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Clinical Chemistry 46: 313-318, 2000;
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(Clinical Chemistry. 2000;46:313-318.)
© 2000 American Association for Clinical Chemistry, Inc.

Articles

Real-Time Quantification of Human Telomerase Reverse Transcriptase mRNA in Tumors and Healthy Tissues

Jacques B. de Kok1,a, Theo J.M. Ruers2, Goos N.P. van Muijen3, Adrie van Bokhoven4, Hans L. Willems1 and Dorine W. Swinkels1

Departments of
1 Clinical Chemistry,
2 Surgery,
3 Pathology, and
4 Urology, University Hospital Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.
a Author for correspondence. Fax 31-243541743; e-mail J.dekok{at}ckcl.azn.nl


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Expression of the hTERT gene, which codes for the catalytic subunit of telomerase, is associated with malignancy. We recently developed a real-time reverse transcription-PCR assay, based on TaqMan technology, for accurate and reproducible determination of hTERT mRNA expression (Lab Investig 1999;79:911–2). This method may be of interest for molecular tumor diagnostics in tissues and corresponding body fluids, washings, or brushes.

Methods: In this study, we measured hTERT expression in a subset of healthy tissues and tumors to select those tumor types with the best potential for quantification of hTERT in corresponding body fluids. To demonstrate the use of the method in body fluids, we quantified hTERT expression in voided urine of patients with bladder cancer and controls.

Results: Real-time measurement of hTERT expression could discriminate between all healthy and malignant tissue samples from pancreas, lung, esophagus, and bladder, but not for colon tissues. Moreover, in five of nine (55%) urine samples, hTERT could be quantified.

Conclusions: The present study demonstrates that accurate quantitative measurement of hTERT expression has high potential for discrimination between healthy and tumor cells in tissues and urine and supports future measurements in pancreatic fluid, bronchoalveolar lavage fluid, esophageal brushings, and urine or bladder washings.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Body fluids, excrements, washings, or brushings (further summarized as bodily samples) are of interest in diagnostics because they can be obtained with minimally invasive or noninvasive techniques. For cancer diagnostics, mutant DNA derived from tumor cells has been found in feces (1), urine (2), sputum (3), pancreatic fluid (4), bile (5), cerebrospinal fluid (6), and plasma/serum (7)(8) and currently is being evaluated for use in early detection, prognosis, and follow-up studies of malignant processes. Unfortunately, not all tumors have readily detectable DNA aberrations such as K-ras mutations. In contrast, they may contain mutations scattered over the gene, microsatellite instability, or loss of heterozygosity, which are more difficult to detect, especially when a marked background of DNA from healthy tissue is present. As an alternative to the detection of DNA aberrations, quantification of gene expression in cells shed in bodily samples has been used as a tumor marker (9)(10).

Telomerase activity is the most general molecular marker for the identification of human cancer and can be detected in 85% of all tumors, whereas most healthy tissues exhibit little or no telomerase expression (11)(12). The enzyme needs at least two components to be functional: the RNA component, coded by the human telomerase RNA (hTR) gene (13), and the human telomerase reverse transcriptase subunit (hTERT1 gene), which codes for the catalytic subunit of the enzyme (14)(15)(16)(17)(18). A method for the quantitative measurement of hTERT mRNA expression may be of interest for molecular diagnosis in tumors and corresponding bodily samples (19).

We recently developed a method for the accurate measurement of hTERT expression, using real-time quantitative PCR (20). The method was applied to several cell lines, primary cell cultures, and healthy tissues, and quantification of hTERT expression showed good correlation with semiquantitative telomerase activity measurements. In the present study, we applied the novel method to several tumor and healthy tissue types to select those tumor types that have the best potential to be detected by hTERT measurements in corresponding bodily samples. As an example, we quantified hTERT expression in the urine of patients with bladder cancer.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
tissues
Frozen tissues (-80 °C) from five different types of tumors (pancreas, lung, colon, esophagus, and bladder) were randomly selected from the tissue bank of the Department of Pathology (University Hospital Nijmegen). Samples of healthy tissue were obtained from autopsies with a postmortem delay of <4 h. For healthy bladder tissue, samples were enriched for urothelial cells by selective removal of nonepithelial tissue.

