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The further course of prostate cancer (PC) after radical prostatectomy (RPX) is decisively influenced by the local tumor stage. Although it is now possible to assess the risk of local recurrence from the histopathology, precise predictions cannot be made. A more accurate evaluation would be desirable, mainly for early planning of adjuvant therapy. We assessed the detection of telomerase activity using the Telomeric Repeat Amplification Protocol in surgical margins compared to RT-PCR-supported prostate-specific antigen (PSA) mRNA detection and conventional histopathological examination. We examined 95 patients with local PC during RPX and 16 patients with muscle-invasive transitional bladder cancer who underwent radical cystectomy. After RPX or RCX biopsies were obtained from 4 or 3 defined areas of the prostatic fossa and processed by TRAP assay for telomerase activity and by RT-PCR for PSA using a standard protocol. Five of 48 patients (10.4%) with organ-confined prostate cancer (pT2) and 7 of 47 patients (14.9%) with locally advanced PC (>pT2) had positive detection of telomerase activity in at least one positive specimen. In contrast to RT-PCR for PSA mRNA and histological findings for organ-confined and locally advanced disease, detection of telomerase activity yielded no statistically significant correlation to clinical parameters. Only PC-patients with positive histopathological margins had a positive correlation between detection of telomerase activity and high Gleason scores (r=0.65, p=0.022). Based on the results obtained and the current state of knowledge, measurement of telomerase activity must be considered less sensitive than RT-PCR for PSA mRNA in surgical margins, although tumor specificity should theoretically be higher. It is not even sure at the present time whether a combination of both methods offers any recognizable advantage over PSA mRNA detection alone. The value of the results obtained in this study will have to be assessed in a further follow-up to determine whether patients with positive molecular detection have an increased risk of local recurrence.

The aim of the study was to evaluate the quantitative detection of human telomerase reverse transcriptase (hTERT) mRNA and telomerase activity as new molecular diagnostic parameters for a subclassification of spermatogenesis disorders. Telomerase activity was detected by a quantitative telomerase PCR ELISA, and hTERT mRNA expression was quantified by fluorescence real-time RT-PCR in a LightCycler in cryopreserved testicular tissue specimens. This was paralleled by a histological workup. The discriminant analysis showed that detection of normalized hTERT expression was able to correctly classify 89.0% of the investigated tissue specimens into the subgroups of full spermatogenesis, maturation arrest or Sertoli-cell-only syndrome. In contrast, discriminant analysis revealed an only 58% accuracy of telomerase activity for the investigated tissue specimens. This study shows that the quantification of hTERT expression in testicular tissue by real-time fluorescence RT-PCR is well suited for correctly classifying spermatogenesis disorders and proved to be markedly superior to the determination of telomerase activity.

Background: The objective of the present study was to evaluate the quantitative detection of human telomerase reverse transcriptase (hTERT) mRNA as a new molecular diagnostic parameter in the work-up of testicular tissue specimens from patients presenting with non-obstructive azoospermia. M ETHODS: hTERT mRNA expression was quantified in 49 cryopreserved testicular tissue specimens by fluorescence real-time RT-PCR in a LightCycler. This was paralleled by conventional histological work-up in all tissue specimens and additional semithin sectioning preparation in cases with maturation arrest (n = 20) and Sertoli cell-only syndrome (SCOS; n = 12)., Results: The average normalized hTERT expression (N(hTERT)) was 136.1 +/- 41.7 copies (mean +/- standard deviation) in tissue specimens with presence of haploid germs cells, N(hTERT) = 48.2 +/- 21.0 copies in those with maturation arrest and N(hTERT) = 2.7 +/- 2.8 copies in those with SCOS. The discriminant analysis showed that detection of N(hTERT) was able correctly to classify 89.0% of the investigated tissue specimens., Conclusions: Our results demonstrate that quantitative detection of hTERT mRNA expression in testicular tissue enables a molecular-diagnostic subclassification of spermatogenesis disorders. Quantitative detection of hTERT in testicular biopsies is thus well suited for predicting successful sperm recovery in patients with azoospermia and is a useful molecular diagnostic parameter for supplementing the histopathological evaluation.

