Telomerase: Its clinical relevance in the diagnosis of bladder cancer

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Oncogene (2002) 21, 650 ± 655 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00 www.nature.com/onc

Telomerase: Its clinical relevance in the diagnosis of bladder cancer Markus MuÈller*,1 1 Department of Urology, UniversitaÈtsklinikum Benjamin Franklin, Freie UniversitaÈt Berlin, Hindenburgdamm 30, 12200 Berlin, Germany

More than 50 years ago, Papanicolaou recognized the importance of a non-invasive technique for the diagnosis and follow-up of patients with carcinoma of the urinary bladder. Cystoscopy, however, has remained the `gold standard' since no currently available non-invasive method can compete with cystoscopy's sensitivity and speci®city. The detection of the ribonucleoprotein telomerase or the telomerase subunits human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT) in urine samples oer new diagnostic perspectives. The present article presents a review of publications in the literature and evaluates their clinical relevance. The experimental studies reported to date are very promising and show that telomerase exactly ful®ls the requirements for a good diagnostic marker for carcinoma of the urinary bladder. The diagnostic application remains in an experimental stage and telomerase is still several steps away for routine use as a clinical parameter. The remaining steps leading to its routine clinical application will be discussed. Oncogene (2002) 21, 650 ± 655. DOI: 10.1038/sj/onc/ 1205071 Keywords: telomerase; bladder cancer; diagnosis; hTR; hTERT Bladder cancer Carcinoma of the urinary bladder occurs with an incidence of 15 cases per 100 000 persons in the general population. It is the most common malignant tumour of the urinary tract and, after prostatic carcinoma, the second most common malignancy of the urogenital system. Incidence peaks in the ®fth to seventh decades and males are about three times more frequently aected than females. Histologically, about 90% of bladder carcinomas are urothelial carcinomas, characterized by malignant proliferation of the transitional epithelium (transitional cell carcinomas). In about 25% of patients, bladder carcinoma is a multifocal disease. Of particular importance in the aetiology is a history of exposure to various chemical substances, which, as carcinogens or cocarcinogens, may lead to the *Correspondence: M MuÈller; E-mail: [email protected]

development of bladder carcinoma with a latency period of up to 30 years. Implicated substances include recreational poisons, such as tobacco, as well as industrial toxins (Morrison et al., 1984). The role of industrial carcinogens has been recognized since 1895 (Rehn, 1895). Besides chemical substances, bladder carcinoma may have iatrogenic causes, such as medical radiation exposure to the lesser pelvis (Duncan et al., 1977). Chronic cystitis has also been implicated as a possible causative factor (Yamamoto et al., 1992). Schistosoma haematobium infections are considered a cause of squamous cell carcinoma of the urinary bladder. The cardinal ± and, usually, initial ± symptom of bladder carcinoma is macrohaematuria. Every instance of painless macrohaematuria should be considered evidence of malignant disease in the urinary tract until the opposite has been proven. The staging of urothelial carcinoma of the bladder follows the TNM classi®cation for urological tumours of the International Union Against Cancer (UICC) and the tumour cells' degree of histological dierentiation (grading). Most relevant clinically is whether a given patient's tumour is super®cial or has already invaded underlying muscular layers. At the time of ®rst diagnosis, bladder carcinoma has spread to the muscle in about 30% of cases: in these patients, curative treatment requires radical cystectomy. Problems complicating the diagnosis and follow-up of bladder carcinomas Due to factors such as size or localization, the great majority of carcinomas of the urinary bladder are either undetectable with standard imaging techniques, such as ultrasound (US), computed tomography (CT) or magnetic resonance imaging (MRI), or cannot be de®nitively dierentiated for non-malignant, reactive processes. Thus, the invasive method of instrumental cystoscopy remains a part of the standard work-up. Bladder carcinoma shows a very high recurrence rate (50 ± 70%) and recurrent disease, when it does occur, is associated in 15 ± 25% of patients with progression to a more advanced tumour stage. Thus, careful and frequent follow-up is of prime importance (Abel, 1988; Heney et al., 1983). Current practice in the follow-up of patients with carcinoma of the urinary bladder favours cystoscopic examinations every 3