percentage of tumor cells in tissue sections
Frozen 4-µm sections of tumor lesions were stained with hematoxylin and eosin. These sections were taken midway along the sections used for RNA isolation. A pathologist determined the percentage of tumor cells in the stained sections.

urine
The second morning urine of nine patients with urothelial cell carcinoma was obtained before transurethral resection of the tumor. Urine was also obtained from four patients with nonmalignant bladder diseases; 50 mL of the urine was centrifuged, and cell pellets were washed two times with ice-cold phosphate-buffered saline. Cell pellets were snap frozen in liquid nitrogen and stored at -80 °C until use.

rna isolation
Total RNA from tissues was isolated by disruption of 20 frozen 20-µm sections in 1 mL of Trizol (Life Technologies), using a sterile pestle. After the manufacturer’s protocol was completed, RNA was further purified using the RNeasy kit (Qiagen), according to the RNA clean-up protocol, and eluted in 50 µL of RNase-free distilled H2O (Life Technologies). The amount of RNA was measured spectrophotometrically. RNA from urine was isolated using Trizol with the addition of 2 µg of poly(A) RNA as carrier. RNA was dissolved in 20 µL of RNase-free H2O.

cDNA SYNTHESIS
Purified RNA (0.2–1.0 µg) from tissue samples was supplemented with RNase-free H2O to a final volume of 10 µL; 10 µL of RNA isolated from the urine samples was used. All samples were denatured for 5 min at 90 °C and cooled immediately on ice. Reverse transcription mixture (10 µL) was added, containing first strand buffer (Life Technologies), 200 units of Moloney murine leukemia virus (Life Technologies), 20 U of RNasin (Promega), 10 mmol/L dithiothreitol, 4.75 µmol/L random hexamers, and 600 µmol/L deoxynucleotides. After the hexanucleotides were annealed for 10 min at 20 °C, cDNA synthesis was performed for 45 min at 42 °C, followed by an enzyme inactivation step for 5 min at 95 °C. cDNA was stored at -20 °C until use.

real-time quantitative pcr
The principle of the real-time quantitative PCR has been described by Heid et al. (21). For both hTERT and 18S ribosomal RNA (rRNA) expression measurements in tissue samples, 1 µL of cDNA was used for amplification by the real-time quantitative PCR system (ABI PrismTM 7700 Sequence Detection System; Perkin-Elmer Applied Biosystems). For the measurement of rRNA and hTERT in urine, 1 and 5 µL were used, respectively. Reaction components (including TaqMan Universal Master Mix) and cycling conditions were identical to those described previously (20).

Emission spectra of all samples were collected every cycle during the last 30 s of the primer annealing/elongation step. The system was linked directly to a Power Macintosh 7200/120 containing software to analyze data. For hTERT, calculation of the fluorescence threshold by the computer was at the default setting (10 SD above the mean base fluorescence from all samples, calculated for cycles 3–15). Because rRNA is expressed abundantly in tissue samples and already interferes with measurement of the base fluorescence during the early PCR cycles, the mean base fluorescence was calculated from PCR cycles 2–6. The number of PCR cycles to reach the fluorescence threshold was the cycle threshold (Ct). The Ct value for each sample was proportional to the log of the initial amount of input cDNA. For the calibration curves for both hTERT and rRNA, we used cDNA from a T24 bladder carcinoma cell line to make the transformations from Ct values to nanograms of cDNA, as described previously (20). The rRNA expression was used to normalize hTERT expression for sample-to-sample differences in RNA input, RNA quality, and reverse transcriptase efficiency.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
interexperiment variation
With the development of a TaqMan Universal Master Mix (Perkin-Elmer) to which only primers, probe, and sample must be added, PCR efficiencies became highly reproducible, producing similar slopes for the calibration curves between experiments (20). In the present experiment, the functions of the calibration curves were: Ct = -3.72 log [hTERT] + 32.84; and Ct = -3.60 log [rRNA] + 12.94; slopes did not differ significantly from the previous experiments (20). However, the height of the curves (especially for rRNA) varied between experiments, partly because of sample dilution in the previous experiments.

