Clinical Chemistry Siemens Point of Care - Urinalysis
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Clinical Chemistry 46: 313-318, 2000;
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow A correction has been published
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (41)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by de Kok, J. B.
Right arrow Articles by Swinkels, D. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by de Kok, J. B.
Right arrow Articles by Swinkels, D. W.
Related Collections
Right arrow Molecular Diagnostics and Genetics
Right arrow Proteomics and Protein Markers
(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.


View this table:
[in this window]
[in a new window]
  		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.


View this table:
[in this window]
[in a new window]
  		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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Sidransky D, Tokino T, Hamilton SR, Kinzler KW, Levin B, Frost P, Vogelstein B. Identification of ras oncogene mutations in the stool of patients with curable colorectal tumors. Science 1992;256:102-105. [Abstract/Free Full Text]
  2. Steiner G, Schoenberg MP, Linn JF, Mao L, Sidransky D. Detection of bladder cancer recurrence by microsatellite analysis of urine. Nat Med 1997;3:621-624. [Web of Science][Medline] [Order article via Infotrieve]
  3. Takeda S, Ichii S, Nakamura Y. Detection of K-ras mutation in sputum by mutant-allele-specific amplification (MASA). Hum Mutat 1993;2:112-117. [Web of Science][Medline] [Order article via Infotrieve]
  4. Kondo H, Sugano K, Fukayama N, Kyogoku A, Nose H, Shimada K, et al. Detection of point mutations in the K-ras oncogene at codon 12 in pure pancreatic juice for diagnosis of pancreatic carcinoma. Cancer 1994;73:1589-1594. [Web of Science][Medline] [Order article via Infotrieve]
  5. Lee JG, Leung JW, Cotton PB, Layfield LJ, Mannon PJ. Diagnostic utility of K-ras mutational analysis on bile obtained by endoscopic retrograde cholangiopancreatography. Gastrointest Endosc 1995;42:317-320. [Web of Science][Medline] [Order article via Infotrieve]
  6. Swinkels DW, de Kok JB, Hanselaar A, Lamers K, Boerman RH. Early detection of leptomeningeal metastasis by PCR examination of tumor-derived K-ras DNA in cerebrospinal fluid [Letter]. Clin Chem 2000;46:132-133. [Free Full Text]
  7. Yamada T, Nakamori S, Ohzato H, Oshima S, Aoki T, Higaki N, et al. Detection of K-ras gene mutations in plasma DNA of patients with pancreatic adenocarcinoma: correlation with clinicopathological features. Clin Cancer Res 1998;4:1527-1532. [Abstract]
  8. de Kok JB, van Solinge WW, Ruers TJM, Roelofs RH, van Muijen GN, Willems JL, Swinkels DW. Detection of tumour DNA in serum of colorectal cancer patients. Scand J Clin Lab Investig 1997;57:601-604. [Web of Science][Medline] [Order article via Infotrieve]
  9. Yamao T, Matsumura Y, Shimada Y, Moriya Y, Sugihara K, Akasu T, et al. Abnormal expression of CD44 variants in the exfoliated cells in the feces of patients with colorectal cancer. Gastroenterology 1998;114:1196-1205. [Web of Science][Medline] [Order article via Infotrieve]
  10. Okamoto I, Morisaki T, Sasaki J, Miyake H, Matsumoto M, Suga M, et al. Molecular detection of cancer cells by competitive reverse transcription-polymerase chain reaction analysis of specific CD44 variant RNAs. J Natl Cancer Inst 1998;90:307-315. [Abstract/Free Full Text]
  11. Shay JW, Bacchetti S. A survey of telomerase activity in human cancer. Eur J Cancer 1997;33:787-791.
  12. Meeker AK, Coffey DS. Telomerase: a promising marker of biological immortality of germ, stem, and cancer cells. A review. Biochemistry 1997;62:1323-1331. [Medline] [Order article via Infotrieve]