Telomerase and bladder cancer M MuÈller

months. Exophytic tumours of the bladder are easily diagnosed at cystoscopy. Tumour growth that does not project above the mucosal surface, such as in carcinoma in situ, however, may escape cystoscopic detection. Also, nonspeci®c areas of redness may be incorrectly interpreted. Due to these factors, cystoscopy, though remaining the best available method, shows rates of sensitivity and speci®city in the range of only 70 ± 80% (Kriegmair et al., 1996). More than 50 years ago, Papanicolaou and Marshall (1945), recognized the importance of a non-invasive technique for the diagnosis and follow-up of patients with carcinoma of the urinary bladder. Cystoscopy, however, has remained the `gold standard' since no currently available non-invasive method can compete with cystoscopy's sensitivity and speci®city. A noninvasive method of comparable diagnostic quality would signi®cantly facilitate the initial diagnosis of carcinoma of the urinary bladder and greatly simplify the follow-up of treated patients. If such a method could also be made cost-ecient, its introduction as a screening method in at-risk population groups, including persons employed in certain branches of the textile and rubber industry, tannery workers and employees of the chemical and pharmaceutical industries, as well as smokers of tobacco, could be useful. Any new, non-invasive technique should meet the following requirements: (1) the method's sensitivity and speci®city must both reach at least 70%; (2) the marker must detect well-dierentiated tumours and tumours in early stages; (3) as far as possible, the method should not be examiner-dependent and its application should be ubiquitous; (4) the marker and detection method must be practicable in routine clinical situations: i.e., the marker must be stable in urine samples and its detection must be reproducible. The method must also be simple to use and suciently inexpensive to facilitate the analysis of a large number of urine samples in a reasonable period of time. Over the past years, numerous attempts have been made to establish various non-invasive methods. Due to inadequacies in terms of their sensitivity or speci®city or to diculties adversely aecting their practicability in routine clinical application, none of these methods was able to gain general acceptance. Urine cytology, despite its high speci®city, was not suciently sensitive in the diagnosis of well-dierentiated carcinomas of the urinary bladder and was quite examiner-dependent, all of which resulted in its becoming less important in recent years (Gamarra and Zein, 1984). The introduction and, in some cases, commercial availability of a number of new markers have resulted in only slight improvements in comparison to the results of conventional urine cytology. Microsatellite analysis of urine samples have shown very high sensitivity and speci®city in the detection of malignant cells. The complexity of the method, however, limited its application in routine clinical use (Mao et al., 1996). Thus, the urgent need for a new, non-invasive technique for the diagnosis and follow-up of carcinoma of the urinary bladder remains.

New diagnostic perspectives are oered by the detection of the ribonucleoprotein telomerase. The activity of this enzyme, with only a few exceptions, is limited to malignant cells and tissues (Kim, 1997; Shay and Bacchetti, 1997). Since 1994, the TRAP-assay (telomeric repeat ampli®cation protocol), a polymerase chain reaction (PCR) based method for detection of telomerase activity, has been available (Kim et al., 1994). The introduction of this method was an important milestone in telomerase research and has become the standard technique for studying the diagnostic capabilities of this enzyme. The TRAPassay, however, is not suitable for routine clinical use. Alternative methods include the detection of the telomerase subunits human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT) in urine samples using the reverse transcriptase polymerase chain reaction (RT ± PCR). New, real-time PCR methods permit a quantitative detection of these subunits.

651

TRAP-assay detection of telomerase activity in the diagnosis of bladder carcinoma Tissue samples As a ®rst step, tissue samples were derived from bladder carcinomas of various tumour stages and degrees of dierentiation and analysed for telomerase activity using the TRAP-assay. It was shown that, regardless of tumour stage or degree of dierentiation, telomerase activity could be identi®ed in nearly all tissue samples derived from bladder carcinomas (Table 1) but not in normal urothelium. Thus, the fundamental prerequisites for a diagnostic method with high sensitivity and speci®city are ful®lled (Gelmini et al., 2000; Ito et al., 1998a; Kamata et al., 1996; Kinoshita et al., 1997; Kyo et al., 1997; Lancelin et al., 2000; Lee et al., 1998; Lin et al., 1996; Linn et al., 1997; May®eld et al., 1998; MuÈller et al., 1996; Rahat et al., 1999; Yokota et al., 1998; Yokota et al., 1998; Yokota et al.,

