Detection of Urothelial Bladder Cancer Based on Urine and Tissue Telomerase Activity Measured by Novel RT-TRAP-2PCR Method.

Alexander Glukhov,Ekaterina Laukhtina,Shahrokh F Shariat,Dmitry Enikeev,Konstantin Polyakovsky,Marco Moschini,Petr Glybochko,Sergey Gordeev,Mark Taratkin,Mohammad Abufaraj,Natalya Potoldykova,Marina Sekacheva

doi:10.3390/jcm10051055

Abstract

Purpose: To assess the diagnostic performance of urine telomerase activity (TA) in detecting bladder cancer (BCa) using the modified Telomeric Repeat Amplification Protocol (TRAP) and the Real Time Telomeric Repeat Amplification Protocol with double Polymerase Chain Reaction (RT-TRAP-2PCR). Methods: In this case-control study, matching urine (in the pre- and post-surgical period) and tissue samples from 68 patients with BCa were assessed for TA. As a control, 45 urine samples were examined from non-BCa patients. TA levels were measured using TRAP and RT-TRAP-2 PCR methods. Results: Preoperative urinary TA was elevated in 64 (94.1%) of the 68 BCa patients. Urine TA was undetectable in 44 control patients, while TA was detected in one patient with histologically verified cystitis. Sensitivity for BCa detection of 94.1% and specificity of 97.8% were observed for urinary TA, while tissue TA had 100% sensitivity and 97.8% specificity. Both urine and tissue TA levels were not significantly higher in patients with muscle-invasive disease compared to those with non-muscle invasive BCa (p > 0.05). Urine and tissue TA levels were not associated with higher tumor grade, stage, and number of tumors (p > 0.05). However, the association was found between higher urinary and tissue TA levels with tumor size ≥ 3 cm (p = 0.02 and p = 0.01, respectively). During the first postoperative year, 17 BCa patients experienced disease recurrence, and urinary TA was present in 14 (82.4%) of these patients. The sensitivity and specificity of urinary TA levels for BCa recurrence in patients with non-muscle invasive bladder cancer (NMIBC) during follow-up were 82% and 94.4%, respectively. Conclusions: This pilot study demonstrates a high diagnostic performance of urinary and tissue TA levels measured by a new RT-TRAP-2PCR method for detecting and monitoring BCa. Additionally, the association was found between higher urinary and tissue TA levels with tumor size ≥ 3 cm; however, higher TA levels failed for significant correlation with advanced tumor stage and grade. Our study could serve as a benchmark for the evaluation of novel biomarkers using the RT-TRAP-2PCR method.

Highlights

Despite extensive research efforts, there are no convincing data to support a reproducible clinical benefit of adding urinary biomarkers beyond cytology into the routine diagnosis of bladder cancer (BCa), its follow-up, and the decision-making process [1,2].Telomerase has been shown to have diagnostic and prognostic value in various malignancies [3,4]
These approaches require a large number of polymerase chain reaction (PCR) cycles during one stage significantly changing the dependence between the rate of amplicon synthesis and the reaction time resulting in nonspecific products synthesis
This study aimed to investigate the feasibility of the relative telomerase activity (TA) level assessment using the novel RT-Telomeric Repeat Amplification Protocol (TRAP)-2PCR method in the urine and tissue samples obtained from

Summary

Introduction

There are no convincing data to support a reproducible clinical benefit of adding urinary biomarkers beyond cytology into the routine diagnosis of bladder cancer (BCa), its follow-up, and the decision-making process [1,2].Telomerase has been shown to have diagnostic and prognostic value in various malignancies [3,4]. The currently used methods for telomerase activity identification based on a real time polymerase chain reaction (PCR) are a modification of the classical TRAP method, involving fluorescent dyes to visualize reaction products, for example, SYBR Green I or SYBR Gold, fluorescent-labeled primers, luminescent-labeled primers, radioactive-isotope labeled primers, and biotinylated primers [8,9,10,11]. These approaches require a large number of PCR cycles during one stage significantly changing the dependence between the rate of amplicon synthesis and the reaction time resulting in nonspecific products synthesis. Their sensitivity is low if the sample lysate contains the reaction inhibitors and/or

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