Telomerase Activity Detected by Quantitative Assay in Bladder Carcinoma and Exfoliated Cells in Urine

Abstract

Early diagnosis is one of the most determining factors for patient survival. The detection of telomerase activity is a potentially promising tool in the diagnosis of bladder and other types of cancer due to the high expression of this enzyme in tumor cells. We carried out a quantitative evaluation of telomerase activity in urine samples in an attempt to determine a cut-off capable of identifying cancer patients. Telomerase activity was quantified by fluorescence TRAP assay in urine from 50 healthy volunteers and in urine and bioptic tumor samples from 56 previously untreated bladder cancer patients and expressed in arbitrary enzymatic units (AEU). Telomerase activity in urine ranged from 0 to 106 AEU (median 0) in healthy donors and from 0 to 282 AEU (median 87) in patients with cancer. A telomerase expression higher than the cut off value determined by receiver operating characteristic (ROC) analysis was observed in 78% of cases, regardless of tumor grade and in 71% (15/21) of cases of nonassessable or negative cytology. The quantitative analysis of telomerase activity in urine enabled us to define cut-off values characterized by different sensitivity and specificity. Cytologic and telomerase determination, used sequentially, enabled us to detect about 90% of tumors.

Introduction

Early diagnosis of bladder cancer is one of the most determining factors for patient survival; the frequency of recurrence and tumor progression depends to a great extent on tumor grade and stage at time of diagnosis [1,2].

Cystoscopy is the standard procedure routinely used for the detection of bladder cancer. However, it represents an invasive, uncomfortable, and expensive procedure and cannot be used for screening programs.

Cytology is a cheaper, noninvasive procedure routinely used for diagnosis, but it has only a 50% sensitivity and many tumors, mainly of low grade, may be missed [3].

Therefore, noninvasive methods for bladder cancer diagnosis are warranted.

Immunohistochemical evaluation of anti-Lewis X antigen has revealed a good overall sensitivity and specificity, especially when combined with cytology, but it has yet to receive widespread acceptance [4]. Alternative markers, such as Bard bladder tumor antigen (BTA), have not shown satisfactory sensitivity and specificity [5,6].

In recent years, a great deal of information has been accumulated on the molecular alterations that take place during the development of bladder tumors, such as gene mutations or genomic rearrangements. Pilot studies on p53 and ras gene mutations or CD44 variant have highlighted the possibility to detect tumor alterations in exfoliated urine cells [7–9]. Moreover, Mao et al. [10], Linn et al. [11], and Steiner et al. [12] have detected microsatellite alterations in urine samples, and new methodological approaches have recently been developed [13].

However, individual tumors harbor specific mutations and the overall analysis of more than one gene mutation or microsatellite loci is needed to reach a high sensitivity.

A potentially promising tool for the diagnosis of bladder cancer is the detection of telomerase activity. The telomerase enzyme is a ribonucleoprotein reverse transcriptase that synthesizes the telomeric repeats located at the ends of chromosomes [14,15]. The majority of somatic cells do not have telomerase activity, and thus these repetitive sequences decrease with subsequent cell divisions due to the incomplete replication of linear DNA molecules. The progressive shortening of telomeres finally reaches a critical stage, probably correlated with cell senescence and death. The enzyme activity is assumed to favor telomere length maintenance and, as a consequence, to play an important part in cell immortalization and also tumor progression [16,17]. Telomerase reactivation has, in fact, been observed in immortalized cell lines and in many tumor histotypes [18].

The widespread association of telomerase activity with tumor cells has induced researchers to investigate and define the role of the enzymatic activity present in tumor tissue or biologic fluids as a diagnostic or prognostic marker [19,20].

In patients with bladder cancer, telomerase activity has been determined in bladder washings and urine [21–33].

In the present study we determined telomerase activity in urine from patients with cancer and healthy donors by using a quantitative evaluation.

Materials and Methods

Biologic Material

Exfoliated cell pellets from the urine of 56 patients were collected by centrifugation, washed once in 1 ml of PBS, and pelleted at 10,000xg for 5 minutes. A fraction of urine samples was sent to the pathologist for cytologic diagnosis and a fraction to the laboratory for the determination of telomerase activity. In parallel, exfoliated cell pellets from the urine of 50 healthy volunteers were collected and processed in the same way. All samples were stored at -70°C for a maximum of 2 months.

Bioptic tumor samples were taken from all the patients at the time of surgery. Diagnosis of tumor was histologically confirmed (WHO score). Three were grade I, 24 grade II, and 29 grade III.

