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1
Program in Human Genetics, and
2
Department of Pathology, University of Maryland at Baltimore, 737 W. Lombard St., Baltimore, MD 21201
aAddress correspondence to this author at: Department of Pathology, University of Maryland at Baltimore, 7-22 MSTF, 10 South Pine St., Baltimore, MD 21201. Fax 410-706-8414; e-mail whighsmi{at}umaryland.edu.
Abstract
Background: Telomerase is a ribonucleoprotein that maintains chromosomal telomere length. Telomerase is not active in nonmalignant somatic cells, but is activated in most human cancers. Telomerase activity in easily obtainable body fluids that bathe tumors may be a useful cancer marker, especially when used in conjunction with conventional cytology.
Approach: Results from studies that assayed telomerase activity in easily obtainable body fluids are reviewed.
Content: The telomerase repeat amplification protocol (TRAP) assay has been used to measure telomerase activity in body fluids, including ascites, pleural effusions, pelvic washes, bronchial washings, bronchial lavage, urine, bladder washings, oral rinses, and plasma. Telomerase activity has sensitivities of 60–90% as a tumor marker with clinical specificities for cancer of 
90%. Telomerase activity is more sensitive than conventional cytology, the sensitivity of which was 40–65% in various studies.
Summary: Telomerase activity in body fluids, as measured by the TRAP assay, is a sensitive potential tumor marker that might help increase the cancer detection rate and the cancer treatment success rate when combined with conventional cytology.
The ends of chromosomes are stabilized and protected from degradation by intercellular nucleases by specialized nucleoprotein structures, the telomeres. The DNA component of telomeres consists of many hundreds to thousands of simple repeat sequences. In mammals, this sequence is 5'-TTAGGG-3' (1)(2)(3). Because the DNA replication machinery cannot fully replicate the ends of linear DNA, telomeres progressively shorten with each round of cell replication (4). Eventually, a critically short telomere length is reached, and the cell stops dividing and senesces (5)(6)(7). This phenomenon is thought to function as a type of "molecular clock", limiting the lifespan of individual cells. However, cell types that, as part of their normal biology, must replicate for many rounds of cell division, e.g., germ cells, embryonic cells, and stem cells, must posses a mechanism to "reset the clock" (8)(9). This mechanism is provided by the enzyme telomerase, a ribonucleoprotein that recognizes the ends of telomeres and adds additional repeats (1)(2)(3). In most postembryonic cell types, telomerase activity is down-regulated; in cancer cells, however, telomerase expression is activated (8)(9)(10)(11). Therefore, telomerase has been the focus of much research over the past several years, with increasing interest in the role of this enzyme in carcinogenesis, as a target for anticancer chemotherapy, and as a biomarker for the detection of cancer.
Telomerase is a ribonucleoprotein consisting of three components: two protein subunits and an RNA subunit. The catalytic core consists of hTERT, the catalytic subunit that possesses reverse transcriptase activity, and hTR, the coding RNA component (12)(13)(14). The second telomerase protein, hTP, may play a structural role. hTR and hTP are expressed ubiquitously, with hTERT expression being the rate-limiting step for telomerase activity (15).
Kim et al. (8) demonstrated that telomerase activity can be detected in various solid tumors and in hematologic malignancies. Further work in many laboratories worldwide has shown that telomerase is active in 80–90% of human cancers (8)(9)(10)(11). For recent reviews on the detection of telomerase in tumor tissue, see Refs. (16)(17)(18). This review compares and contrasts the findings of studies that attempted to evaluate the utility of telomerase as a tumor marker in body fluids (Table 1
).
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Assays for Telomerase Activity
Several methods have been developed for the detection of telomerase activity. To measure telomerase activity, not only must all telomerase components be expressed in the cell or tissue being studied, but they also must be correctly assembled into a functional unit that is capable of adding 5'-TTAGGG-3' repeats to the end of a telomere. The telomere repeat amplification protocol (TRAP) 1 assay and variations on this assay are the most widely used methods for monitoring telomerase activity. The TRAP assay, first described by Kim et al. (8), is a two-stage, PCR-based assay. In the first stage, telomerase adds 5'-TTAGGG-3' repeats to the end of a synthetic primer (termed the TS primer) that has a telomere-like sequence. In the second stage, the extended oligonucleotide products are amplified using a reverse primer (termed the CX primer) that is complimentary to the repeat sequences. When visualized by autoradiography, a positive test by TRAP assay shows a ladder of bands differing by 6 bp, the length of the hexameric 5'-TTAGGG-3' repeat (8). The band volume can be quantified by densitometry, by phosphoimaging, or by fluoroimaging after incorporation of 32P during amplification or after staining with SYBR GreenTM (Molecular Probes).
