Novel method for simultaneous analysis of p53 and K-ras mutations and p53 protein expression in single histologic sections

Background and Objective.-Abnormal protein expression and gene mutation sho uld be examined on exactly identified lesions. To perform simultaneous anal yses of oncogene or tumor suppressor gene mutations and related protein exp ression in single histologic sections, we have developed a novel method usi ng an antigen-retrieval solution for a polymerase chain reaction template b efore immunohistochemical staining. Methods.-Using 20 cases of sporadic colorectal carcinoma, several kinds of antigen-retrieval solutions were tested after heating rehydrated, 4-mum-thi ck, formalin-fixed, paraffin-embedded histologic sections at 96 degreesC fo r 20 minutes, Polymerase chain reaction-single-strand conformation polymorp hism analysis was conducted for p53 (exons 5 through 9) and K-ras (exons 1 and 2), and the histologic sections were then immunostained with monoclonal antibody against p53. Results.-DNA analysis of antigen-retrieval solutions was possible in all 20 cases and revealed completely consistent results (100%) with fresh cancer tissue and microdissected cancer tissue of paraffin-embedded histologic sec tions. With this method, K-ras mutations were positive in 10 of 20 cases (e xon 1 in 9 cases and exon 2 in 1 case) and p53 mutations were positive in 9 of 20 cases (exon 5 in 4 cases, exon 6 in 1, exon 7 in 3, and exon 8 in 1 case), with 8 of the 9 p53 mutation cases showing diffuse p53 protein expre ssion on immunostaining. Base alterations of all abnormal conformers were c onfirmed with direct sequencing. For polymerase chain reaction-single-stran d conformation polymorphism analysis, sodium citrate buffer (pH 6.0) was fo und to be the optimal antigen-retrieval solution. Conclusions.-This newly developed method can be used for routine immunostai ning and genetic analysis with single histologic sections.