Aim: To evaluate the quantitative detection of human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT) mRNA as diagnostic parameters in the workup of testicular tissue specimens from patients presenting with non-obstructive azoospermia., Methods: hTR and hTERT mRNA expression were quantified in 38 cryopreserved testicular tissue specimens by fluorescence real-time reverse transcription-polymerase chain reaction (RT-PCR) in a LightCycler(r). This was paralleled by conventional histological workup in all tissue specimens and additional semithin sectioning preparation in cases with maturation arrest (n = 12) and Sertoli-cell-only syndrome (n = 12)., Results: The average normalized hTERT expression (N(hTERT)) was 131.9 +/- 48.0 copies (mean +/- SD) in tissue specimens with full spermatogenesis, N(hTERT) = 51.2 +/- 17.2 copies in those with maturation arrest and N(hTERT) = 2.7 +/- 2.4 copies in those with Sertoli-cell-only syndrome (SCOS). The discriminant analysis showed that detection of N(hTERT) (N(hTR)) had a predictive value of 86.8% (55.3%) for correct classification in one of the three histological subgroups., Conclusion: Our results demonstrate that quantitative detection of hTERT mRNA expression in testicular tissue enables a molecular-diagnostic classification of gametogenesis. Quantitative detection of hTERT in testicular biopsies is thus well suited for supplementing the histopathological evaluation.

Background: The further course of prostate cancer (PC) after radical prostatectomy (RPX) is decisively influenced by the local tumor stage. Although it is thus far possible to assess the risk of local recurrence from the pathohistology, precise predictions cannot be made. A more precise evaluation would be desirable, mainly for early planning of adjuvant therapy. Other authors have shown that telomerase activity may be a marker for malignant potential. We assessed the detection of telomerase activity using the telomeric repeat amplification protocol (TRAP) in surgical margins compared to conventional histopathological examination., Methods: Ninety-two patients with local PC who underwent RPX were examined. After RPX biopsies were obtained from four defined areas of the prostatic fossa and processed by TRAP assay for telomerase activity using a standard protocol., Results: In 5 of 48 patients (10.4%) with organ-confined prostate carcinoma (pT2) telomerase activity could be detected. Seven of 47 patients (14.9%) with locally advanced PC (> pT2) had at least one positive specimen., Conclusions: The results obtained in our study indicate that detection of telomerase activity by TRAP assay may be a suitable parameter for molecular staging of surgical margins, because of the high tumor-specificity. Further follow-up must clarify whether patients with positive molecular detection have an increased risk of local recurrence., (Copyright 2001 Wiley-Liss, Inc.)

In contrast to human primary fibroblasts, mouse embryonic fibroblasts have telomerase activity, immortalize spontaneously in culture, and can be neoplastically transformed by oncogenic insult. Ectopic expression of the human telomerase catalytic subunit, human telomerase reverse transcriptase (hTERT), in human primary cells allows both spontaneous immortalization and neoplastic transformation by oncogenes. This suggests that telomerase activity, as well as the fact that mouse telomeres are longer than human telomeres, may explain some of the differences in cellular control between human and murine cells. Telomerase inhibition in immortal or transformed human cells using dominant negative hTERT mutants leads to telomere shortening and cell death. Here we study the effect of expression of a dominant negative mutant of the catalytic subunit of mouse telomerase, mTERT-DN, in a murine kidney tumor cell line, RenCa, whose telomeres are similar in length to human telomeres. After showing initial telomerase activity inhibition and telomere shortening, all clones expressing mTERT-DN reactivated telomerase and showed normal viability, in contrast with that described for human cells. This efficient telomerase reactivation coincided with a significant increase in the endogenous TERT mRNA levels in the presence of mTERT-DN expression. The results presented here reveal the existence of fundamental differences in telomerase regulation between mice and man.

Objective: Determination of telomerase activity and the expression of human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT) in the testicular tissue of patients with infertility arising from various causes., Design: Prospective observational study., Setting: A university hospital., Patient(s): Thirty-three patients with azoospermia arising from various causes. There were 12 testicular biopsy specimens from patients with Sertoli cell-only syndrome, 9 from patients with maturation arrest, and 12 from patients with obstructive azoospermia and normal histologic findings., Intervention(s): Thirty-three testicular biopsies., Main Outcome Measure(s): Correlation of histologic findings at testicular biopsy with telomerase activity, hTERT, and hTR., Result(s): All 12 biopsy specimens from patients with obstructive azoospermia were positive for telomerase activity, hTR, and hTERT. Biopsy specimens from the 9 patients with maturation arrest were positive for telomerase activity in 8 cases, hTR in 9 cases, and hTERT in 5 cases. None of the patients with Sertoli cell-only syndrome showed either telomerase activity or hTERT, but all of them showed hTR., Conclusion(s): Telomerase activity and evidence of hTERT in testicular tissue are highly sensitive and highly specific markers of gametogenesis, which could gain in importance as part of the fertility workup before microinjection procedures.

