P1-07-23: Absolute Quantification of Estrogen Receptor alpha in Breast Cancer.
Authors: Britton D , Scott G , Russell C , Held J , Ward M , Benz C , Pike I 
Published: June 2012

Abstract
Estrogen receptor alpha (ER) is the first biomarker to have been clinically validated as a predictor of cancer therapy response. Measurements of tumor ER expression were based on radiolabeled ligand binding to receptor present in tumor lysates. Despite substantial tissue requirement, lack of specificity for ER isoforms (alpha versus beta) or receptor integrity, quantitative determination of tumor ER content (fmol/mg total protein) was possible. Introduction of anti-ER antibodies later permitted immunohistochemical (***lHC) evaluation of ER expression. IHC determination of ER status in newly diagnosed breast cancer is now a standard of care. While these IHC assays have been shown to be as predictive of endocrine responsiveness as ligand binding assays, they remain semi-quantitative at best reporting tumor ER status either categorically (e.g. + or -) or as a numeric score which is subjective and lacks a linear relationship with endocrine responsiveness. Thus the lack of precision for quantifying ER as a predictive biomarker is one of the most important unresolved issues in breast cancer. We are working to develop a proteomic liquid chromatography-mass spectrometry (LC-MS) assay to help resolve this issue. Samples included recombinant ER (rER), immunoprecipitated (IP) rER, and IP ER from MCF7 cells. ER was digested with trypsin, lyophilised and solubilised in 5 femto-mol/microliter (100 μl) heavy peptide internal standard mix. ER peptides were resolved by LC (100 μl/minute) and detected by selected reaction monitoring MS. The area under the total ion chromatogram for each peptide were used to quantify the amount of analyte present in each sample as a single point reference to the signal of the heavy peptide spike. An 11 point calibration curve (0.1-1000 fmol on column (o/c)) of light peptides with each point in the curve spiked with 100 fmol heavy peptide was also produced to determine assay characteristics such as limits of detection (LOD), limits of quantification (LOQ), linearity, accuracy and precision. Three ER peptides were selected for quantification as they gave the greatest LOD, LOQ, linearity as well as reasonable intra- and inter-assay precision following multiple digestions of rER (intra = 3 digestions in 1 day; inter = 9 digestions over 3 weeks).

Following IP of ER from four replicate MCF7 cell lysates (1mg/ml total protein) and measurement of ion intensities of the three ER peptides the mean concentration of ER was calculated to be 52 fmol (S.D of 7.5 fmol; n=4) per mg of total cell lysate after normalising for IP efficiency. We continue to develop the method to improve sensitivity and normalise for variability in IP and digestion. With the inclusion of reference peptides to known ER phosphorylation sites we are also in the process of quantifying ER phosphorylation. We aim to accurately determine ER concentration and phosphorylation status in tumor lysates and assess how these correlate with responsiveness to antiestrogen therapies.
Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-07-23.