Novel method for simultaneous analysis of p53 and K-ras mutations and p53 protein expression in single histologic sections.
OBJECTIVE
Abnormal protein expression and gene mutation should be examined on exactly identified lesions. To perform simultaneous analyses of oncogene or tumor suppressor gene mutations and related protein expression in single histologic sections, we have developed a novel method using an antigen-retrieval solution for a polymerase chain reaction template before immunohistochemical staining.


METHODS
Using 20 cases of sporadic colorectal carcinoma, several kinds of antigen-retrieval solutions were tested after heating rehydrated, 4-microm-thick, formalin-fixed, paraffin-embedded histologic sections at 96 degrees C for 20 minutes. Polymerase chain reaction-single-strand conformation polymorphism 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 sections. With this method, K-ras mutations were positive in 10 of 20 cases (exon 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 expression on immunostaining. Base alterations of all abnormal conformers were confirmed with direct sequencing. For polymerase chain reaction-single-strand conformation polymorphism analysis, sodium citrate buffer (pH 6.0) was found to be the optimal antigen-retrieval solution.


CONCLUSIONS
This newly developed method can be used for routine immunostaining and genetic analysis with single histologic sections.
Yamashita
K
K
Department of Pathology, Kitasato University East Hospital, Sagamihara, Kanagawa, Japan.
Yoshida
T
T
Shinoda
H
H
Okayasu
I
I
D000230:Adenocarcinoma / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D017931:DNA Primers / Q000737:chemistry; D004273:DNA, Neoplasm / Q000032:analysis; D004210:Dissection; D005260:Female; D016158:Genes, p53 / Q000235:genetics*; D011905:Genes, ras / Q000235:genetics*; D006652:Histological Techniques; D006801:Humans; D008297:Male; D008846:Micromanipulation; D008875:Middle Aged; D009154:Mutation; D009367:Neoplasm Staging; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
D017931:DNA Primers; D004273:DNA, Neoplasm; D016159:Tumor Suppressor Protein p53