Objectives: An important characteristic of malignant cells is their unlimited replicative potential, their immortality. In conferring this immortality, the enzyme telomerase is believed to play a crucial role. The detection of telomerase activity provides new knowledge regarding the biologic growth behavior of tumors and offers new diagnostic and therapeutic tools., Methods: In the present study the sensitive TRAP assay (telomeric repeat amplification protocol) was used to examine 44 malignant renal tumors and 8 benign tumors of the kidney and 52 specimens of normal renal tissue for telomerase activity., Results: Telomerase activity was detected in 63% of tissue samples obtained from histologically confirmed renal cell carcinomas. In cases of renal cell carcinoma restricted to the kidney, telomerase activity was detected in 58%. In cases in which tumor growth has progressed beyond the limits of the organ, telomerase activity was found in 69%. This stage dependence, however, did not reach statistical significance. No correlation to tumor grading was observed. Telomerase activity was found less frequent in chromophobe renal cell carcinomas. Neither the 8 benign renal tumors (4 oncocytomas and 4 angiomyolipomas) nor the specimens of normal kidney showed any evidence of telomerase activity., Conclusions: The proportion of remarkable slow-growing renal cell carcinomas showing telomerase activity is less than in other malignancies and may correlate with biologic growth behavior. Possible explanations include the presence of an alternative pathway, called ALT (alternative lengthening of telomeres) and an association with the loss or presence of the telomerase suppressor on the short arm of chromosome 3. Prolonged follow-up will be of special interest to determine whether lack of telomerase activity predicts favorable outcome.

New, noninvasive methods for the early detection of urothelial carcinomas of the urinary bladder are needed for the diagnosis, follow-up, and screening of patients with bladder cancer. Detection of the enzyme telomerase in urine could offer these new diagnostic possibilities. The standard technique for detecting telomerase activity is the telomeric repeat amplification protocol (TRAP assay). Because of the instability of the ribonucleoprotein telomerase in an aggressive medium, such as urine, investigations conducted to date have yielded nonuniform or even contradictory findings. This study compares the detection of human telomerase RNA (hTR) by reverse transcriptase-PCR (RT-PCR) with detection of telomerase activity by the TRAP assay in the diagnosis of urothelial carcinoma of the urinary bladder. Sedimented cells obtained from urine of 30 patients with urothelial carcinoma, 15 patients with benign urological disorders, 3 patients as part of follow-up for malignant disease, and 20 healthy subjects were examined for the presence of hTR and for telomerase activity (TRAP). In patients with bladder cancer, telomerase activity was detected by the TRAP assay in only 2 of 30 specimens (7%). However, increased levels of hTR were detected by RT-PCR in 25 of the same 30 cases (83%). For patients with benign urological disorders, such as urolithiasis or urinary tract infections, hTR was detected in samples obtained from 4 of 15 patients (27%). Low hTR expression levels were found in 15% of the healthy controls. The detection of hTR by RT-PCR represents a promising new method for detecting malignant cells in urine.

Early diagnosis is still the most important prerequisite for successful cancer treatment and this holds true for bladder cancer. Urine cytology is commonly used as a non-invasive screening procedure for the detection of bladder carcinoma, but this method is labour-intensive and often generates false-negative results. The ribonucleoprotein telomerase appears to be promising new cancer marker, since its activity has been reported to correlate with indefinite growth. The aim of this study was to investigate whether telomerase activity can be detected in bladder cancer and in corresponding bladder washings. For this purpose, a sensitive non-radioactive TRAP (telomeric repeat amplification protocol) detection system was developed. With this technique, telomerase activity was found in 95 per cent of the carcinomas (n = 20), in 70 per cent of the corresponding bladder washings, but in none of the urine samples obtained from patients with bladder carcinoma. No telomerase activity was detectable in normal urothelium or in samples from dysplastic urothelium. The data obtained from bladder washings show that superficial carcinoma cells released into the bladder still harbour telomerase activity. The absence of telomerase activity in voided urine is thus most likely due to degradation or inactivation under these conditions. The high rate of telomerase activity in bladder carcinoma indicates that the activation of telomerase in a common step in the tumourigenesis of bladder cancer.