Table 1

Telomerase activity in tissue samples from patients with bladder carcinoma

Reference MuÈller et al., 1996 Lin et al., 1996 Kamata et al., 1996 Kinoshita et al., 1997 Yoshida et al., 1997 Linn et al., 1997 Kyo et al., 1997 Lee et al., 1998 Yokota et al., 1998 Ito et al., 1998a Mayfield et al., 1998 Rahat et al., 1999 Gelmini et al., 2000 Lancelin et al., 2000

Sample size (n)

Detectable telomerase activity (sensitivity)

75 40 13 42 56 12 13 23 26 22 52 29 33 14

96% 97,5% 100% 98% 86% 92% 100% 96% 100% 100% 81% 90% 94% 78% Oncogene


652

1998; Yoshida et al., 1997). Contrary to our own ®ndings (MuÈller et al., 1996), low levels of telomerase activity were identi®ed in dysplastic urothelium by Lin et al. (1996) in one of two cases and by Yoshida et al. (1997) in two of two cases. In a very small number of cases, telomerase activity was detected in histologically normal urothelial tissue immediately adjacent to con®rmed tumours. This was probably due to the presence of individual tumour cells or tumour precursors that had escaped detection with conventional light microscopy (Kyo et al., 1997; Lancelin et al., 2000). Bladder washings The second step considered whether the sensitivity of the TRAP-assay and the stability of telomerase were adequate to identify the presence of malignant cells by the determination of telomerase activity in bladder washings and urine samples. To maximize the number of cells in a physiologic environment, bladder washings were conducted using a physiologic saline solution. In diagnostic use, telomerase activity can be reliably detected in bladder washings with sensitivities averaging 70 ± 80% (Table 2) and a speci®city of 100% (Dalbagni et al., 1997; Gelmini et al., 2000; Kinoshita et al., 1997; Lee et al., 1998; MuÈller et al., 1996). This method, which is not examiner-dependent, reliably detects bladder carcinoma cells in bladder washings. Bladder washings, however, are obtained through a catheter or cystoscope; hence, the objective of a noninvasive technique is not met. Urine samples The option of detecting telomerase activity in samples of spontaneous urine would have high clinical relevance. Since telomerase activity can be detected in bladder washings obtained from patients with bladder carcinoma with a high degree of sensitivity and counting of cells using trypan blue staining results in a suciently high number of tumour cells, the sensitivity of the TRAP-assay should conceivably be adequate in the case of urine analysis. Because the TRAP-assay detects telomerase activity and not simply the presence of the enzyme, positive ®ndings at TRAP-assay require the presence of living cells. Bladder washings are obtained by mechanical irrigation of the empty urinary bladder using physiological saline solution at a physiologic pH level. Mechanical Table 2 Telomerase activity in bladder washings in patients with bladder carcinnoma Reference MuÈller et al., 1996 Kinoshita et al., 1997 Dalbagni et al., 1997 Lee et al., 1998 Gelmini et al., 2000

Oncogene

Sample size (n)


