Detection of telomerase status by semiquantitative and in situ assays, and by real-time reverse transcription-polymerase chain reaction (telomerase reverse transcriptase) assay in bladder carcinomas.

Abstract

To investigate whether telomerase activity could be used as a diagnostic and/or prognostic marker of bladder carcinoma, by assessing telomerase activity using semiquantitative and in situ methods, and quantifying telomerase reverse transcriptase (hTERT) mRNA expression in a series of bladder carcinomas. Telomerase activity was detected by the telomeric repeat amplification protocol (TRAP) assay using a telomerase polymerase chain reaction (PCR) enzyme-linked immunosorbent assay (ELISA) kit on a series of 29 bladder carcinomas and on three normal bladder samples. hTERT mRNA levels were quantified using real-time quantitative reverse transcription (RT)-PCR. For the in situ detection of telomerase activity, the same telomerase PCR ELISA kit was used and applied to frozen-tissue sections only for discordant cases between telomerase activity and hTERT mRNA status. Telomerase activity was positive in 15 of the 29 bladder carcinomas (52%) and negative for the three normal bladder samples. hTERT was detected and quantified in all tumour samples, with major differences in hTERT values. None of the three normal bladder samples had quantifiable hTERT mRNA, giving complete sensitivity and specificity for the method in diagnosing bladder carcinoma. Comparing the results of RT-PCR and TRAP assay showed a significant association between the enzyme activity and levels of hTERT mRNA expression, with only five discordant cases, most of them being TRAP-negative and hTERT-positive. Among these cases the in situ results of telomerase activity were concordant with hTERT mRNA levels by RT-PCR and not with TRAP assay results, as nuclear fluorescence of malignant epithelial cells. The semiquantitative evaluation of positive cell numbers showed a heterogeneity of telomerase activity within the tumour tissue. There was a significant correlation between RT-PCR and histopathological variables (grade and stage), and a significant correlation between TRAP assay results and histopathological grade. Detecting hTERT mRNA by RT-PCR seems to be a promising method, with a much better sensitivity than the TRAP assay in diagnosing bladder carcinomas, and a better correlation with histopathological variables. False-negative cases on the TRAP assay are explained by the heterogeneity of telomerase activity within tumour cells. Thus evaluating hTERT gene expression levels could be used as a marker of malignant progression, useful in the early diagnosis and follow-up of bladder carcinomas.