P53 determination in intestinal metaplasia by immunohistochemistry, chip and conventional sequencing technology

Background: The mutations of p53 gene play an important role in the pathogenesis of malignant diseases. Data about localization of specific p53 mutations are not well known in intestinal metaplasia.

Aims: Our aim was to examine if there were any differences between the DNA isolated from blood, from the altered tissue biopsy and from the healthy part of the stomach. Our aims were also to find disease specific p53 mutations, and to find relation between the results of immunohistochemistry and sequencing data. To compare the traditional sequencing technique with DNA sequencing microarray technology is also an important question. The traditional sequencing is the gold standard to determinate DNA sequence, but it is time consumable, so the microarray technology should be a good substitute method.

Materials and Methods: After informed consent, peripheral blood was drawn and gastric biopsy samples were taken from the pathological and healthy part of the stomach. Genomic DNA was extracted from altogether 21 biopsy and blood samples from patients with intestinal metaplasia. From the 11 exons of p53 ten (2–11) were amplified by PCR and examined by GeneChip p53 Assay (Affymetrix), and four (5–8) exons with traditional gel sequencing method (LICOR IR2 and ABI 310 Genetic Analyser). We could examine the intestinal metaplasia biopsies with wild type and mutant p53 immunohistochemistry method in 3 patients.

Results: Some heterozygote mutations were found with microarray technology what we could not confirm by traditional sequencing. We did not find any mutations of the samples with traditional sequencing. In the three cases where we could examine the protein expression, we found correlation with the sequencing results. In two cases the wild type p53 immunohistochemistry has positive result, but none of the samples were positive to the mutant p53 stain.

Conclusions: It seems that this chip technology is not reliable enough to give a new opportunity for the genetic diagnosis. Our results are in good relation with the preceding observations of our research group, that in intestinal metaplasia, as in a premalignant state, the level of wild type p53 could be elevated.