Abstract 2494: Liquid chromatography-multiple reaction monitoring mass spectrometry biomarker quantification for breast cancer patient assessment.
Authors: Chen Yi , Britton David J. , Wood Elizabeth R. , Magliocco Anthony M. , Pike Ian , Koomen John M. 
Published: August 2014

Abstract
Introduction: A quantitative mass spectrometry platform has been developed for measuring biomarkers of protein expression and phosphorylation in breast cancer. The quantification of numerous biomarkers from a single tissue specimen has the potential to impact patient care. Specifically, the focus of initial development was on the expression and phosphorylation of estrogen receptor alpha (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2). However, immunohistochemical assessments of Ki-67 and vimentin are also widely used for monitoring proliferation in breast cancer. The integration of tissue quality control measurements, quantification of proliferation biomarkers, and ER/PR/HER2 evaluation could improve patient assessment and open novel avenues for patient classification, prognosis, and selection of therapy in breast cancer.
Experimental Procedures: Breast cancer cell lines MCF7, BT474, and T47D were used to develop peptide-based quantitative assays, including stable isotope-labeled standard peptides, (n = 31) for protein expression and phosphorylation biomarkers using liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) of digested whole cell lysates or SDS-PAGE fractionated proteins followed by immunoprecipitation to examine expression and phosphorylation of ER/PR/HER2 (IP-LC-MRM). Using 2 mg of total protein, the sample is divided into three parts. Tissue QC biomarkers and expression levels of Ki67, vimentin, and HER2 are measured in digests of whole cell lysate or tissue homogenate (1% of sample). Then, parallel IP strategies are used for HER2 (10% of sample) and ER/PR (90%) before LC-MRM analysis of expression and phosphorylation. Multiplex IP of the three proteins was not successful, because the amplification of HER2 could not be accommodated. The sensitivity of each assay was tested using serial dilutions of cell lysate to evaluate requirements for analysis of clinical specimens.
Data Summary: LC-MRM assays for both unmodified and phosphorylated peptides have been developed and characterized for each protein using cell line models. Assays have been implemented in cell lines to study Estradiol/EGF stimulation as well as lapatinib inhibition of HER2. Data has been generated for pre-treatment frozen tissue specimens from breast cancer patients that are either HER2+ or ER+ (n = 12 per group). The analysis of a larger group of patients and in clinical scenarios (e.g. comparison of naïve and drug resistant tissues from the same patient) will establish the clinical utility of these assays.
Conclusions: The ability to examine protein expression as well as phosphorylation status in these biomarkers with quantitative mass spectrometry has the potential to improve the selection of targeted therapeutics.
Citation Format: Yi Chen, David J. Britton, Elizabeth R. Wood, Anthony M. Magliocco, Ian Pike, John M. Koomen. Liquid chromatography-multiple reaction monitoring mass spectrometry biomarker quantification for breast cancer patient assessment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2494. doi:10.1158/1538-7445.AM2013-2494