ABSTRACT
Paired colorectal liver metastases (CLM) and normal tissue samples from a consecutive series of 36 patients were studied prospectively. MIB-1 expression was studied by immunohistochemistry on paraffin-embedded sections. DNA ploidy and S-phase fraction (SPF) measurements were performed by flow cytometry on frozen tissues. Mutations within the p53 (exons 5-8) and c-Ki-ras (codons 12 and 13) genes were detected by PCR single-strand conformation polymorphism analysis followed by sequencing. A high correlation was observed between the MIB-1 LI and SPF value (rho=0.81; P<0.01). Moreover, p53 gene mutations were associated with either high MIB-1 LI and high SPF. In univariate analysis, SPF and MIB-1 levels were related to risk of death. The association between overall survival and DNA-ploidy or p53 mutations did not reach statistical significance, but a slightly better survival was observed for patients either with DNA-diploid tumours or without mutations (P=0.05 and P=0.06, respectively). SPF was shown by multivariate Cox model analysis to be an independent prognostic variable and thus it might be a useful prognostic factor in patients with CLM.
Subject(s)
Biomarkers, Tumor/analysis , Colorectal Neoplasms/pathology , Genes, p53/genetics , Genes, ras/genetics , Liver Neoplasms/secondary , Point Mutation , Adult , Aged , Antigens, Nuclear , Colorectal Neoplasms/chemistry , Colorectal Neoplasms/genetics , Female , Flow Cytometry , Humans , Immunohistochemistry , Ki-67 Antigen , Liver Neoplasms/chemistry , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Male , Middle Aged , Multivariate Analysis , Nuclear Proteins/analysis , Nuclear Proteins/immunology , Ploidies , Polymorphism, Single-Stranded Conformational , Prognosis , Prospective Studies , S Phase , Survival RateABSTRACT
We compared the SSCP behaviour of the DNA fragments containing c-ki-ras 2 wild type 12 and 13 codons or each of the 12 possible point mutated sequences in these two codons. We found that a single electrophoresis condition was sufficient to distinguish each specific mutation from the other 11 and from the wild type sequence. This observation makes it possible to identify each specific mutation directly by SSCP without any need for reamplification and sequencing.
