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[Cancer Research 59, 3166-3170, July 1, 1999]
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[Cancer Research 59, 3166-3170, July 1, 1999]
© 1999 American Association for Cancer Research

Molecular Biology and Genetics

Telomerase Activity and Human Telomerase Reverse Transcriptase mRNA Expression in Soft Tissue Tumors

Correlation with Grade, Histology, and Proliferative Activity

Pu Yan, Jean-Michel Coindre, Jean Benhattar, Fredrik T. Bosman and Louis Guillou1

University Institute of Pathology, CH-1011 Lausanne, Switzerland [P. Y., J. B., F. T. B., L. G.]; and Bergonié Institute and University of Bordeaux II, 33076 Bordeaux, France [J-M. C.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Telomerase activity (TA) is detected in most human cancers but, with few exceptions, not in normal somatic cells. Little is known about TA in soft tissue tumors. We have examined a series of benign and malignant soft tissue tumors for TA using the telomerase repeat amplification protocol assay. Analysis of the expression of the human telomerase reverse transcriptase was also carried out using RT-PCR. TA was undetectable in benign lesions (15 of 15) and low-grade sarcomas (6 of 6) and was detectable in 50% (19 of 38) of intermediate-/high-grade sarcomas. Although the presence of TA in soft tissue tumors is synonymous with malignancy, it is neither a reliable method in making the distinction between reactive/benign and malignant (especially low-grade) lesions nor a reliable marker of tumor aggressiveness. Leiomyosarcomas and storiform/pleomorphic malignant fibrous histiocytomas rarely showed TA, irrespective of their grade. A strong correlation between human telomerase reverse transcriptase mRNA expression and TA was observed, supporting the close relationship between both parameters. No significant relationship was observed between proliferative activity (as assessed by MIB-1 immunolabeling) and TA. We verified that the absence of telomerase expression was not due to the presence of telomerase inhibitors and therefore alternative mechanism(s) for cell immortalization, yet to be determined, seem to be involved in the development and/or maintenance of some soft tissue sarcomas.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Telomerase is a complex enzyme containing both protein components and a RNA component. Its RNA subunit acts as a template for the synthesis of telomeric DNA, whereas a protein component, the hTERT2 (hEST2), catalyzes this process to make up for the inability of conventional DNA polymerase to replicate completely the ends of linear DNA (1 , 2) . TA has been detected in most human cancers as well as in some precancerous lesions and benign tumors (3, 4, 5) . It is usually not detected in normal somatic cells, except for normal human leukocytes (e.g., activated B and T lymphocytes), human germ-line tissues (adult testes and ovaries but not mature spermatozoa and oocytes), and proliferating stem cells (5) . Recently, based on a large number of samples, Meeker et al. (3) summarized the current data on TA in human cancers using the PCR-based TRAP assay. With the apparent exception of retinoblastoma, which is often telomerase negative (6) , the prevalence of TA in human cancers appeared to vary between 82 and 100% (3) .

Because of their rarity, ubiquitous location, and significant morphological diversity (>150 different histological types described thus far), soft tissue neoplasms are often a source of great difficulty in diagnostic pathology, especially when it comes to differentiating between benign and malignant lesions. TA has been shown to be a potential useful diagnostic tool for the detection of cancer (5) as well as a potential prognostic marker for selected tumors (7, 8, 9) . Apart from four sarcoma cases examined by Kim et al. (10) , TA has, thus far, not been systematically examined in soft tissue tumors.

Here, we examined a series of benign and malignant soft tissue lesions for TA using the TRAP assay and for hTERT mRNA expression. Because telomerase activation and hTR expression generally correlate with growth rate (11, 12, 13) , we also examined sarcomas for proliferative activity using Mib-1 immunolabeling. We showed that reactive lesions, benign tumors, low-grade sarcomas, and ~50% of intermediate-/high-grade sarcomas were devoid of TA. When present, the latter correlated with hTERT expression but did not correlate with proliferative activity. We also showed that leiomyosarcomas and storiform-pleomorphic malignant fibrous histiocytomas are predominantly telomerase-negative neoplasms.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tissue Samples.
From 54 patients, 59 frozen tissue samples, including 15 benign soft tissue lesions, and 44 STSs were retrieved from the tumor banks of the University Institute of Pathology of Lausanne, Switzerland, and from that of the Department of Pathology of the Bergonié Institute, Bordeaux, France. Histological typing and subtyping was performed on formalin-fixed, paraffin-embedded sections stained with H&E, using the histological classification of soft tissue tumors of the WHO (14) . Additional techniques, including immunohistochemistry and electron microscopy, were used for diagnostic purposes, if necessary. Histological grade was established using the updated version of the French Federation of Cancer Centers grading system (15) .