  13. Feng J, Funk WD, Wang SS, Weinrich SL, Avilion AA, Chiu CP, et al. The RNA component of human telomerase. Science 1995;269:1236-1241. [Abstract/Free Full Text]
  14. Meyerson M, Counter CM, Eaton EN, Ellisen LW, Steiner P, Caddle SD, et al. hEST2, the putative human telomerase catalytic subunit gene, is up-regulated in tumor cells and during immortalization. Cell 1997;90:785-795. [Web of Science][Medline] [Order article via Infotrieve]
  15. Nakamura TM, Morin GB, Chapman KB, Weinrich SL, Andrews WH, Lingner J, et al. Telomerase catalytic subunit homologs from fission yeast and human. Science 1997;277:955-959. [Abstract/Free Full Text]
  16. Kilian A, Bowtell DD, Abud HE, Hime GR, Venter DJ, Keese PK, et al. Isolation of a candidate human telomerase catalytic subunit gene, which reveals complex splicing patterns in different cell types. Hum Mol Genet 1997;6:2011-2019. [Abstract/Free Full Text]
  17. Nakayama J, Tahara H, Tahara E, Saito M, Ito K, Nakamura H, et al. Telomerase activation by hTRT in human normal fibroblasts and hepatocellular carcinomas. Nat Genet 1998;18:65-68. [Web of Science][Medline] [Order article via Infotrieve]
  18. Weinrich SL, Pruzan R, Ma L, Ouellette M, Tesmer VM, Holt SE, et al. Reconstitution of human telomerase with the template RNA component hTR and the catalytic protein subunit hTRT. Nat Genet 1997;17:498-502. [Web of Science][Medline] [Order article via Infotrieve]
  19. Lo YMD. Quantitative assays for telomerase: means for studying the end [Editorial]. Clin Chem 1998;44:2399-2400. [Free Full Text]
  20. de Kok JB, Zendman AJ, van de Locht LT, Ruers TJ, van Muijen GN, Mensink E, Swinkels DW. Real-time hTERT quantification: a promising telomerase-associated tumor marker. Lab Investig 1999;79:911-912. [Web of Science][Medline] [Order article via Infotrieve]
  21. Heid CA, Stevens J, Livak KJ, Williams PM. Real time quantitative PCR. Genome Res 1996;6:986-994. [Abstract/Free Full Text]
  22. Breslow RA, Shay JW, Gazdar AF, Srivastava S. Telomerase and early detection of cancer: a National Cancer Institute workshop. J Natl Cancer Inst 1997;89:618-623. [Free Full Text]
  23. Kim NW, Piatyszek MA, Prowse KR, Harley CB, Wets MD, Ho PL, et al. Specific association of human telomerase activity with immortal cells and cancer. Science 1994;266:2011-2015. [Abstract/Free Full Text]
  24. Yoshida K, Sugino T, Tahara H, Woodman A, Bolodeoku J, Nargund V, et al. Telomerase activity in bladder carcinoma and its implication for noninvasive diagnosis by detection of exfoliated cancer cells in urine. Cancer 1997;79:362-369. [Web of Science][Medline] [Order article via Infotrieve]
  25. Muller M, Krause H, Heicappell R, Tischendorf J, Shay JW, Miller K. Comparison of human telomerase RNA and telomerase activity in urine for diagnosis of bladder cancer. Clin Cancer Res 1998;4:1949-1954. [Abstract]
  26. Dalbagni G, Han W, Zhang ZF, Cordon-Cardo C, Saigo P, Fair WR, et al. Evaluation of the telomeric repeat amplification protocol (TRAP) assay for telomerase as a diagnostic modality in recurrent bladder cancer. Clin Cancer Res 1997;3:1593-1598. [Abstract]
  27. Ramakumar S, Bhuiyan J, Besse JA, Roberts SG, Wollan PC, Blute ML, O’Kane DJ. Comparison of screening methods in the detection of bladder cancer. J Urol 1999;161:388-394. [Web of Science][Medline] [Order article via Infotrieve]
  28. Kinoshita H, Ogawa O, Kakehi Y, Mishina M, Mitsumori K, Itoh N, et al. Detection of telomerase activity in exfoliated cells in urine from patients with bladder cancer. J Natl Cancer Inst 1997;89:724-730. [Abstract/Free Full Text]
