p53 Gene Mutations in Astrtocytoma Detection by Direct DNA Sequencing

Choi, Won Hee; Choi, Kyung Chan; Chi, Je G

Abstract

PURPOSE: Mutations in the p53 gene have been recognized in brain tumor, and clonal expansion of p53 mutant cells has been shown to be associated with glioma progression. However, studies on the p53 gene have been limited by the need for fresh frozen tissues. We have tried a method utilizing polymerase chain reaction (PCR) for the direct DNA sequecing of the p53 gene using a single 10 m paraffin-embedded tissue section. We applied this method to detect for p53 gene mutations in exons 5~8 in human astrocytoma utilizing paraffin-embedded tissues.
MATERIALS AND METHODS
Twenty paraffin blocks containing tumor were selected from surgical specimens from twenty different cases. Tumors included 10 astrocytomas and 10 anaplastic astrocytomas. Ten controls were also selected among autopsy cases showing normal brain in light microscopy. The tissue section on the stained glass slide was used to guide microdissection of an unstained adjacent tissue section to ensure above 90% of the tumor cell population for p53 mutational analysis. RESULT: Mutation in the p53 gene was identified in 1 of 10 (10%) anaplastic astrocytomas. Mutations in the p53 gene were identified in 1 of 10 cases (10%) by PCR and direct DNA sequencing. Mutation in exon 7 resulting in amino acid substitution was found in one anaplastic astrocytoma (codon 245, GGC-->GAC: glycine-->aspartic acid). Ten control cases, ten astrocytomas and nine anaplastic astrocytomas were confirmed to be negative by direct sequencing of amplified DNA.
CONCLUSION
This study demonstrates the feasibility of evaluating p53 gene mutations in archived astrocytoma specimens using PCR and direct DNA sequencing on paraffin sections. Application of this method should facilitate investigation of the role of p53 gene mutations in tumor biology.