ADVANTAGES OF IMMUNOSTAINING OVER DNA ANALYSIS USING PCR AMPLIFICATION TO DETECT P53 ABNORMALITY IN LONG-TERM FORMALIN-FIXED TISSUES OF HUMAN COLORECTAL CARCINOMAS

To study the appropriate period for formalin fixation in order to dete ct p53 abnormalities in formalin-fixed tissue, we used seven surgicall y resected human colorectal cancer specimens. The immunohistochemical reactivity of p53 immunostaining and amplification of DNA by polymeras e chain reaction (PCR) of the p53 gene were compared after various per iods of 10% formalin fixation (1 day, and 1, 2, 4, and 8 weeks). For c omparative immunostaining, we used the monoclonal antibody Ki-67 (MIB- 1), and for comparative polymerase chain reaction (PCR), K-ras at codo n 12 was amplified. Immunostaining was performed by the streptavidin-b iotin method with microwave retrieval, and PCR amplifications were per formed by the nested PCR method. p53 and Ki-67 immunoreactivity did no t change essentially for up to 2 weeks and 1 week, respectively, of fo rmalin fixation. PCR amplification for p53 at exon 8 and K-ms at codon 12 was successful until 1 day and 2 weeks, respectively, of formalin- fixation for the specimens of all seven cases. Thereafter, the amplifi cation tended to worsen as the fixation time lengthened. Further, the DNA was more successfully amplified in the second PCR than in the firs t. These results suggest that to detect p53 abnormality in specimens t hat have been formalin-fixed for long periods, immunohistochemical sta ining may have advantages over DNA analysis with PCR amplification.