All tissue specimens were serially sectioned. Ten sections were cut at 5 µm, and the first and the last were stained with H&E for conventional microscopic examination to assess tissue preservation and the relative amount of tumor tissue in the specimen available. Any specimen containing <30% tumor cells was excluded from the series. Intermediate sections were cut at 12 µm and put into two chilled 1.5-ml Eppendorf tubes, using a tapered glass pipette, for extraction of protein and total RNA. Depending on the size of tissue samples, between one and five tissue sections provided sufficient material for protein extraction in 30 µl of CHAPS lysis buffer for determination of TA. For a sufficient amount of RNA, 5–10 sections were necessary.

Total RNA was extracted, and its quality was assessed as described previously (16) . Following extraction using Trizol (Life Technologies, Inc., Gaithersburg, MD) and ethanol precipitation in presence of 10 µg of glycogen, total RNA from soft tissue lesions was redissolved in 20 µl of RNase-free water. About 0.6–1.0 µg of total RNA was subjected to 1% agarose gel electrophoresis. Preservation of 28S and/or 18S rRNA species were used to assess RNA degradation. Samples in which 28S and/or 18S RNA was no longer detectable were not tested for TA.

TRAP Assay.
TA was determined using the TRAP assay with some modifications (17) . SW480 colorectal carcinoma cells were used as a positive control in the PCR amplification. To this end, cell pellets (~1x 105 cells) were suspended in 400 µl of CHAPS lysis buffer. Frozen tissue sections (one to five sections) were homogenized with 30 µl of CHAPS lysis buffer. After incubation for 30 min on ice, the lysate was centrifuged, and the supernatant was immediately frozen at -80°C and stored until use. Protein concentration of the extract was measured by the bicinchoninic acid protein assay kit (Pierce, Rockford, IL).

In every case, the TRAP assay was performed using three different concentrations of the protein extract: 0.1, 0.5, and 1.5 µg, respectively. The protein aliquot was incubated with 20 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 63 mM KCl, 0.005% Tween 20, 1 mM EGTA, 100 µM dNTPs, and 50 ng of TS primer (5'-AATCCGTCGAGCAGAGTT-3') in a thermocycler for 30 min at 30°C for the generation of telomeric repeats. After heating at 94°C for 2 min and cooling at 72°C, 1 unit of Taq DNA polymerase, 50 ng of ACX return primer [5'-GCGCGG(CTTACC)3CTAACC-3'], 50 ng of NT internal control primer (5'-ATCGCTTCTCGGCCTTTT-3'), and 0.01 amol of TSNT internal control (5'- AATCCGTCGAGCAGAGTTAAAAGGCCGAGAAGCGAT-3') were added to a total reaction volume of 25 µl. Then, 30 PCR cycles (94°C for 30 s, 56°C for 45 s, and 72°C for 45 s) were performed. Five µl of PCR product were electrophoresed on a 8% polyacrylamide nondenaturing gel. The gel was stained with SYBR Gold (Molecular Probes, Eugene, OR) and visualized under UV light using a charge coupled device camera. The TRAP assay included the amplification of an internal control of 36 bp; a false-negative result due to the presence of PCR/Taq DNA polymerase inhibitors was concluded when the 36-bp amplified product was not observed.

