Purpose: The prognostic relevance of p53 protein accumulation in muscle-invasive bladder carcinoma is well documented, but the prognostic relevance of p53 alterations in superficial bladder tumors remains uncertain. Immunohistochemical data are divergent, possibly because of the use of nonstandardized techniques. We therefore investigated the relevance of p53 gene point mutations and loss of heterozygosity (LOH) for tumor recurrence. The results of this molecular analysis were compared with accumulation of the p53 protein as shown by immunohistochemistry.
Material and methods: Representative tumor tissue was selected and microdissected from 40 patients (pTa, 18 patients; pT1, 22 patients; grade I, 7 patients; grade II, 28 patients; grade III, 5 patients). Polymerase chain reaction (PCR) was carried out with exons 5-8. All PCR products were screened for p53 mutations with temperature-gradient gel electrophoresis (TGGE). When mobility shift was observed, direct nucleotide sequencing was performed. Detection of LOH was performed with nonradioactive microsatellite analysis using three markers (TP 53, D17S513 and D17S786) on chromosome 17p. Immunohistochemistry was performed with the DO 7 antibody. Tumor samples with p53 accumulation of 5% or more positive nuclei were classified as positive. Univariate analysis for disease-free survival was performed using Kaplan-Meier analysis and the log-rank test.
Results: TGGE and direct sequencing detected mutations in 10 of 40 patients (2 of 18 pTa and 8 of 22 pT1 patients). LOH was detected in 11 patients. Both a mutation and LOH were detected in 3 patients. p53 immunohistochemistry detected at least 5% positive nuclei in 28 of 40 patients (70%). After a median follow-up of 26 months 14 patients suffered disease recurrence. Whereas disease-free survival did not correlate with a mutation (p = 0.77, log-rank test), LOH (p = 0.2) or a mutation in combination with LOH (p = 0.23), a positive p 53 immunoreaction was significantly associated with short disease-free survival (p = 0.009).
Conclusion: Despite the relatively high percentage of patients with p53 gene alteration in this population no significant correlation between the detection of molecular alteration and disease recurrence could be found. We conclude that, in contrast to immunohistochemical accumulation, gene alterations play only a minor role in tumor recurrence of p53 in patients with superficial transitional cell carcinoma of the bladder, and that immunohistochemical accumulation of the p53 protein has to be explained by mechanisms other than gene mutations.