The TRAP assay is highly sensitive and specific for telomerase activity in tumor samples, but it has several practical limitations. One limitation is that it is time- and labor-intensive, typically requiring a skilled technician 24 h to generate results. Another limitation is that because telomerase contains an RNA component, the activity is labile and easily destroyed by RNase. Therefore, the protocol for preparation of TRAP assay samples is intricate and requires careful attention to sample collection. In addition, the TRAP assay, as originally described by Kim et al. (8), requires the use of radioactivity and polyacrylamide gel electrophoresis. For these reasons, several modifications have been made to the TRAP assay to make it more "user-friendly". One modified method, the TRAP-eze® (Intergen Discovery Products) telomerase detection reagent set, includes the necessary reagents in a convenient form (19); it is said to be more sensitive and cost-effective and less labor-intensive that the conventional TRAP assay as well as having a wider linear range (19). The TRAP-eze still uses radioactivity and polyacrylamide gel electrophoresis and still requires 8–10 h of technician time.
A telomerase assay that replaces the electrophoretic step with an ELISA detection system has been described. In this system, the PCR-amplified product is hybridized to probes that are complementary to the 5'-TTAGGG-3' sequence, followed by ELISA quantification (20). Wu et al. (20) recently compared this protocol with the TRAP-eze assay. This group found that both the TRAP-eze and the TRAP ELISA had linear ranges of 250-5000 cell-equivalents. Ultimately, the authors concluded that neither method appears to have a clear advantage over the other. It was suggested that researchers should use the method with which they feel more comfortable and use the other method as a backup or double-check method (20).
One limitation of the TRAP assay is that it is a solution-phase technique, so information on the cell type expressing telomerase is lost. It is not possible to know whether telomerase activity is coming from tumor cells or from other cells in the sample, such as activated lymphocytes. To overcome this limitation, Ohyashiki et al. (21) developed an in situ TRAP assay that uses fluorescent dyes and microscopy to visualize telomerase activity in the nuclei of the cells of interest.
To eliminate the technical issues inherent in the TRAP assay, several authors have developed in situ hybridization assays for the quantification of both hTR RNA and hTERT mRNA and correlated the results with prognostic factors for the tumors (22). hTR and hTERT expression does not necessarily equate to telomerase activity.
Telomerase Activity in Bodily Fluids: Rationale
Many laboratories have investigated the expression of telomerase activity in tumor vs healthy and nonmalignant tissues (8)(9)(16)(17). However, obtaining samples from most tumors is invasive and not amenable to serial analyses. For this reason, investigators have begun to assay telomerase activity in body fluids that might contain tumor cells that have been released from the tumor site and have begun to compare the results with such variables as tumor progression and tumor stage. These fluid samples, such as ascites, pleural effusions, sputum, bronchial lavage or washings, urine or bladder washings, oral rinses, and plasma, can be obtained largely through noninvasive or minimally invasive means. Although cytologic examination of cells in these fluids remains the accepted standard for detection of many cancers, its sensitivity is typically only 50–75% (Table 1
).
Measuring Telomerase Activity in Ascites/Pleural Effusions
Several investigators have explored the use of telomerase activity measurements for the differentiation of benign and malignant etiologies of ascites. Tangkijvanich et al. (23) measured telomerase activity in nonmalignant ascites and malignancy-related ascites associated with hepatocellular carcinoma (HCC) and peritoneal carcinomatosis (PC). The sensitivity and specificity of the telomerase assay for the detection of malignancy were compared with the sensitivity and specificity of cytology. Telomerase activity was detected in 81% of PC and 67% of HCC ascites samples. Interestingly, PC and HCC were positively diagnosed by cytology in only 56% and 11% of cases, respectively. The false-positive rate for telomerase, or the number of nonmalignant ascites cases in which telomerase activity was detected, was 4.3%; all of these samples had some degree of lymphocytic contamination. For HCC and PC combined, the overall sensitivities of telomerase activity and cytology were 76% and 40%, respectively; the specificities were 95.7% and 100%, respectively. The authors concluded that telomerase activity is more sensitive that cytology in diagnosing PC and HCC and that telomerase activity may be useful as a detector of early intraperitoneal metastasis (23).