Analysis of hTERT Expression by RT-PCR.
Analysis of the expression of hTERT was carried out by RT-PCR. cDNA was synthesized from 5–10 µg of total RNA using random primers. To amplify the reverse-transcribed cDNA, we subjected 2-µl aliquots of cDNA to 40 PCR cycles (95°C for 30 s, 65°C for 45 s, and 72°C for 45 s) in a 20-µl volume containing 10x Taq buffer, 50 ng of upstream primer 5'-TTCCTGCACTGGCTGATGAGTGT-3', 50 ng of downstream primer 5'-CGCTCGGCCCTCTTTTCTCTG-3', 250 µM dNTPs, and 1 unit of Taq DNA polymerase (Boehringer Mannheim). The primers correspond to nucleotides 1686–1708 and 1994–2014 of the published hTERT cDNA sequence (18) . PCR products were analyzed on an 1.5% of agarose gel. The size of the hTERT PCR-amplified product was 329 bp. The quality of cDNA was controlled by PCR amplification of p53 and glyceraldehyde-3-phosphate dehydrogenase transcripts.

Detection of Telomerase Inhibitors.
Fourteen cases were examined for the potential presence of telomerase inhibitors. These included 4 sarcomas (cases 18, 19, 23, and 24; Table 1Citation ) with undetectable TA, despite hTERT mRNA expression, and 10 lesions (cases 16, 21, 22, 33, 42, 44, 49, 51, 54, and 58; Table 1Citation ) that were hTERT and telomerase negative. To this end, the TRAP assay was performed in parallel with cell extracts obtained from tumor tissue alone, tumor tissue mixed with 5 x 105 (5 µl) SW480 colorectal cancer cells, and SW480 cells alone. When only the first cell extract gave a negative TRAP assay (with the 36-bp internal control amplified), this was taken to indicate the absence of telomerase inhibitors in the tumor tissue extract.


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  		Table 1 Telomerase activity and hTERT mRNA expression in benign and malignant soft tissue tumors

 
Immunohistochemistry.
For all sarcoma cases, additional frozen sections were cut and stained with the Mib-1 antibody, recognizing the cell cycle-associated antigen Ki-67 (Immunotech SA, Marseille, France; diluted 1:10). Proliferation index was defined (after semiquantitative assessment of the staining) as low when <5% of the nuclei were stained, moderate when 5–15% of the nuclei were stained, and high when >15% of the nuclei were stained.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Fifteen reactive/benign lesions and 44 STSs were included in our study. Of the latter, six tumors corresponded to local sarcoma recurrences (cases 16, 18, 19, 24, 25, and 54), and nine corresponded to sarcoma metastases located in the lungs (cases 33, 34, 57, 58, and 59) and soft tissues (cases 28, 30, 47, and 55; see Table 1Citation ). From one patient, tissue samples from the primary tumor (case 23) as well as from a locally recurrent lesion (case 24) were available for examination. From another patient, the primary tumor (case 26) and two thoracic wall metastases (cases 30 and 47) were available, and from a third patient, a local recurrence (case 54) and a lung metastasis (case 33) could be analyzed. From one patient, we analyzed two lung metastases (cases 57 and 58). Two patients (cases 23 and 53) received neoadjuvant chemotherapy prior to tumor sampling. The distribution according to histological types and subtypes and histological grade is shown in Table 1Citation .

TA was undetectable in benign soft tissue lesions (including 14 tumors and 1 myositis ossificans), whereas it was observed in 19 of 44 (43%) STSs. None of the low-grade (grade 1), 9 of 16 (56%) of the intermediate-grade (grade 2), and 10 of 22 (45%) of the high-grade (grade 3) STSs showed TA (Table 1Citation ; Fig. 1Citation .). There was no correlation between histological grade and TA using the {chi}2 test (P = 0.06). Only 1 (case 52) of 10 leiomyosarcomas and 1 (case 59) of 7 storiform/pleomorphic malignant fibrous histiocytomas expressed TA, irrespective of tumor grade. Most (five of six) locally recurring sarcomas failed to display TA. Five sarcoma metastases of nine (56%) were telomerase positive. Two sarcoma patients showed concordant TA status between the primary tumor and the corresponding recurrence (cases 23 and 24) and between the primary tumor and the corresponding metastases (cases 26, 30, and 47).



