Impact of biospecimens handling on biomarker research in breast cancer

Background: Gene expression profiling is moving from the research setting to the practical clinical use. Gene signatures able to correctly identify high risk breast cancer patients as well as to predict response to treatment are currently under intense investigation. While technical issues dealing with RNA preparation, choice of array platforms, statistical analytical tools are taken into account, the tissue collection process is seldom considered. The time elapsed between surgical tissue removal and freezing of samples for biological characterizations is rarely well defined and/or recorded even for recently stored samples, despite the publications of standard operating procedures for biological sample collection for tissue banks. Methods: Breast cancer samples from 11 patients were collected immediately after surgical removal and subdivided into aliquots. One was immediately frozen and the others were maintained at room temperature for respectively 2, 6 and 24 hrs. RNA was extracted and gene expression profile was determined using cDNA arrays. Phosphoprotein profiles were studied in parallel. Results: Delayed freezing affected the RNA quality only in 3 samples, which were not subjected to gene profiling. In the 8 breast cancer cases with apparently intact RNA also in sample aliquots frozen at delayed times, 461 genes were modulated simply as a function of freezing timing. Some of these genes were included in gene signatures biologically and clinically relevant for breast cancer. Delayed freezing also affected detection of phosphoproteins, whose pattern may be crucial for clinical decision on target-directed drugs. Conclusion: Time elapsed between surgery and freezing of samples appears to have a strong impact and should be considered as a mandatory variable to control for clinical implications of inadequate tissue handling.