Combining bevacizumab and chemoradiation in rectal cancer. Translational results of the AXEBeam trial.

BACKGROUND: This study characterises molecular effect of bevacizumab, and explores the relation of molecular and genetic markers with response to bevacizumab combined with chemoradiotherapy (CRT). METHODS: From a subset of 59 patients of 84 rectal cancer patients included in a phase II study combining bevacizumab with CRT, tumour and blood samples were collected before and during treatment, offering the possibility to evaluate changes induced by one dose of bevacizumab. We performed cDNA microarrays, stains for CD31/CD34 combined with α-SMA and CA-IX, as well as enzyme-linked immunosorbent assay (ELISA) for circulating angiogenic proteins. Markers were related with the pathological response of patients. RESULTS: One dose of bevacizumab changed the expression of 14 genes and led to a significant decrease in microvessel density and in the proportion of pericyte-covered blood vessels, and a small but nonsignificant increase in hypoxia. Alterations in angiogenic processes after bevacizumab delivery were only detected in responding tumours. Lower PDGFA expression and PDGF-BB levels, less pericyte-covered blood vessels and higher CA-IX expression were found after bevacizumab treatment only in patients with pathological complete response. CONCLUSIONS: We could not support the 'normalization hypothesis' and suggest a role for PDGFA, PDGF-BB, CA-IX and α-SMA. Validation in larger patient groups is needed.

A new approach to the validation of tissue microarrays.

Although tissue microarrays (TMA) have been widely used for a number of years, it is still not clear how many core biopsies should be taken to determine a reliable value for percentage positivity or how much heterogeneity in marker expression influences this number. The first aim of this study was to validate the human visual semi-quantitative scoring system for positive staining of tumour tissue with the exact values determined from computer-generated images. The second aim was to determine the minimum number of core biopsies needed to estimate percentage positivity reliably when the immunohistochemical staining pattern is heterogeneous and scored in a non-binary way. Tissue sections from ten colorectal cancer specimens were stained for carbonic anhydrase IX (CA IX). The staining patterns were digitized and 400 artificial computer-generated images were generated to test the accuracy of the human scoring system. To determine the minimal number of core biopsies needed to account for tumour heterogeneity, 50 (artificial) core biopsies per section were taken from the tumoural region of the ten digitally recorded full tissue sections. Based on the semi-quantitative scores from the 50 core biopsies per section, 2500 x n (n = 1-10 core biopsies) experimental core biopsies were then generated and scores recorded. After comparison with field-by-field analysis from the tumoural region of the whole tissue section, the number of core biopsies that need to be taken to minimize the influence of heterogeneity could be determined. In conclusion, visual scoring accurately estimated the percentage positivity and the percentage tumour present in a section, as judged by comparison with the artificial images. The exact number of core biopsies that has to be examined to determine tumour marker positivity using TMAs is affected by the degree of heterogeneity in the expression pattern of the protein, but for most purposes at least four is recommended.