Simultaneous detection of apoptosis and proliferation in colorectal carcinoma by multiparameter flow cytometry allows separation of high and low-turnover tumors with distinct clinical outcome.

BACKGROUND: Dukes C colorectal carcinoma is treated with adjuvant chemotherapy. Adjuvant treatment is not standard for patients with Dukes B tumors, even though about 20% of patients within this tumor stage die of recurrent disease. The authors investigated whether a novel method of simultaneous detection of apoptosis and proliferation would improve the assessment of prognosis in colorectal carcinoma patients, with the ultimate goal of accurately identifying patients eligible for adjuvant therapy. METHODS: A multiparameter flow cytometric assay with heat pretreatment was performed on 278 paraffin-embedded colorectal adenocarcinomas. After immunochemical isolation of tumor cells, apoptosis and proliferation were assessed simultaneously by immunostaining for the M30 antibody and by quantitative DNA analysis, respectively. Patients were followed for more than 10 years. RESULTS: The mean values of apoptosis (apoptotic fraction [AF], i.e., the percentage of M30-positive cells) and proliferation (S-phase fraction [SPF]) were 11.1% and 13.1%, respectively. The AF and SPF values were correlated positively (P = 0.01) and both increased with advancing tumor stage (P = 0.02). Combining AF and SPF, patients with tumors with a high turnover (i.e., both AF and SPF values were greater than the mean) had an overall survival rate of 13%, whereas patients with low-turnover tumors (i.e., both AF and SPF values were less than the mean) had an overall survival rate of 89% (P < 0.001 by log rank test). Moreover, within Dukes B and C stages, patients with high-turnover tumors had a poorer survival than patients with low-turnover tumors (P < 0.001 for both stages). CONCLUSIONS: The simultaneous detection of apoptosis and proliferation in archival material allows separation of high and low-turnover colorectal adenocarcinomas and improves the assessment of prognosis. This technique could be used to stratify patients for adjuvant chemotherapy.

Target validation for genomics using peptide-specific phage antibodies: a study of five gene products overexpressed in colorectal cancer.

Genomic approaches are providing a wealth of information on differential gene expression in cancer. To identify the most interesting genes amongst the many identified, high-throughput methods for analysis of genes at the translational level are required. We have used a rapid method for the in vitro selection of antibodies to peptide antigens for the generation of probes to 5 gene products that we have found to be overexpressed in colorectal cancer. The rationale of our study was to select a non-immune phage displayed human antibody library on peptides designed from the coding regions of the gene sequences and to verify whether such antibodies would be suitable probes for the parental protein in immunohistochemical and Western blot analysis. After the generation of a profile of genes overexpressed in primary colorectal cancer (CRC) we selected 5 genes, Ese-3b, Fls353, PBEF, SPARC and Smad5 for a more detailed analysis using phage display-derived antibodies. For these 5 antigens we designed 14-20 amino acid peptides predicted to be exposed on the surface of the parental protein. Selection of a large phage displayed antibody library resulted in specific antibodies for 6 of 8 different peptides with between 2 and 15 different antibodies isolated per peptide. Of 20 antibodies tested, 2 antibodies recognized the putative parental protein from primary CRC tissue. An antibody specific for a PBEF-derived peptide (Fab/PBEF-D4) was shown to recognize a protein product of the expected molecular weight in Western blotting and showed overexpression in n = 6/8 matched tumor/normal protein lysates. Furthermore, in immunohistochemistry this antibody showed restricted staining of the tumor stromal compartment with no detectable staining of epithelial cells. The discovery that PBEF is overexpressed in cancer is unexpected given that the normal function of PBEF is as a cytokine required for the maturation of B cell precursors. We also report on the isolation of an antibody (Fab/SMAD-50) specific for a Smad5-derived peptide that showed cytoplasmic staining of epithelial cells in both CRC tumor and matched normal mucosa. Fab/SMAD-50 also bound to a group of proteins in Western blotting with molecular weights consistent with belonging to the Smad family. These antibodies may be suitable probes for further investigation of the roles of PBEF and Smad5 in cancer. The amenability of phage display to automation suggests that this approach may be developed for implementation on a genomics scale. Indeed, the large-scale generation of antibody probes that can be used to study protein expression in situ would be of great value in target validation for functional genomics.