献 】

• [1]Yarden Y: The EGFR family and its ligands in human cancer. Signaling mechanisms and therapeutic opportunities. Eur J Cancer 2001, 37:3-8.
• [2]Schlessinger J: Ligand-induced, receptor-mediated dimerization and activation of EGF receptor. Cell 2002, 110:669-672.
• [3]Pawson T: Specificity in signal transduction: from phosphotyrosine-SH2 domain interactions to complex cellular systems. Cell 2004, 116:191-203.
• [4]Vivanco I, Mellinghoff IK: Epidermal growth factor receptor inhibitors in oncology. Curr Opin Oncol 2010, 22:573-578.
• [5]Fruehauf J: EGFR function and detection in cancer therapy. J Exp Ther Oncol 2006, 5:231-246.
• [6]Mascaux C, Wynes MW, Kato Y, et al.: EGFR Protein expression in non-small cell lung cancer predicts response to an EGFR tyrosine kinase inhibitor - a novel antibody for immunohistochemistry or AQUA technology. Clin Cancer Res 2011, 17:7796-7807.
• [7]Pirker R, Pereira JR, von Pawel J, et al.: EGFR expression as a predictor of survival for first-line chemotherapy plus cetuximab in patients with advanced non-small-cell lung cancer: analysis of data from the phase 3 FLEX study. Lancet Oncol 2012, 13:33-42.
• [8]Aebersold R, Mann M: Mass spectrometry-based proteomics. Nature. 2003, 422:198-207.
• [9]Ong SE, Mann M: Mass spectrometry-based proteomics turns quantitative. Nat Chem Biol 2005, 1:252-262.
• [10]Addona TA, Abbatiello SE, Schilling B, et al.: Multi-site assessment of the precision and reproducibility of selected reaction monitoring-based measurements of proteins in plasma. Nat Biotechnol 2009, 27:633-641.
• [11]Nilsson T, Mann M, Aebersold R, et al.: Mass spectrometry in high-throughput proteomics: ready for the big time. Nat Methods 2010, 7:681-685.
• [12]Rudnick PA, Clauser KR, Kilpatrick LE, et al.: Performance metrics for liquid chromatography-tandem mass spectrometry systems in proteomics analyses. Mol Cell Proteomics 2010, 9:225-241.
• [13]Prieto DA, Hood BL, Darfler MM, et al.: Liquid Tissue: proteomic profiling of formalin-fixed tissues. Biotechniques 2005, Jun(Suppl):32-35.
• [14]Hood BL, Darfler MM, Guiel TG, et al.: Proteomic analysis of formalin-fixed prostate cancer tissue. Mol Cell Proteomics 2005, 4:1741-1753.
• [15]Patel V, Hood BL, Molinolo AA, et al.: Proteomic analysis of laser-captured paraffin-embedded tissues: a molecular portrait of head and neck cancer progression. Clin Cancer Res 2008, 14:1002-1014.
• [16]Cheung W, Darfler MM, Alvarez H, et al.: 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 precursors. Pancreatology 2008, 8:608-616.
• [17]Bateman NW, Sun M, Bhargava R, et al.: Differential proteomic analysis of late-stage and recurrent breast cancer from formalin-fixed paraffin-embedded tissues. J Proteome Res 2011, 10:1323-1332.
• [18]Nishimura T, Nomura M, Tojo H, et al.: Proteomic analysis of laser-microdissected paraffin-embedded tissues: (2) SRM assay for stage-related proteins upon nonmetastatic lung adenocarcinoma. Proteomics 2010, 73:1100-1110.
• [19]DeSouza LV, Krakovska O, Darfler MM, et al.: mTRAQ-based quantification of potential endometrial carcinoma biomarkers from archived formalin-fixed paraffin-embedded tissues. Proteomics 2010, 10:3108-3116.
• [20]Güzel C, Ursem NT, Dekker LJ, et al.: Multiple reaction monitoring assay for preeclampsia related calcyclin peptides in formalin fixed paraffin embedded placenta. J. Proteome Res 2011, 10:3274-3282.
• [21]Wei Y, Tong J, Taylor P, et al.: Primary tumor xenografts of human lung adeno and squamous cell carcinoma express distinct proteomic signatures. J Proteome Res 2011, 10:161-174.
