Abstract 4874: Quantitative comparison of matched normal and tumor breast proteomic profiles
Authors: Sutton Chris , Rustogi Nitin , Scally Andrew , Loizidou Maria , Hadjisavvas Andreas , Kyriacou Kyriacos 
Published: June 2012

Introduction – Identification of markers of different stages of breast cancer remains a challenge. Although biofluids such as urine and plasma are preferred sources to discover potential biomarkers, tissue proteins are diluted compared to their site of expression and difficult to analyse due to degradation or due to the wide dynamic range of plasma proteins. As mastectomy is frequently used as part of treatment, it is possible to get sufficient histologically-defined tissue to perform proteomics profiling at the source of the disease and therefore more accurately identify changes in expression, for subsequent biomarker verification. Two dimensional separation of stable isotope-labelled total peptide mixtures from matched normal and disease biopsies from 10 patients with adenoma, DCIS or invasive carcinoma were compared in order to identify markers of different stages of the disease.

Methods – Two lysis buffers, RIPA and urea, in combination with sonication, were used sequentially to maximise protein extraction from matched normal and diseased breast biopsies of 10 patients. The biopsies were split into three groups each comprising matched normal and diseased protein extracts from 4 patients one of which is common to all the groups to act as an internal control. Each protein extract in each group was trypsin-digested and iTRAQ (isobaric tag for relative and absolute quantitation) labelled. The digests from each group were pooled together and the resulting peptide mixture separated using isoelectric focusing followed by reverse phase nanoHPLC. Fractions from nanoHPLC were analysed by MALDI (matrix assisted laser desorption/ionisation) MS and MS/MS, and the data searched against a sequence database to identify proteins in the original sample.

Results – The proteins identified in each group of 4 patients were compared and those which were observed in all three data sets, were subjected to further quantitative analysis. For each protein from each patient, a normal:tumor ratio was determined from the iTRAQ data. Those proteins for each patient that were significantly changed in expression (a ratio of &lt;0.77 equates with a significant decrease and a ratio of &gt;1.3 equates with a significant increase) were clustered together for further processing. Those proteins whose response was changed across a patient group (adenoma, DCIS or invasive carcinoma) were investigated for associations with cancer biology. Those which may represent novel disease stage-specific biomarkers were verified using immunohistochemistry and Western blotting. Discussion – A number of proteins significantly increased or decreased have previously been associated with breast cancer. In addition, there were a number of new proteins identified which were associated with adenoma, DCIS and invasive carcinoma stages of disease, and these may be suitable targets for drug development as well as for developing potential biomarkers.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4874. doi:10.1158/1538-7445.AM2011-4874