Cell Extract Preparation

Tissue samples were suspended in 200 µl of ice-cold TRAP lysis buffer (Tris-HCl pH 7.5 10 mM, MgCl₂ 1 mM, EGTA 1 mM, phenyl methylsulfonyl fluoride 0.1 mM, β-mercaptoethanol 5 mM, 3-[(3-cholamidopropyl) dimethylamino]-1-propanesulfonate (CHAPS) 0.5% and glycerol 10%), homogenized and incubated on ice for 1 hour. The lysate was centrifuged for 20 minutes at 10,000xg at 4°C. The supernatant was removed, snap frozen, and stored at -70°C [34]. Urine samples were washed with ice-cold wash buffer (Hepes KOH pH 7.5 10 mM, MgCl₂ 1.5 mM, KCl 10 mM, and dithiothreitol 1 mM) and resuspended in ice-cold TRAP lysis buffer. The suspension was incubated on ice for 1 hour and centrifuged for 20 minutes at 10,000xg at 4°C. The supernatant was collected and stored as above.

The human bladder cancer cell line, MCR, established in our laboratory and expressing high telomerase activity, was used as positive control. Cells were isolated and washed with PBS and wash buffer. The cells (10⁶) were lysed in 200 µl of ice-cold TRAP lysis buffer and collected and stored as above.

Protein concentrations of each lysate were measured with Bio-Rad protein assay (Bio-Rad, Hercules, CA).

Fluorescence TRAP Assay

Detection of telomerase activity was performed as described previously [34,35]. A quantity of 0.1 µg of each protein extract was assayed in 45 µl of mix containing 200 µM dNTPs, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, 0.004% Tween 20, 0.004 Nonidet P-40, 0.4% glycerol, 0.1 µg TS primer end-labeled with HEX (Applied Biosystems, Foster City, CA), 0.5 µg of T4 gene 32 protein (Roche Molecular Biochemicals, Mannheim, Germany). After incubation in a thermal cycler for 30 minutes at 23°C and an inactivation of 2 minutes at 90°8C, 5 µl of mix containing 2 U of ExTaq (Takara Shuzo, Kyoto, Japan), 0.1 µg of CX primer, and 25 ag of an internal telomerase assay standard of 150 bp (ITAS) [35] were added.

An amplification was immediately run in a thermal cycler using 32 cycles at 94°C for 30 seconds, 50°C for 30 seconds, and 72°C for 30 seconds.

Four microliters of PCR product with 3 µl loading buffer (blue dextran in deionized formamide and EDTA 10 mM) were loaded onto a 5% w/v denaturing polyacrylamide gel (acrylamide:bisaclylamide, 19:1, 7 Murea, 1 x TBE). Electrophoresis was carried out on an Applied Biosystems 373A DNA Sequencer equipped with GeneScan 672 Collection and Analysis software.

Telomerase products were evaluated on fluorescence electropherograms and the area underlying the different peaks was calculated.

Serial dilutions of protein extract of MCR cell line corresponding to 10, 30, 100, 300, 1000, and 3000 cells were analyzed in each assay and telomerase activity in MCR cells was relatively expressed to activity of 100 cell equivalents and normalized to peak of ITAS (Figure 1A and B).

Figure 1.

Representative electropherograms of serial dilution of MCR bladder cancer cell line and quantitation of telomerase ladder. (A) Peaks corresponding to telomeric repeats synthesized by telomerase in a serial dilution of cell extracts (10, 30, 100, 300, 1000, and 3000 cell equivalents). Cell extract dilutions were assayed in the presence of the internal standard (ITAS). (B) All telomeric peak areas corresponding to a dilution were summed. This data was normalized to area of ITAS and expressed as relative to the signal of normalized 100 cell dilution.

To obtain quantitative evaluations, the areas of each sample were also normalized to the ITAS signal. The relative telomerase activity was correlated to corresponding mcr cell number and expressed in arbitrary enzymatic units (AEU).

All experiments were performed in duplicate and only variations of less than 15% for individual samples were accepted.

Statistical Analysis

Telomerase activity was considered as a continuous variable.

The relationship between tissue telomerase level and histologic grade was analyzed using a nonparametric ranking statistic.

Spearman's correlation coefficient was used to investigate the relationship between telomerase activity in urine and tumor tissue.

The most accurate cut-off value to discriminate between healthy donors and tumor patients was calculated using the receiver operating characteristic (ROC) curve. In the ROC curve the true positive rates (sensitivity) were plotted against the false positive rates (1-specificity) for all classification points.

Results

Telomerase activity determined in cell pellets from voided urine and tumor tissue of 56 patients as well as in urine samples from 50 healthy donors was visualized as electropherograms (Figure 2).

Figure 2.

TRAP assay of urine samples. Lanes 1, 2, and 3 show a representative electropherogram of positive urine cell extracts. Lane 4 shows the result of negative control, only one peak corresponding to ITAS is observed.

Only two urine samples and two tumor samples from 56 patients contained telomerase inhibitors and were thus classified as unassessable. Conversely, telomerase activity was evaluable in urine samples from all 50 healthy controls.

Telomerase levels ranged from 0 to 282 AEU (median 87) in voided urine and from 0 to 421 AEU (median 105) in tumor tissue. Lower levels of telomerase activity from 0 to 106 AEU (median 0) were detected in urine samples from healthy donors. Furthermore, value distributions of telomerase activity showed that urine levels below 40 AEU belonged to healthy donors in 85% of cases and to patients with cancer in only 15% of cases. However, values above 120 AEU were detected only in patients with cancer. An overlapping in controls and patients with cancer was observed for values from 41 to 120 AEU, albeit with a progressive decrease in high values in the former subset (Figure 3).

