cDNA sequencing improves the detection of P53 missense mutations in colorectal cancer

Abstract

Background: Recently published data showed discrepancies between P53 cDNA and DNA sequencing in glioblastomas. We hypothesised that similar discrepancies may be observed in other human cancers.

Methods: To this end, we analyzed 23 colorectal cancers for P53 mutations and gene expression using both DNA and cDNA sequencing, real-time PCR and immunohistochemistry.

Results: We found P53 gene mutations in 16 cases (15 missense and 1 nonsense). Two of the 15 cases with missense mutations showed alterations based only on cDNA, and not DNA sequencing. Moreover, in 6 of the 15 cases with a cDNA mutation those mutations were difficult to detect in the DNA sequencing, so the results of DNA analysis alone could be misinterpreted if the cDNA sequencing results had not also been available. In all those 15 cases, we observed a higher ratio of the mutated to the wild type template by cDNA analysis, but not by the DNA analysis. Interestingly, a similar overexpression of P53 mRNA was present in samples with and without P53 mutations.

Conclusion: In terms of colorectal cancer, those discrepancies might be explained under three conditions: 1, overexpression of mutated P53 mRNA in cancer cells as compared with normal cells; 2, a higher content of cells without P53 mutation (normal cells and cells showing K-RAS and/or APC but not P53 mutation) in samples presenting P53 mutation; 3, heterozygous or hemizygous mutations of P53 gene. Additionally, for heterozygous mutations unknown mechanism(s) causing selective overproduction of mutated allele should also be considered. Our data offer new clues for studying discrepancy in P53 cDNA and DNA sequencing analysis.

Figure 1

Molecular analyses of P53 gene. (A) cDNA sequencing showing mutation (arrow) and DNA analysis confirming presence of wild type nucleotide only (case no. 3 in Table 1). (B) cDNA and DNA sequencing showing both bands equally (case no. 1 in Table 1). (C) cDNA sequencing showing prelevance of mutated nucleotide, and DNA presenting prelevance of wild type band (case no. 9 in Table 1). (D) estimation of sequencing sensitivity for sample presenting only mutated band, see Materials and Methods (case no. 3 in Table 1). (E) example of LOH analysis for microsatellite marker D17S675. The lost allele is marked with an arrow (case no.13, left and 10, right in Table 1). LOH analysis confirms a high amount of nontumor cells (cells without LOH on 17 p) in sample no. 13. N, normal tissue (blood); T, tumor sample.

Figure 2

P53 expression level in tumor samples with mutation and tumor samples without mutation versus control tissue. Significance of difference according to Mann-Whitney U test, P < 0.05.

Figure 3

P53 immunohistochemistry. (A) case no. 2 with nuclear expression of the protein. (B) case no. 20 without immunopositivity for P53. Magnification 200×.