40 43 63 23 33

73% 86% 50% 96% 82%

irrigation releases living cells from intact tumour-cell masses and immediately suspends them in a physiological medium. In native urine, however, suspended tumour cells are exposed for various lengths of time to destructive substances, such as proteases, urea, salts and, usually, acid pH values. All of these factors may cause early inactivation or degradation of the enzyme, thus limiting telomerase's stability in urine samples. Due to this problem, various research groups have been unable to reach consistent results. Sensitivities for urine samples have ranged between 0% and 100% (Table 3) (Arai et al., 2000; Bialkowska-Hobrzanska et al., 2000; Dalbagni et al., 1997; Gelmini et al., 2000; Kavaler et al., 1998; Kinoshita et al., 1997; Kitsukawa et al., 1999; Linn et al., 1997; MuÈller et al., 1996, 1998; Rahat et al., 1999; Ramakumar et al., 1999; Yokota et al., 1998; Yoshida et al., 1997). The reasons for these widely divergent ®ndings remain unclear, particularly because methodical dierences, such as in the treatment of the samples, are not apparent. Although the TRAP-assay with direct detection of telomerase activity certainly represents the biologically most relevant method, its practicability for the non-invasive detection of tumour cells in urine samples obtained from patients with carcinoma of the urinary bladder is severely limited by the enzyme's vulnerability to inactivation by external factors. This opinion, ®rst postulated by us as early as 1996 (MuÈller et al., 1996) and thereafter controversially discussed in the literature, has, in the meantime, been generally accepted (Arai et al., 2000; Cassel et al., 2001; de Kok et al., 2000a; Wu et al., 2000). Furthermore, the TRAP-assay, despite the assertions of certain authors (Gelmini et al., 2000), is not truly quantitative, but only semiquantitative at best (Yokota et al., 1998) Due to the instability of the enzyme in urine samples and the resulting poor sensitivity of the detection of telomerase activity, alternatives to the TRAP-assay were sought. As alternatives to detection of telomerase activity using the TRAP-assay, use of RT ± PCR to detect telomerase subunits such as its RNA portion

Table 3

Telomerase activity in urine samples of bladder carcinoma

Reference MuÈller et al., 1996 Kinoshita et al., 1997 Yoshida et al., 1997 Linn et al., 1997 Dalbagni et al., 1997 Kavaler et al., 1998 Yokota et al., 1998 MuÈller et al., 1998 Ramakumar et al., 1999 Kitsukawa et al., 1999 Rahat et al., 1999 Gelmini et al., 2000 Arai et al., 2000 Bialkowska-Hobrzanska et al., 2000

Sample size (n)


30 42 26 12 63 104 29 30 57 26 21 33 19 35

0% 55% 62% 0% 35% 85% 86% 7% 70% 85% 81% 94% 21% 49%


(hTR) or its catalytic protein component (hTERT) received increasing attention. Because of the clinical importance of urine samples, the remaining portion of the present discussion will be limited to the detection of these substances in urine. RT ± PCR detection of hTR in the diagnosis of carcinoma of the urinary bladder The RNA component of human telomerase (hTR) was ®rst cloned in 1995 by Feng et al. (1995). In early investigations, it was assumed that the expression of hTR does not correlate with telomerase activity (Avilion et al., 1996), since hTR is also detectable in benign cells and tissues. Further studies, however, were able to show that while low hTR levels were detectable in all cells, hTR levels were signi®cantly increased in malignant cells (Heine et al., 1998; Hiyama et al., 1999; Kuniyasu et al., 1997; Sallinen et al., 1997; Soder et al., 1997; Yashima et al., 1997). In an early investigation, we used RT ± PCR to detect hTR in the urine of 30 patients with con®rmed urothelial carcinoma of the bladder (MuÈller et al., 1998). In these experiments examining hTR in urine samples we obtained a sensitivity of 83% and a speci®city of 80% in the diagnosis of urothelial carcinomas of the urinary bladder (Table 4). That hTR is detectable in about one-®fth of urine samples obtained from patients with benign urologic disease or healthy subjects is not surprising, since, unlike telomerase activity, hTR is also present in normal cells and tissues. The levels of hTR in malignant cells, however, are much higher than in benign cells. Maitra et al. (2001) were able to con®rm the overexpression of hTR in bladder tumour cells using in-situ hybridization. Further improvement in results can be expected from quantitative analyses using real-time RT ± PCR. Since 1999, we have been conducting a prospective study investigating the ecacy of this method in about 200 patients with bladder tumours and corresponding controls. Results will be available in the near future. RT ± PCR detection of hTERT in the diagnosis of carcinoma of the urinary bladder The second alternative to the TRAP-assay is detection of the mRNA of the catalytic protein component of human telomerase (hTERT) using RT ± PCR (Harrington et al., 1997; Kilian et al., 1997; Meyerson et al., 1997; Nakamura et al., 1997). Of particular importance is the fact that the catalytic protein component of Table 4 Human telomerase RNA (hTR) in urine samples of bladder carcinoma Reference MuÈller et al., 1998

Sample size (n)