Duggan et al. (24) found telomerase activity to be more sensitive than cytology in ascitic samples from ovarian cancer patients. Telomerase activity was detected in 88% of ascitic samples from ovarian cancer patients, whereas a positive cytologic diagnosis was made in only 64% of patients. The difference in specificities was statistically significant. The telomerase activity false-positive rate was 5%. However, one reason to regard these data with caution is that this group detected telomerase activity in samples that were left unprocessed at room temperature for 5 days (24). In the experience of many telomerase research laboratories, telomerase activity is sensitive to degradation. Samples therefore usually require immediate treatment with RNase inhibitors and flash freezing to maintain detectable telomerase activity.
The sample-processing issue also affects the reliability of the study performed by Cunningham et al. in 1997 (25). This group assayed for telomerase activity in pelvic wash samples from patients with various tumor types at various sites. Pleural effusions and pelvic washes are fluids that, when sampled, can contain cells from several tumor sites, including lung, breast, ovary, and the gastrointestinal tract, because carcinomas in any organ can metastasize to the pleura. These results were compared with fine-needle aspirate samples from the same patients. This study had a low sample number (n = 24). Only six of the wash samples were cytologically positive, and only four of these had detectable telomerase activity. However, the fluid samples were not processed for 2–3 days, so the two samples that were cytologically positive and telomerase negative might have been degraded positive samples and therefore were false negatives. In addition, the telomerase activity detection specificity was higher in fine-needle aspirates than in fluid samples (25). It should be noted, however, that this work was performed in 1997 when the TRAP assay was in its research-utility infancy.
Yang et al. (26) showed more convincing evidence for the clinical utility of telomerase in pleural effusions in three groups: (a) malignant pleural effusions as diagnosed by cytology; (b) nonmalignant pleural effusions; and (c) pleural effusions that were suspected to be malignant, but that were inconclusive by cytology. Of 144 samples, telomerase activity was detected in 91% (64 of 70) of malignant samples, 91% (20 of 22) suspicious samples, and only 5.8% (3 of 52) of nonmalignant samples. Overall, telomerase activity had a sensitivity of 91%, a specificity of 94%, a positive predictive value of 96%, and a negative predictive value of 89%. Interestingly, the three false-positive results in this study were in samples of effusions from patients with tuberculosis. The authors point out that the prevalence of tuberculosis is increased among cancer patients and concluded that measurement of telomerase activity is a useful adjunct to cytology in the evaluation of malignant pleural effusions (26).
Telomerase Activity in Bronchial Washings/Lavage Samples
Bronchial lavage and bronchial washing samples have been used to assess the utility of telomerase activity measurements in lung cancer patients. Yahata et al. (27) compared cytologic diagnosis for lung cancer with telomerase activity measured by both the solution-phase TRAP and the novel in situ TRAP assay. For the in situ assay, cells from bronchial washings were immobilized on glass slides, the TRAP protocol was performed, and a sample was considered telomerase positive when the fluorescent signal was present in the nucleus. For the two versions of the TRAP assay combined, sensitivity for lung cancer was 82%, whereas cytology was only 41% sensitive; there was a 77% concordance between the two versions of the TRAP assay. Telomerase activity could be detected irrespective of lung tumor localization. The authors concluded that the combination of cytology and TRAP assays is useful for the early diagnosis and monitoring of lung cancer (27). This group published a second study (28) in which telomerase activity measured by the in situ method in various types of lung cancer was investigated. The in situ TRAP assay had a high sensitivity in squamous cell carcinoma, adenocarcinoma, large cell cancer, and metastasis from colon carcinoma, but was not sensitive in small cell cancer. Furthermore, all cells that were telomerase positive showed morphologic features of malignancy (28). However, this study had a small sample number (n = 18). It would be interesting and useful if these observations were reproduced with a larger sample set.
Xinarinanos et al. (29) found telomerase activity in bronchial lavage samples to be 70% sensitive in patients with non-small cell lung cancer. Bronchial lavage samples from patients with non-small cell lung cancer were also compared with samples obtained from healthy controls, and the telomerase activity detection rate was compared with the detection rate of cytologic examination. Telomerase activity was detected more frequently in squamous cell carcinoma than in adenocarcinoma. Ninety percent of samples that were positive by cytology were also positive for telomerase activity; furthermore, 54% of samples that were negative by cytology possessed telomerase activity. The sensitivity of cytology by itself was 43%, but when cytology was combined with the TRAP assay, the combined sensitivity was 74% (29). The authors noted that this combined rate of sensitivity was higher than the sensitivity routinely being achieved at present for lung cancer diagnostic testing.
