In addition to observations in pancreatic carcinomas, Smad4 selleckchem is also known as a gene involved in juvenile polyposis tumour predisposition syndrome (Howe et al, 1998; Huang et al, 2000). Mutations of the Smad4 gene have been detected in some colorectal cancers, but its role in this specific cancer remains unclear. The frequencies of mutations (5�C45%) have been found to be low (Takagi et al, 1996; MacGrogan et al, 1997; Ohtaki et al, 2001), but data originated from relatively small studies, and the tumour populations examined were inhomogeneous explaining the broad range of incidences found. The aim of this study was to further expand these data by Smad4 mutation analysis of a large set of early-stage (I�CIII) colorectal cancer patients treated in a randomised multicentre trial of 5-fluorouracil (5-FU)/Mitomycin C adjuvant chemotherapy of the Swiss Group for Clinical Cancer Research (SAKK study 40/81).

Owing to the significance of LOH in colorectal cancer and the role of the remaining gene, this study was focused on patients with an allelic loss of one Smad4. METHODS Patients Patients from whom biopsies were isolated, were part of a previous randomised multicentre study of the SAKK on the benefit of treatment with adjuvant chemotherapy between 1981 and 1987 (Laffer, 1995). Deoxyribo nucleic acid (DNA) samples of these patients were extracted from tumour as well as from healthy tissue derived from the same patient in order to perform genetic analyses. Paraffin-embedded material was available from 329 of the 505 patients.

To investigate genetic alterations in the 18q21 region in these tumours, a gene dosage study of the tumour-suppressor genes Smad2, Smad4 and DCC was performed (Boulay et al, 1999). For technical reasons, high-quality DNA for analysis was available from 294 patients only. Individual dosage of the Smad4 gene showed a total deletion frequency (one or both alleles) of 68% when compared to normal tissue. In total, 167 patients (=57%) were detected with an allelic loss of one Smad4 copy. In this study, we randomly chose 73 of these 167 patients to search for the presence of point mutations in the remaining gene. After analysis of these 73 out of 167 patients, two point mutations of Smad4 had been detected, and for statistical reasons, further mutation analysis in the remaining 94 out of 167 patients did not seem necessary to substantiate our finding.

Gene copy status scoring Genomic samples from 294 patients were tested for copy dosage of the Smad4 gene using TaqMan quantitative real-time PCR (Perkin-Elmer, GSK-3 Huenenberg, Switzerland). Copy status of the Smad4 gene was determined by comparing tumour DNA to DNA from normal tissue derived from the same patient as described previously (Boulay et al, 1999). Duplex PCR Polymerase chain reaction (PCR) amplification on DNA was performed in 15��l reaction volume, containing 1.