Breast cancer is by far the most common diagnosed form of
cancer and the leading cause of
cancer death in women today. Clinically useful
biomarkers for early detection of
breast cancer could lead to a significant reduction in mortality. Here we describe a detailed analysis using gel-based proteomics in combination with mass spectrometry and immunohistochemistry (IHC) of the tumour interstitial fluids (TIF) and normal interstitial fluids (NIF) collected from 69 prospective
breast cancer patients. The goal of this study was to identify abundant
cancer up-regulated
proteins that are externalised by cells in the tumour microenvironment of most if not all these lesions. To this end, we applied a phased
biomarker discovery research strategy to the analysis of these samples rather than comparing all samples among each other, with inherent inter and intra-sample variability problems. To this end, we chose to use samples derived from a single tumour/benign tissue pair (patient 46, triple negative tumour), for which we had well-matched samples in terms of epithelial cell numbers, to generate the initial dataset. In this first phase we found 110
proteins that were up-regulated by
a factor of 2 or more in the TIF, some of which were confirmed by IHC. In the second phase, we carried out a systematic computer assisted analysis of the 2D
gels of the remaining 68 TIF samples in order to identify TIF 46 up-regulated
proteins that were deregulated in 90% or more of all the available TIFs, thus representing common
breast cancer markers. This second phase singled out a set of 26
breast cancer markers, most of which were also identified by a complementary analysis using LC-MS/MS. The expression of
calreticulin,
cellular retinoic acid-binding protein II,
chloride intracellular channel
protein 1, EF-1-beta,
galectin 1, peroxiredoxin-2,
platelet-derived endothelial cell growth factor,
protein disulfide isomerase and
ubiquitin carboxyl-terminal
hydrolase 5 were further validated using a tissue microarray containing 70 malignant
breast carcinomas of various grades of atypia. A significant number of these
proteins have already been detected in the blood/plasma/secretome by others. The next steps, which include
biomarker prioritization based on the hierarchal evaluation of these markers, antibody and
antigen development, assay development, analytical validation, and preliminary testing in the blood of healthy and
breast cancer patients, are discussed.