Measuring Telomerase Activity in Urine/Bladder Washings
bladder cancer
Assaying for telomerase activity can increase the detection of bladder cancer in urine and bladder-washing samples. Kinoshita et al. (30) found that telomerase activity was 89% sensitive when results on urine and bladder-washing samples from the same patients were combined. Cytologic examination of these patients was only 42% sensitive. The TRAP assay was 100% specific for cancer patients. Perhaps most importantly, the TRAP assay was markedly more sensitive than cytology in detecting the most treatable (grade 1) tumors; the percentages of positive samples detected by the TRAP assay and cytology were 75% and 8%, respectively (30).
Yoshida et al. (31) obtained similar results for urine samples. TRAP assay sensitivity in urine was 62%; TRAP assay sensitivity in primary tumor samples was 86%; and TRAP assay specificity was 96%. [This study did not provide comparison with cytology (31).] Similarly, Kalavier et al. (32) compared the TRAP assay, in this case the TRAP-eze version, with cytology in urine samples of bladder cancer patients. All cancer patients had been diagnosed but not yet treated. A statistically significant difference was seen between the sensitivity of the TRAP assay (85%) and that of cytology (51%), but the TRAP assay had a 39% false-positive rate, which could most probably be explained by these patients having severe inflammation and their inflammatory cells contaminating the samples (32).
Ramakumar et al. (33) analyzed voided urine samples from bladder cancer patients and controls to compare the sensitivities and specificities of several bladder cancer screening methods, including the bladder tumor antigen (BTA), nuclear matrix protein 22 (NMP22), fibrin/fibrinogen degradation products (FDP), chemiluminescent hemoglobin, and the TRAP assays. Of all of the methods analyzed, the TRAP assay had the highest sensitivity (67%) and specificity (99%). For all tumors and within all tumor grades and stages, telomerase had the strongest association with bladder cancer; the association was especially strong between telomerase activity and grade 1 and noninvasive tumors (33).
prostate cancer
Meid et al. (34) investigated the use of TRAP analysis on voided urine after prostate massage for the detection of prostate cancer. The authors noted that cytologic examination of exfoliated cells in urine sediments, even after prostate massage, had a relatively poor sensitivity for the detection of prostate cancer. Telomerase activity was detected in 14 of 24 samples from patients with prostate cancer and in none of the 12 controls without histologic evidence of neoplasia (sensitivity, 58%; specificity, 100%). The telomerase test had greater sensitivity in 9 patients with poorly differentiated cancer (8 of 9 positive) than in 15 patients with well- or moderately differentiated tumors (6 of 15 positive). To address the issue of telomerase stability in urine, PC-3 or LNCaP cells were mixed with freshly voided urine and incubated at room temperature or 4 °C. As a surrogate for telomerase stability, RNA was extracted from these cells, and the integrity of the RNA was judged by the appearance of 18S and 28S RNA bands on agarose gel electrophoresis. Even after a 30-min incubation, the extracted RNA was markedly degraded. The authors found that the addition of EDTA to a final concentration of 20 mmol/L stabilized the RNA for up to 2 h at 4 °C. Although these authors did not measure telomerase activity in urine samples with added cells for a direct determination of telomerase stability, they were the first to address this critical issue. The authors concluded that telomerase activity measurements may be useful for the detection of prostate cancer, but noted that more work had to be done to address stability.
Measuring Telomerase Activity in Other Fluids: Oral Rinses and Plasma
In oral rinses from head and neck cancer patients, the sensitivity for the TRAP assay was only 32%, although it was positive in 80% of the tumors (35).
One of the most routinely obtained bodily fluid samples is blood plasma, which is produced after centrifugation of whole blood. At first glance plasma does not seem to be a useful sampling medium for telomerase because there would ordinarily be no tumor cells circulating in the blood. However, if even a small number of tumor cells undergo necrosis and release their contents into the plasma, there is the possibility of detecting tumor-specific analytes. Although several studies have addressed the detection of tumor-specific mRNA in plasma, this broader topic is beyond the scope of this review. There is one report of the detection of hTERT and hTR mRNA in plasma. Chen et al. (22) reported the detection of these transcripts by reverse-transcription PCR. hTR was expressed in 94% of tumors, but was found in only 28% of plasma samples from the same patients. Ninety-four percent of tumors, although not the same 94% that had hTR expression, expressed hTERT, but only 25% of patient plasma samples were positive for hTERT mRNA (22). Once again, it is critical to note that hTR and hTERT expression in a cell does not necessarily correlate with telomerase activity in that cell. This study, unlike most of the previously discussed studies, was not measuring telomerase activity.