As an additional control for reproducibility between experiments, hTERT and rRNA expression were measured in the cDNA of two calibrators: the bladder carcinoma cell lines RT4 and SCaBER. The normalized hTERT values were 122.03 for RT4 and 89.53 for SCaBER (Table 1 ) and were not significantly different from previous measurements: 111.99 ± 13.8 and 92.74 ± 8.06, respectively (20). Because the method is very reproducible, normalized hTERT values between experiments could be compared directly.


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  		Table 1. Expression of hTERT, rRNA, and normalized hTERT in tumors, corresponding healthy tissues, and cell lines.

hTERT IN TUMORS AND HEALTHY TISSUES
Normalized hTERT expression was measured in five different tumor types and compared with the corresponding healthy tissues (Table 1Up ). Healthy pancreas tissue (n = 3) did not express hTERT above the detection limit, whereas all three tumors did. Sample PT1 had low hTERT expression, which is partly attributable to the relatively low amount of tumor tissue (30%) in the sample. All three samples of healthy lung tissue contained low hTERT expression; whereas expression in four of five lung tumors was at least fivefold higher. When sample LT1 was corrected for tumor percentage (0.53 x 2.5), the expression was also higher than in healthy tissues. Both samples of healthy colonic tissue expressed high concentrations of hTERT within the range of the colorectal adenocarcinomas. Two of three samples of healthy esophagus tissue did express hTERT. However, all six esophageal carcinomas expressed higher concentrations, especially considering the low percentage of tumor tissue in most samples. Three samples of healthy bladder wall tissue did not express hTERT, whereas all six bladder carcinomas expressed hTERT, with normalized values ranging from 1.37 to 25.3.

hTERT IN URINE
Normalized hTERT expression was measured in nine urine samples of patients with tumors of various stages and grades. hTERT expression was also measured in urine from four donors with nonmalignant bladder diseases as specificity controls (Table 2 ). In the urine of five patients (55%), hTERT could be quantified. In the urine of four patients and in the specificity controls, no hTERT was detected. The urine of both patients with a carcinoma in situ and a superficial tumor had much higher normalized hTERT expression than patients with solely a superficial tumor.


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  		Table 2. Normalized hTERT expression in urines of patients with bladder cancer and in controls.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Telomerase activity is associated with the acquisition of malignancy and may have potential as a biomarker for the early detection of cancer (22). It is of importance that the detection of cancer is achievable in specimens obtained by relatively noninvasive procedures. For example, telomerase activity measured with the telomeric repeat amplification protocol (TRAP) assay (23) has been detected in urine or bladder washings (24)(25)(26)(27)(28), bronchoalveolar lavage fluid (29), pancreatic juice (30), and colonic luminal washings (31). Because the TRAP assay needs the functional ribonucleoprotein, the presence of protein activity inhibitors, proteases, or RNases in an aggressive medium may complicate detection and subsequently lower sensitivity.

With the cloning of both genes coding for the RNA (hTR) and protein component (hTERT) of telomerase, it soon became clear that only the expression of hTERT was closely correlated with telomerase activity (18)(32). Screening of hTERT expression using Northern blotting, reverse transcription-PCR (RT-PCR), and in situ hybridization in various tumors and healthy tissues confirmed these initial findings (33)(34)(35). However, an interesting exception to the relationship between hTERT expression and telomerase activity was found in Wilms tumor, where only hTERT expression but not telomerase activity significantly correlated with recurrence (36). Moreover, several splicing variants of hTERT mRNA have been found with still unknown functions (16). In addition to tissues, the presence of hTERT mRNA was tested in bodily samples, but without accurate quantitative methods (37)(38).

We recently developed an assay for the accurate quantification of hTERT expression (20). An advantage of this assay compared with the TRAP assay is that it needs only a 144-base fragment of hTERT mRNA, making the assay less sensitive to RNase activity and insensitive to proteases and protein inhibitors. Moreover, the inclusion of an endogenous control (rRNA) in the assay normalizes the hTERT expression for cDNA input and assay affectors. The real-time quantitative PCR method is very reproducible between experiments, with equal slopes of the calibration curves but different heights. If the PCR efficiency remains constant in successive experiments, the measurement of a calibration curve will become superfluous and can be replaced by a calibrator that corrects solely for height differences between experiments.

