1. Application of selected reaction monitoring for multiplex quantification of clinically validated biomarkers in formalin-fixed, paraffin-embedded tumor tissue
- Author
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Jon Burrows, Stephen M. Hewitt, Marlene Darfler, Todd Hembrough, Richard A. Bender, Wei-Li Liao, David B. Krizman, Joseph Abdo, Kathleen Bengali, and Sheeno Thyparambil
- Subjects
medicine.medical_specialty ,Tissue Fixation ,Formalin fixed paraffin embedded ,Receptor, ErbB-3 ,Receptor, ErbB-2 ,Breast Neoplasms ,Enzyme-Linked Immunosorbent Assay ,Biology ,Bioinformatics ,Mass Spectrometry ,Pathology and Forensic Medicine ,Receptor, IGF Type 1 ,Breast cancer ,Formaldehyde ,medicine ,Biomarkers, Tumor ,Humans ,Multiplex ,Epidermal growth factor receptor ,Paraffin Embedding ,Clinical pathology ,Selected reaction monitoring ,Receptors, Somatomedin ,medicine.disease ,ErbB Receptors ,Cancer research ,biology.protein ,Molecular Medicine ,Immunohistochemistry ,Female ,Quantitative analysis (chemistry) - Abstract
One of the critical gaps in the clinical diagnostic space is the lack of quantitative proteomic methods for use on formalin-fixed, paraffin-embedded (FFPE) tissue. Herein, we describe the development of a quantitative, multiplexed, mass spectrometry-based selected reaction monitoring (SRM) assay for four therapeutically important targets: epidermal growth factor receptor, human EGF receptor (HER)-2, HER3, and insulin-like growth factor-1 receptor. These assays were developed using the Liquid Tissue-SRM technology platform, in which FFPE tumor tissues were microdissected, completely solubilized, and then subjected to multiplexed quantitation by SRM mass spectrometry. The assays were preclinically validated by comparing Liquid Tissue-SRM quantitation of FFPE cell lines with enzyme-linked immunosorbent assay/electrochemiluminescence quantitation of fresh cells (R2 > 0.95). Clinical performance was assessed on two cohorts of breast cancer tissue: one cohort of 10 samples with a wide range of HER2 expression and a second cohort of 19 HER2 IHC 3+ tissues. These clinical data demonstrate the feasibility of quantitative, multiplexed clinical analysis of proteomic markers in FFPE tissue. Our findings represent a significant advancement in cancer tissue analysis because multiplexed, quantitative analysis of protein targets in FFPE tumor tissue can be tailored to specific oncological indications to provide the following: i) complementary support for anatomical pathological diagnoses, ii) patient stratification to optimize treatment outcomes and identify drug resistance, and iii) support for the clinical development of novel therapies.
- Published
- 2012