Sensitivity

Specificity

30

83%

80%

human telomerase (hTERT) correlates with telomerase activity (Nakamura et al., 1997). This has been con®rmed for the speci®c case of tissue samples obtained from carcinomas of the urinary bladder (Ito et al., 1998a). In addition, using a quantitative, realtime PCR method, it could be shown that hTERT levels also correlate with both the tumour stage and its degree of dierentiation (de Kok et al., 2000b). In an early investigation on urine samples obtained from patients with carcinoma of the urinary bladder, Ito et al. (1998b) documented a sensitivity of 80% and a speci®city of over 95%. One diculty with the detection of hTERT relates to the fact that, per cell, only a few copies of hTERT are present. Also, the mRNA of hTERT is less stable than hTR. In a more recent study, however, the researchers demonstrated an even higher sensitivity (Bialkowska-Hobrzanska et al., 2000). In an early, small study of nine urine samples, quantitative, real-time PCR detected hTERT in 55% of cases (de Kok et al., 2000c) (Table 5). Since 1999, we have been conducting a prospective study in about 200 patients with bladder tumours and corresponding controls investigating the ecacy of this method in detecting hTERT by real-time RT ± PCR in urine samples. Results will be available by the end of this year.

653

Current clinical relevance Experimental studies conducted to date are very promising and clearly show that telomerase, in principle, satis®es the requirements postulated above for markers useful in the diagnosis of carcinoma of the urinary bladder: (1) With only a few exceptions, telomerase activity and hTERT are found only in samples obtained from bladder carcinoma, but not in normal controls. (2) hTR is detected more often and at higher levels in samples obtained from bladder carcinomas than in normal controls. (3) It identi®es both well-dierentiated and early-stage tumours. (4) The methods are not examiner-dependent. The diagnostic bene®t, however, remains in an experimental stage and is still several steps removed from application as a routine clinical procedure. While there are a large number of studies investigating telomerase activity, the number of samples and controls is mostly quite small and, as mentioned above, the results are sometimes contradictory. For these reasons and because of the limited practicability of the current TRAP-assay test format, it is unlikely that detection of telomerase activity in urine samples

Table 5 Human telomerase reverse transcriptase (hTERT) in urine samples of bladder carcinoma Reference Ito et al., 1998b Bialkowska-Hobrzanska et al., 2000 de Kok et al., 2000c

Sample size (n)

Sensitivity

Specificity

33 35

80% 94%

96% 92%

9

55%

100% Oncogene


654

using the TRAP-assay will ®nd routine clinical use. Investigations of the diagnostic relevance of hTR and hTERT in urine samples are far fewer and are also based on small patient collectives. The results of the ®rst larger prospective study are expected by the end of this year. Main prerequisites for the practical application of these concepts to routine clinical practice include a standardized test format and a standardized treatment of urine samples. In our opinion, the only currently available test formats deserving of consideration are the real-time PCR techniques, which actually quantify levels of the telomerase subunits hTERT and hTR. As is the case with other diagnostic laboratory methods, studies of larger patient collectives will be necessary to determine the validity of cut-o points for hTR-positive and negative ®ndings. An additional re®nement could be a combined detection of both hTR and hTERT. Also, real-time PCR methods allow for the reproducible analysis of a large number of samples in a short time period with standardized assays. The necessary equipment is already available in routine laboratories in most larger hospitals.

Detection of hTR and/or hTERT using real-time PCR methods requires qualitatively adequate RNA derived from the cell pellets of urine samples. Currently available equipment permits automated standardized RNA processing, though these devices have yet to be widely distributed. A truly ubiquitous application would require any method to be available for use in a private urological practice: for this, PCR-based test formats are not suitable. Ubiquitous application of any method for the detection of telomerase subunits in urine samples in the diagnosis, follow-up and, perhaps, screening of carcinoma of the urinary bladder will only become feasible when good antibodies become commercially available. Because of the weak expression of hTERT, the development of such antibodies has been lagging. Such antibodies would permit development of the next intermediate step, which would be ELISA based test formats. The dream of every urologist, however, is and remains the development of a simple dip stick test that would permit examination of urine samples for bladder tumour cells in a few seconds. It is hoped that every advance in this ®eld brings us one step closer to ful®lling this dream.

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