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.