Figure 3.

Distribution of telomerase activity values in urine from healthy donors and tumor patients.

The ROC curve was traced to define the sensitivity and specificity of different enzymatic levels (Figure 4). The relative sensitivity and specificity of the most relevant cut-off values are listed in Table 1.

Figure 4.

ROC curve of telomerase activity in urine.

Table 1.

Sensitivity and Specificity of Telomerase Activity in Urine.

Cut-off (AEU)	Overall Series			Patients with Nonassessable or Negative Cytologic Examination
	No. Cases Positive/Negative	Sensitivity (%)	Specificity (%)	No. Cases Positive/Negative	Sensitivity (%)	Specificity (%)
55	48/6	89	68	16/5	76	68
60	46/8	85	70	16/5	76	70
65	42/12	78	82	15/6	71	82
70	39/15	72	86	15/6	71	86
75	39/15	72	90	15/6	71	90
80	32/22	59	92	13/8	62	92

In 9 of the 56 cancer patients, cytologic examination was not performed because of a lack of exfoliated cells. Of the remaining 47 patients, malignant cells were detected in 34. Telomerase activity assay performed in urine from 21 of the 22 patients with nonassessable or negative cytology confirmed the good specificity and a slightly lower but nonetheless high sensitivity observed for the TRAP assay on the overall series (Table 1).

Side analyses were performed to investigate the relation between telomerase activity in urine and in tumors or as a function of tumor grade. Matched values in urine and tumors from individual patients showed no correlation (r_s=0.34). In our case series 24 and 27 (94%) tumors showed grade 2 and grade 3 scores, respectively, and grade 1 was observed in only three cases. The median values of telomerase activity in urine were 80, 83.5, and 92 for patients with grades 1, 2, and 3 tumors, respectively, that is, AEU values showed only a trend but did not diminish with increasing tumor differentiation. It is worthy of note that the three patients with grade 1 tumors showed very high telomerase levels (77, 80, 141 AEU; mean 99.33 AEU and standard deviation 36.11 AEU) in urine.

Discussion

The simplest and most widely used approach for the detection of bladder cancer is the cytomorphologic examination of cells in urine. However, this method shows a low sensitivity for low-grade tumors and a variability in sensitivity and specificity, depending on the expertise of the cytopathologist [36]. At present, cystoscopy is the most sensitive and specific method but it is also invasive, extremely uncomfortable, and is not applicable for screening or disease-monitoring purposes.

Recently, telomerase activity has been proposed as a marker of bladder tumor cells as it is present in a large number of different tumor histotypes [19,20].

The study of this enzyme activity in bladder tissue has been marked by different steps characterized by different objectives and by an evolution of methods from qualitative to quantitative. Initially, the studies aimed to analyze telomerase activity in bladder cancer and surrounding macroscopically healthy tissue in relation to that present in washings and urine samples [22,25,37,38]. In a second step, the main objective was to verify whether this enzyme activity could offer a reliable and noninvasive diagnostic tool [23,24,26–33]. Because not only cancer cells but also some cells from healthy tissue or benign lesions exhibit telomerase activity, quantitative methods have been developed to define threshold values. Telomerase activity in washings and urine has also been investigated for its potential role in monitoring postsurgical residual disease or in predicting tumor recurrence [27,30,39,40].

The information from the first body of studies clearly shows that telomerase activity is virtually absent in normal bladder tissue, rare in benign lesions when not associated with a serious inflammatory situation, and frequent in tumors. In particular, telomerase activity has been observed in 85% to 100% of bladder tumors and in a variable percentage (0% to 70%) of microscopically healthy adjacent tissue, probably as a consequence of the extent of surgery or of multifocality [21,22,28,31,33].

Furthermore, the results from different studies have almost always shown that tumor telomerase activity is not related to grade, size, or stage and that it is less frequently present in biologic fluids. Among these, a lower frequency is observed in urine with respect to washings [21,22,33,38].

In prospect of using telomerase activity for diagnostic purposes, sensitive and quantitative approaches are needed to avoid false-negative and false-positive results. In the present study we evaluated telomerase activity in voided urine from an adequate series of healthy volunteers and patients with bladder cancer. The presence of telomerase activity was also observed in the former group, but the quantitative determination and statistical analysis we used enabled us to identify different cut-off values characterized by different sensitivity and specificity. Equally as important, telomerase activity higher than the best cut-off value determined by ROC analysis was observed in 71% of cases of unassessable or negative cytology.

In conclusion, the method we are proposing is highly reproducible, does not miss low-grade tumors, and, more importantly, if performed after cytologic evaluation, succeeds in unmasking the presence of tumors in cytologically negative cases.

Our final goal is to improve the potential of such a noninvasive approach by enlarging the case series to include more grade I tumors and by investigating the alternative or additional information that the expression of hTERT protein can provide.