Summary and Future Directions
Measurement of telomerase activity in easily obtained fluids may be a useful tool for the diagnosis and monitoring of cancer progression. The commonly used TRAP assay is sensitive, but it is time- and labor-intensive and requires careful sample preparation and a carefully trained technician.
The specificity of the TRAP assay for the detection of malignancies is 94–100% (Table 1
). The majority of reports cited in this review noted that false-positive TRAP results were most likely attributable to lymphocyte contamination. This implies that TRAP assay results may be best interpreted in conjunction with cytologic examination, but the performances of combined cytology/telomerase screening algorithms have not been defined. Similarly, testing for telomerase with multiple assay systems (TRAP, in situ TRAP, hTERT expression analysis) may increase the sensitivity of cancer detection, but potentially at the expense of decreased specificity. The optimal testing strategy, using one or a combination of telomerase detection methods, has yet to be defined.
Alternative detection methods, such as ELISA assays for the detection of the hTERT subunit, would potentially address some of the technical difficulties. Using the TRAP assay, researchers have measured telomerase activity in ascites, pleural effusions, bronchial lavage and washings, urine, bladder washings, and oral rinses. In all cases but the last, the TRAP assay was more sensitive than standard cytology in identifying patients with cancer. In many cases, early cancer detection leads to more effective treatment options and therefore a higher patient survival rate.
Telomerase activity assays have yet to make an impact in the routine clinical laboratory. It is reasonable to ask why. One reason is technical. Few investigators have rigorously defined the stability of telomerase activity in clinical specimens. Hou et al. (36) found telomerase half-lives of 5–11 h in three cell lines; the half-life in body fluids is likely to be less. From an operational perspective, issues regarding sample transport and processing are likely to be critical. In addition, the available studies have not demonstrated that telomerase detection provides independent diagnostic or prognostic information. To date, decision algorithms incorporating telomerase assays have not been developed for any tumor type. Outcome studies will also be needed in this area.
It appears that telomerase measurements in easily obtained body fluids can play a useful role in the clinical laboratory for the diagnosis of cancer. Technical hurdles remain to be overcome, and the clinical utility of telomerase testing must be more rigorously defined before it can become routine in the clinical laboratory.
Footnotes
1 Nonstandard abbreviations: TRAP, telomere repeat amplification protocol; HCC, hepatocellular carcinoma; and PC, peritoneal carcinomatosis. 
References
The following articles in journals at HighWire Press have cited this article:
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  L. Terrin, E. Rampazzo, S. Pucciarelli, M. Agostini, R. Bertorelle, G. Esposito, P. DelBianco, D. Nitti, and A. De Rossi Relationship Between Tumor and Plasma Levels of hTERT mRNA in Patients with Colorectal Cancer: Implications for Monitoring of Neoplastic Disease Clin. Cancer Res., November 15, 2008; 14(22): 7444 - 7451. [Abstract] [Full Text] [PDF]
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V. D'Arcy, Z. K. Abdullaev, N. Pore, F. Docquier, V. Torrano, I. Chernukhin, M. Smart, D. Farrar, M. Metodiev, N. Fernandez, et al. The Potential of BORIS Detected in the Leukocytes of Breast Cancer Patients as an Early Marker of Tumorigenesis. Clin. Cancer Res., October 15, 2006; 12(20): 5978 - 5986. [Abstract] [Full Text] [PDF]
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 J. P. Jakupciak, P. E. Barker, W. Wang, S. Srivastava, and D. H. Atha Preparation and Characterization of Candidate Reference Materials for Telomerase Assays Clin. Chem., August 1, 2005; 51(8): 1443 - 1450. [Abstract] [Full Text] [PDF]
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G. I. Botchkina, R. H. Kim, I. L. Botchkina, A. Kirshenbaum, Z. Frischer, and H. L. Adler Noninvasive Detection of Prostate Cancer by Quantitative Analysis of Telomerase Activity Clin. Cancer Res., May 1, 2005; 11(9): 3243 - 3249. [Abstract] [Full Text] [PDF]
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 J. P. Jakupciak, W. Wang, P. E. Barker, S. Srivastava, and D. H. Atha Analytical Validation of Telomerase Activity for Cancer Early Detection: TRAP/PCR-CE and hTERT mRNA Quantification Assay for High-Throughput Screening of Tumor Cells J. Mol. Diagn., August 1, 2004; 6(3): 157 - 165. [Abstract] [Full Text]
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