Novel Polymerase Chain Reaction Approach for Full-Coding p53 Mutation Detection in Microdissected Archival Tumors

Abstract

Evidence suggests that up to 25% of p53 mutations are outside of exons 5-8 and that insertions, deletions, and polymorphic sites in the p53 gene may play a significant role in the process of carcinogenesis. A novel polymerase chain reaction (PCR) approach for the analysis of the entire p53 coding and splice site regions from microdissected, formalin-fixed, paraffin-embedded tumor tissues has been developed which allows multiple genetic analyses to be performed from one primary amplification reaction. The method was initially evaluated using well-characterized cell lines. In addition to confirming the published p53 mutations for HT29, Molt 4, A431, and HN5, a 16 base pair (bp) duplication within intron 3 was detected in both the A431 and HT29 cell lines. Analysis of archival samples of ovarian cancer identified the same 16-bp duplication and coding region variations. In all samples, using GenBank submission U94788 as a reference, a C-insertion was detected at nucleotide positions 11818 and 11874 within intron 2. At nucleotide position 14168, within intron 7, a T-to-G base change was found. This novel PCR approach has the potential to reduce the amount of clinical material required by up to 95%, thus facilitating retrospective studies on archival tumor collections. Furthermore, a wider analysis of the p53 gene, including splice sites and intronic regions, may yield additional information regarding cancer predisposition, response to therapy, and progression.

EGFR induces expression of IRF‐1 via STAT1 and STAT3 activation leading to growth arrest of human cancer cells

“…It has been reported that IRF-1 cooperate with the tumor suppressor p53-a cooperation necessary for some cells to undergo apoptosis. 31 Interestingly, both HN5 and A431 cell lines are known to carry mutations in the gene encoding p53, 32,33 suggesting that IRF-1 may be unable to induce apoptosis because of the lack of functional p53. A number of studies have reported IFN-associated genes or ''IFN signatures'' associated with human cancers.…”

Clinical use of genotype to predict chemosensitivity in oligodendroglial tumors

“…28,34 p53 mutations (exons 5 through 8) were detected by direct sequence analysis of PCR products. 34,35 Statistics. Statistical analysis was performed using SPSS.…”

Characterisation of molecular alterations in microdissected archival gliomas

“…In comparison to the GenBank TP53 gene sequence used as reference (EM:U94788), two somatic intronic sequence variations were identified. This duplication has been identified in our laboratory in HN5 and A431 cell lines and in an ovarian cancer sample [46], but not in any of the gliomas analysed in this study. The intron 7 variation was found in only one Gen-Bank submission, in a sequence also derived from an astrocytoma (EM:AF136270).…”

“…TP53 mutation detection used a novel PCR methodology, which, by use of multiple simultaneous amplification of all TP53 amplicons within a single first round reaction, reduces markedly the amount of clinical material required for full analysis [46]. More extensive sequence data, including some intronic sequence, is obtained using this primer set compared with most other TP53 methodologies.…”

“…For TP53 mutation detection, a novel methodology has been developed for use with small amounts of microdissected paraffin-embedded tissue [46]. PCR products (n=15) were obtained to cover exon 1, all of the coding sequence in exons 2-11, and flanking intronic sequence (given in [46]). Inclusion of all first round primers in a single reaction enables multiple simultaneous amplification of all TP53 amplimers in the first round.…”