  29. Arai T, Yasuda Y, Takaya T, Ito Y, Hayakawa K, Toshima S, et al. Application of telomerase activity for screening of primary lung cancer in broncho-alveolar lavage fluid. Oncol Rep 1998;5:405-408. [Web of Science][Medline] [Order article via Infotrieve]
  30. Suehara N, Mizumoto K, Tanaka M, Niijama H, Yokohata K, Tominaga Y, et al. Telomerase activity in pancreatic juice differentiates ductal carcinoma from adenoma and pancreatitis. Clin Cancer Res 1997;3:2479-2483. [Abstract/Free Full Text]
  31. Yoshida K, Sugino T, Goodison S, Warren BF, Nolan D, Wadsworth S, et al. Detection of telomerase activity in exfoliated cancer cells in colonic luminal washings and its related clinical implications. Br J Cancer 1997;75:548-553. [Web of Science][Medline] [Order article via Infotrieve]
  32. Nakayama JI, Tahara H, Tahara E, Saito M, Ito K, Nakamura H, et al. Telomerase activation by hTRT in human normal fibroblasts and hepatocellular carcinomas. Nat Genet 1998;18:65-68.
  33. Ramakrishnan S, Eppenberger U, Mueller H, Shinkay Y, Narayanan R. Expression profile of the putative catalytic subunit of the telomerase gene. Cancer Res 1998;58:622-625. [Abstract/Free Full Text]
  34. Ito H, Kyo S, Kanaya T, Takakura M, Inoue M, Namiki M. Expression of human telomerase subunits and correlation with telomerase activity in urothelial cancer. Clin Cancer Res 1998;4:1603-1608. [Abstract]
  35. Kolquist KA, Ellisen LW, Counter CM, Meyerson M, Tan LK, Weinberg RA, et al. Expression of TERT in early premalignant lesions and a subset of cells in normal tissues. Nat Genet 1998;19:182-186. [Web of Science][Medline] [Order article via Infotrieve]
  36. Dome JS, Chung S, Bergemann T, Umbricht CB, Saji M, Carey LA, et al. High telomerase reverse transcriptase (hTERT) messenger RNA level correlates with tumor recurrence in patients with favorable histology Wilms’ tumor. Cancer Res 1999;59:4301-4307. [Abstract/Free Full Text]
  37. Ito H, Kyo S, Kanaya T, Takakura M, Inoue M, Namiki M. Detection of human telomerase reverse transcriptase messenger RNA in voided urine samples as a useful diagnostic tool for bladder cancer. Clin Cancer Res 1998;4:2807-2810. [Abstract]
  38. Morales CP, Burdick JS, Saboorian MH, Wright WE, Shay JW. In situ hybridization for telomerase RNA in routine cytologic brushings for the diagnosis of pancreaticobiliary malignancies. Gastrointest Endosc 1998;48:402-405. [Web of Science][Medline] [Order article via Infotrieve]
  39. Hisatomi H, Nagao K, Kanamaru T, Endo H, Tomimatsu M, Hikiji K. Levels of telomerase catalytic subunit mRNA as a predictor of potential malignancy. Int J Oncol 1999;14:727-732. [Web of Science][Medline] [Order article via Infotrieve]
  40. Liu K, Schoonmaker MM, Levine BL, June CH, Hodes RJ, Weng NP. Constitutive and regulated expression of telomerase reverse transcriptase (hTERT) in human lymphocytes. Proc Natl Acad Sci U S A 1999;96:5147–52..
  41. Hardwick RH, Morgan RJ, Warren BF, Lott M, Alderson D. Brush cytology in the diagnosis of neoplasia in Barrett’s esophagus. Dis Esophagus 1997;10:233-237. [Medline] [Order article via Infotrieve]
  42. Tahara H, Yasui W, Tahara E, Fujimoto J, Ito K, Tamai K, et al. Immuno-histochemical detection of human telomerase catalytic component, hTERT, in human colorectal tumor and non-tumor tissue sections. Oncogene 1999;18:1561-1567. [Web of Science][Medline] [Order article via Infotrieve]
  43. Spiessl B, Beahrs OH, Hermanek P, Hutter RV, Scheibe O, Sobin LH, et al. TNM atlas: illustrated guide to the TNM/pTNM classification of malignant tumours, 3rd ed., 2nd revision. Berlin: Springer-Verlag; 1992:274–9..
  44. Mostofi FK, Sobin LH, Torloni H. International histological classification of tumors., No. 10. Histologic typing of urinary bladder tumors. Geneva: World Health Organization, 1973:36 pp..



