Abstract 535: Selected reaction monitoring (SRM)-based assay for the quantification of biomarkers in human breast cancer tissue
Authors: Tchafa Alimatou M. , Lemoff Andrew S. , Mirzaei Hamid 
Published: August 2015

Abstract
Background: Breast cancer is a heterogeneous disease that has remained the second leading cause of cancer-related death in women despite the advances in research over the past decades. Numerous studies have shown that just like in other cancers, early detection drastically improves survival rates. Breast cancer diagnosis is currently dependent on the differential expression of key proteins (biomarkers) that allow for classification, staging and identification of proper therapies. However, because of its heterogeneity, accurate measurement of these biomarkers remains challenging and subjective. Antibody-based techniques such as immunohistochemistry are currently the standard methods of biomarker measurements in breast cancer. With the improvement of genomic and proteomic technology, the number of validated biomarkers is increasing at a faster pace than the number of corresponding clinical assays, preventing the quick translation from the bench to the clinic. Selected reaction monitoring (SRM) is a robust and accurate mass spectrometry-based method that can be used to simultaneously quantify subgroups of protein-specific peptides within any complex protein mixture. The goal of this study is to develop an SRM-based assay for simultaneous detection of 40 biomarkers in tissues.
Method: Our panel of biomarkers consists of well-established proteins whose expression correlates with breast cancer aggressiveness and therapy, such as estrogen receptor (ER), HER2 and progesterone receptor (PR). It also includes cellular characterization markers like E-cadherin, fibronectin and vimentin that can aid in better categorization of cancer types. Peptides specific to beta-actin and GAPDH are used as internal controls. We have access to over 2000 characterized breast tissue samples available at the UT Southwestern Medical Center tissue repository. These paraffin-embedded tissues are laser-capture micro-dissected and digested prior to SRM measurements. For validation, we compare the SRM measurements to current bench-top techniques such as immunohistochemistry and Western blot.
Results: Using our current method, we have successfully quantified the levels of ER, PR, Her2, and fibronectin on 2.4mm2 breast tissues. Additional experiments are underway to quantify the levels of these and additional proteins in additional samples and to compare their expression to their clinical and pathology reports.
Conclusion: Development of high quality antibody assays is time consuming and requires extensive resources and efforts. In addition immunohistochemistry assays cannot be multiplexed. This project will provide a faster, cost-effective, and multiplexable method for breast cancer biomarkers detection and quantification. Future studies will focus on measuring the levels of the same biomarkers in serum samples. Altogether, it will allow for a quick validation platform as new biomarkers are identified.
Citation Format: Alimatou M. Tchafa, Andrew S. Lemoff, Hamid Mirzaei. Selected reaction monitoring (SRM)-based assay for the quantification of biomarkers in human breast cancer tissue. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 535. doi:10.1158/1538-7445.AM2015-535