The impact of delay in cryo-fixation on biomarkers of Src tyrosine kinase activity in human breast and bladder cancers.

Demonstration of pharmacodynamic activity of new, targeted cancer drugs in tumour tissue is potentially important in guiding early drug development. However, delays between tumour sampling and sample fixation may result in variability of pharmacodynamic biomarkers. The aim of this study, was to assess the impact of delays in fixation on biomarkers of Src kinase activity. A total of 20 patients with locally advanced breast cancer and 5 with early bladder cancer had multiple tissue samples taken which were fixed at documented time points up to 60 min after biopsy. These were examined to determine if the amount of Paxillin, phospho-Paxillin, phospho-focal adhesion kinase (FAK) and total phospho-Tyrosine changed over time, using a quantitative lysate immunoassay. In breast cancer, there was an increase in the amount of phospho-Paxillin (60% per h; P = 0.019) up to 60 min after biopsy. The amount of total Paxillin decreased (28% per h; P = 0.034) over the same time course. In early bladder cancer, no changes were noted in any endpoints up to 45 min. Standardisation of the time taken between biopsy and fixation may be critical, particularly in studies using phosphorylated protein biomarkers.