The following articles in journals at HighWire Press have cited this article:


Home page
 J. Leukoc. Biol.Home page
A. Webb, A. Johnson, M. Fortunato, A. Platt, T. Crabbe, M. I. Christie, G. F. Watt, S. G. Ward, and L. A. Jopling
Evidence for PI-3K-dependent migration of Th17-polarized cells in response to CCR2 and CCR6 agonists
J. Leukoc. Biol., October 1, 2008; 84(4): 1202 - 1212.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
M. Hanke, I. Kausch, G. Dahmen, D. Jocham, and J. M. Warnecke
Detailed Technical Analysis of Urine RNA-Based Tumor Diagnostics Reveals ETS2/Urokinase Plasminogen Activator to Be a Novel Marker for Bladder Cancer
Clin. Chem., December 1, 2007; 53(12): 2070 - 2077.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Pathol.Home page
B Luzar, M Poljak, A Cor, U Klopcic, and V Ferlan-Marolt
Expression of human telomerase catalytic protein in gallbladder carcinogenesis
J. Clin. Pathol., August 1, 2005; 58(8): 820 - 825.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
R. Rej
Clinical Chemistry through Clinical Chemistry: A Journal Timeline
Clin. Chem., December 1, 2004; 50(12): 2415 - 2458.
[Abstract] [Full Text] [PDF]

Home page
 Ann OncolHome page
A. Tchirkov, C. Chaleteix, C. Magnac, Y. Vasconcelos, F. Davi, A. Michel, F. Kwiatkowski, O. Tournilhac, G. Dighiero, and P. Travade
hTERT expression and prognosis in B-chronic lymphocytic leukemia
Ann. Onc., October 1, 2004; 15(10): 1476 - 1480.
[Abstract] [Full Text] [PDF]

Home page
 JCOHome page
R. Gertler, R. Rosenberg, D. Stricker, J. Friederichs, A. Hoos, M. Werner, K. Ulm, B. Holzmann, H. Nekarda, and J.-R. Siewert
Telomere Length and Human Telomerase Reverse Transcriptase Expression As Markers for Progression and Prognosis of Colorectal Carcinoma
J. Clin. Oncol., May 15, 2004; 22(10): 1807 - 1814.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
A. Stahlberg, J. Hakansson, X. Xian, H. Semb, and M. Kubista
Properties of the Reverse Transcription Reaction in mRNA Quantification
Clin. Chem., March 1, 2004; 50(3): 509 - 515.
[Abstract] [Full Text] [PDF]

Home page
 JCOHome page
F. Buttitta, C. Pellegrini, A. Marchetti, A. Gadducci, S. Cosio, L. Felicioni, F. Barassi, S. Salvatore, C. Martella, G. Coggi, et al.
Human Telomerase Reverse Transcriptase mRNA Expression Assessed by Real-Time Reverse Transcription Polymerase Chain Reaction Predicts Chemosensitivity in Patients With Ovarian Carcinoma
J. Clin. Oncol., April 1, 2003; 21(7): 1320 - 1325.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
T. Masui, R. Hosotani, S. Tsuji, Y. Miyamoto, S. Yasuda, J. Ida, S. Nakajima, M. Kawaguchi, H. Kobayashi, M. Koizumi, et al.
Expression of METH-1 and METH-2 in Pancreatic Cancer
Clin. Cancer Res., November 1, 2001; 7(11): 3437 - 3443.
[Abstract] [Full Text] [PDF]

eLetters:

Read all eLetters

Erratum table 1
Jacques de Kok
Clinical Chemistry Online, 13 Mar 2000 [Full text]
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow A correction has been published
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (41)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by de Kok, J. B.
Right arrow Articles by Swinkels, D. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by de Kok, J. B.
Right arrow Articles by Swinkels, D. W.
Related Collections
Right arrow Molecular Diagnostics and Genetics
Right arrow Proteomics and Protein Markers

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS