Immunohistochemical measurement of endothelial cell apoptosis and proliferation in formalin-fixed, paraffin-embedded...

There is a need for simple, reproducible methodology for measurement of endothelial cell (EC) apoptosis and proliferation in formalin-fixed, paraffin-embedded (FFPE) human tissue obtained in clinical trials of potential anti-angiogenic agents. Therefore, we developed colorimetric, dual-label immunohistochemical techniques for use on FFPE tissue, based on the use of single-stranded (ss) DNA and Ki-67 as markers of EC apoptosis and proliferation, respectively. Digital image analysis was used to obtain the total tumour microvessel density (MVD), from which the EC apoptosis index (AI) and proliferation index (PI) were derived manually as the number of positive ECs per vessel. Immunohistochemical measurement of EC apoptosis and proliferation was validated on human colorectal cancer liver metastases from a randomized, placebo-controlled trial of the selective cyclooxygenase-2 inhibitor rofecoxib. Proliferating and apoptotic ECs clustered in discrete areas of the tumour vasculature. ECAI (median [inter-quartile range, IQR] 0.0018 [0.0003-0.0064]) and ECPI (median [IQR] 0.0043 [0.002-0.014]) values were low and highly variable in tumours from both placebo- and rofecoxib-treated patients. Our novel dual-label immunohistochemical methods will be generally applicable in FFPE human cancer tissue and should prove invaluable for measuring the anti-angiogenic activity of experimental therapies in clinical trials. The low absolute level of and variability in EC apoptosis and proliferation detectable in human colorectal cancer liver metastases indicates that similar intervention studies using these end-points will need to ensure adequate size in order to be able to detect anti-angiogenic activity by immunohistochemical methods.