Crypt organization in the progression to colorectal neoplasia: Computerized three dimensional (3D) crypt reconstruction and quantitative analysis of bcl-2, bas, mitosis and apoptosis within the crypt

Abstract

AIMS: Alterations in epithelial proliferation and apoptosis in colonic mucosa are associated with an increased risk of colon cancer. Deregulation of bcl-2 and bax are important factors in the control of cell turnover. The aim of this study was to analyze patterns of bcl-2 and bax expression and in mitosis and apoptosis in the progression to colorectal neoplasia, and to reconstruct colorectal crypts to visually demonstrate this deregulation. METHODS: 12 patients with a sigmoid adenomatous polyp and 12 low risk controls were studied. Mucosal biopsies were taken from the polyp, tissue adjacent to and distant from the polyp and from the sigmoid colon of controls. Apoptotic and mitotic figures, and cells expressing bcl-2 or bax were counted, and labeling indices calculated for each within each crypt. Results are expressed as total labeling index (TLI) and labeling index (L1) for each of the five compartments (cpts) in which colonic crypts were divided for mitotic index (MI), apoptotic index (AI), and bd-2 and bax expression. Sample individual crypts that were complete within serially sections were idemified. The appropriate cells were marked, the images captured and stored, and then whole crypts were reconstructed in 3D using Analyze3.1 to assess the intra-erypt relationships of each variable. RESULTS: There was no significant difference between TLI and LI for bcl-2 and bax for controls vs. distant mucosa. The TL1 and L1 were significantly higher for polyp vs adjacent vs. distant mucosa for both bcl-2 (cpts 1-3, p<O.02) and bax (cpts 2-5, p<0.01). There was a significam increase in MI in polyp vs. distant mucosa (cpts 1-3, p<0.05). No difference in AI was seen between distant and adjacent mucosa, but there was a trend to a reduction in AI in polyp mucosa vs. all other sites. A total of 56 crypts were available for reconstruction. In reconstructed crypts, deregulation of bcl-2 and bax, increased mitosis, and the presence of apoptotic bodies in the basal cpts of polyps where bd-2 expression was highest, was easily visualized in still images and video sequences. CONCLUSIONS: Mitosis, expression ofbcl-2 and bax, and apoptosis maybecome deregulated within the colorectal crypt in the progression to colorectal neoplasia. In addition to quantitative analysis, computerized reconstruction with still images or video sequences of whole crypts can be useful in advancing our understanding of this process.