For our analyses, we made a selection of tumor types of which cells may be found in bodily samples. All tumors in our study expressed hTERT. Healthy pancreas and bladder tissues did not express hTERT. The results for bladder tissues were in agreement with the results obtained by Ito et al. (34), who detected hTERT expression in only 3 of 18 healthy bladder tissue samples but in 30 of 33 urothelial cancers by use of RT-PCR. Possibly, quantitative measurement of hTERT expression in these tissues could have discriminated these three positive healthy bladder tissue samples from the tumors when a cutoff concentration was determined. This advantage of real-time RT-PCR was shown by Hisatomi et al. (39), who determined a cutoff concentration using real-time quantification of hTERT expression to differentiate between malignant and benign hepatocellular tissues.

In healthy lung, very low background hTERT expression was detected in all samples. Although in a recent study no hTERT mRNA was found in individual cells from healthy lung by in situ hybridization (35), our results are not in conflict with these results because PCR detection is more sensitive. Moreover, low hTERT expression in lung tissue can also be caused by the presence of (inflammatory) leukocytes (40).

In healthy esophagus tissue, hTERT expression has been found by us and others and is predominantly localized to the basal cell layers of the columnar epithelium (35). These basal cells are not likely to be removed during superficial esophageal brushings and therefore are not expected to interfere with the detection of neoplasia (in contrast to biopsies). In patients with progressive Barretts esophagus, hTERT quantification in brushings may be of interest for the detection of onset of malignancy (41).

Both samples of healthy colonic tissues expressed high concentrations of hTERT. This is in agreement with other reports in which hTERT expression was detected in colonic crypt epithelial cells (35)(42). Although no hTERT expression was present in cells at the top of the crypts, which are predominantly shed into the lumen of the colon, the ubiquity of inflammatory lymphocytes expressing high concentrations of hTERT may interfere with hTERT quantification in feces or colonic effluent samples (35)(40).

Our results show that real-time measurement of hTERT expression discriminates between healthy and malignant tissues for pancreatic, lung, esophageal, and bladder tissues. This discrimination would have been even more distinct if the percentage of tumor cells was higher in the selected tissues. In future studies, using higher numbers of healthy tissue samples and neoplasms with high percentages of tumor cells, accurate cutoff concentrations may be chosen to determine the sensitivity and specificity for each type of tissue. Even for discrimination between healthy colonic and tumor tissues, a cutoff may be determined to increase specificity, although sensitivity will probably be compromised.

Overall, the different hTERT expression ranges between tumor tissues and healthy tissues suggested promising applications for the detection of tumor cells in pancreatic fluid, bronchoalveolar washings, esophageal brushings, and urine or bladder washings. To demonstrate this potential use, we collected the voided urine of nine patients scheduled for transurethral resection of a superficial bladder carcinoma and quantified hTERT after RNA isolation. In five urine samples, hTERT could be quantified, with the most aggressive tumors (carcinoma in situ) expressing the highest concentrations of hTERT, whereas all control urines were negative. It would be interesting to determine whether the quantification of hTERT expression in urine would be useful in the diagnosis and follow-up of patients with bladder cancer.


   Acknowledgments
 
We thank Prof. D.J. Ruiter (Department of Pathology, University Hospital Nijmegen) for determining the percentage of tumor cells in the tissues and Prof. J. Schalken (Urology Research Department, University Hospital Nijmegen) for donation of the bladder tissues. We also thank T. Aalders (Urology Research Department, University Hospital Nijmegen) and H. Zendman (Department of Pathology, University Hospital Nijmegen) for technical assistance; Dr. J. Klein Gunnewiek (Department of Clinical Chemistry, Canisius Wilhelmina Hospital, Nijmegen) for collection of the urine samples; and Dr. B. Giesendorf (Department of Clinical Chemistry, University Hospital Nijmegen) for constructive comments on experimental set up.


   Footnotes
 
1 Nonstandard abbreviations: hTERT, human telomerase reverse transcriptase; Ct, cycle threshold value; rRNA, 18S ribosomal RNA; TRAP, telomeric repeat amplification protocol; and RT-PCR, reverse transcription-PCR.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Jacques de Kok
Clinical Chemistry Online, 13 Mar 2000 [Full text]
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