• [22]Lara-Guerra H, Waddell TK, Salvarrey MA, et al.: Phase II study of preoperative gefitinib in clinical stage I non-small-cell lung cancer. J Clin Oncol 2009, 27:6229-6236.
• [23]Pegram MD, Lipton A, Hayes DF, et al.: Phase II study of receptor-enhanced chemosensitivity using recombinant humanized anti-p185HER2/neu monoclonal antibody plus cisplatin in patients with HER2/neu-overexpressing metastatic breast cancer refractory to chemotherapy treatment. J Clin Oncol 1998, 16:2659-2671.
• [24]Druker BJ, Talpaz M, Resta DJ, et al.: Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. N Engl J Med 2001, 344:1031-1037.
• [25]Cregger M, Berger AJ, Rimm DL: Immunohistochemistry and quantitative analysis of protein expression. Arch Pathol Lab Med 2006, 130:1026-1030.
• [26]Taylor CR: Standardization in immunohistochemistry: the role of antigen retrieval in molecular morphology. Biotech Histochem 2006, 81:3-12.
• [27]Walker RA: Quantification of immunohistochemistry–issues concerning methods, utility and semiquantitative assessment. Histopathology 2006, 49:406-410.
• [28]Leong TY, Leong AS: How does antigen retrieval work? Adv Anat Pathol 2007, 14:129-131.
• [29]Anagnostou VK, Welsh AW, Giltnane JM, et al.: Analytic variability in immunohistochemistry biomarker studies. Cancer Epidemiol Biomarkers Prev 2010, 19:982-991.
• [30]Daneshtalab N, Doré JJ, Smeda JS: Troubleshooting tissue specificity and antibody selection: Procedures in immunohistochemical studies. J Pharmacol Toxicol Methods 2010, 61:127-135.
• [31]Mason JT, O’Leary TJ: Effects of formaldehyde fixation on protein secondary structure: a calorimetric and infrared spectroscopic investigation. J Histochem Cytochem 1991, 39:225-229.
• [32]Rait VK, O'Leary TJ, Mason JT: Modeling formalin fixation and antigen retrieval with bovine pancreatic ribonuclease A: I-structural and functional alterations. Lab Invest 2004, 84:292-299.
• [33]Rait VK, Xu L, O’Leary TJ, Mason JT: Modeling formalin fixation and antigen retrieval with bovine pancreatic RNase A II. Interrelationship of cross-linking, immunoreactivity, and heat treatment. Lab Invest 2004, 84:300-306.
• [34]Fowler CB, Cunningham RE, O'Leary TJ, Mason JT: ‘Tissue surrogates’ as a model for archival formalin-fixed, paraffin-embedded tissues. Lab Invest 2007, 87:836-846.
• [35]Huang SK, Darfler MM, Nicholl MB, et al.: LC/MS-based quantitative proteomic analysis of paraffin-embedded archival melanomas reveals potential proteomic biomarkers associated with metastasis. PLoS One 2009, 4:e4430.
• [36]Sprung RW: Brock JW, Tanksley JP, et al: Equivalence of protein inventories obtained from formalin-fixed paraffin-embedded and frozen tissue in multidimensional liquid chromatography tandem mass spectrometry shotgun proteomic analysis. Mol Cell Proteomics 2009, 8:1988-1998.
• [37]Bell LN, Saxena R, Mattar SG, et al.: Utility of formalin-fixed, paraffin-embedded liver biopsy specimens for global proteomic analysis in nonalcoholic steatohepatitis. Proteomics Clin Appl 2011, 5:397-404.
• [38]Nirmalan NJ, Hughes C, Peng J, et al.: Initial development and validation of a novel extraction method for quantitative mining of the formalin-fixed, paraffin-embedded tissue proteome for biomarker investigations. J Proteome Res 2011, 10:896-906.
• [39]Ikeda K, Monden T, Kanoh T, et al.: Extraction and analysis of diagnostically useful proteins from formalin-fixed, paraffin-embedded tissue sections. J Histochem Cytochem 1998, 46:397-403.
• [40]Becker KF, Schott C, Hipp S, Metzger V, et al.: Quantitative protein analysis from formalin-fixed tissues: implications for translational clinical research and nanoscale molecular diagnosis. J Pathol. 2007, 211:370-378.