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  		Fig. 1. TA using TRAP assay in benign and malignant soft tissue tumors. Selected examples of grade 3, grade 2, grade 1, and benign soft tissue tumors include an angiosarcoma (case 43), a round cell liposarcoma (case 29), an unclassified spindle cell sarcoma (case 21), and an hemangioma (case 5), respectively. The TRAP assay was performed using three different concentrations of the protein extract: 0.1, 0.5, and 1.5 µg. Lane 1, pGEM DNA size markers (Promega, Madison, WI).

 
hTERT mRNA was detected in 19 (44%) of 43 STSs, including 15 of 18 (83%; case 26 not available) telomerase-positive and 4 of 26 (15%) telomerase-negative cases. A strong correlation between hTERT mRNA expression and TA was demonstrated using the {chi}2 test (P < 0.0001). None of the benign lesions expressed hTERT mRNA. In three cases (cases 34, 38, and 41), marked TA was observed in the absence of detectable hTERT mRNA. A lack of expression of both enzyme activity and hTERT mRNA was observed in 21 of 43 sarcomas (49%; case 26 not available), recurrences and metastases included. Ten of these cases (cases 16, 21, 22, 33, 42, 44, 49, 51, 54, and 58) were examined for the presence of telomerase inhibitors, and all proved to be negative (Fig. 2.Citation ). In none of the four telomerase-negative, hTERT-positive sarcomas (cases 18, 19, 23, and 24) was the lack of TA due to the presence of telomerase inhibitors.



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  		Fig. 2. Detection of telomerase inhibitors. Protein extracts from a leiomyosarcoma (LMS; case 51) were successively examined prior to and after complementation with extracts from SW480 colorectal cancer cells. The latter showed TA and was used as a control (Lane 2). TA was not detected in the protein extract from the leiomyosarcoma when it was taken in isolation (Lanes 3–5). After complementation with SW480, TA was restored (Lanes 6–8), indicating that the lack of telomerase expression in the leiomyosarcoma was not due to the presence of telomerase inhibitors. The amplified 36-bp internal control acknowledged the absence of PCR/Taq DNA polymerase inhibitors. The TRAP assay was performed using three different concentrations of the protein extract: 0.1, 0.5, and 1.5 µg. Lane 1, pGEM DNA size markers (Promega).

 
The Mib-1 (Ki-67) labeling index was assessed in 41 STSs. Low and intermediate Mib-1 labeling indices were detected in 34 STSs (83%). High labeling indices (>15%) were observed in the high-grade (G3) tumor category only. Local recurrences and sarcoma metastases showed a Mib-1 labeling index of <=15% (see Table 1Citation .). There was no relationship between Mib-1 indices and TA (P = 0.33).


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
TA has been shown to be activated in the majority of epithelial cancers (5) and may provide a useful diagnostic or prognostic indicator not only in epithelial but also glial neoplasms (7 , 9 , 11 , 19) . Little is known about TA and its potential diagnostic and prognostic implications in soft tissue lesions. The purpose of this study was to clarify this issue.

TA was undetectable not only in all benign lesions but also in all low-grade sarcomas as well as in ~50% of the intermediate-/high-grade sarcomas. This indicates that, in all likelihood, a telomerase-positive tumor is a sarcoma, but when telomerase is negative, it has no value as a parameter for predicting behavior.

Before concluding, one must rule out the possibility of false-negative results. The TRAP assay used for the detection of TA is subject to limitations (16 , 17 , 20) and requires positive and negative controls. In this study, every frozen tissue specimen was histologically controlled before submission to be sure that it was qualitatively and quantitatively representative of the tumor. Tissue quality is of vital importance for the success of the telomerase detection assay. Recently, we developed a quality test in which the preservation of 28S and/or 18S rRNAs species is used to assess total RNA degradation (16) . Of the 99 mesenchymal lesions originally selected for this study, 59 only met the quality test requirements and were subsequently retained for analysis. A second reason for false-negative results might be the presence of Taq DNA polymerase inhibitors or telomerase inhibitors as illustrated in Hodgkin’s disease. Until recently, Hodgkin’s disease was thought to be a predominantly telomerase-negative lesion (21) . However, a recent study (22) showed that this apparent lack of TA was due to the presence of telomerase inhibitors, more specifically, eosinophil-associated RNases. The potential presence of inhibitors was carefully examined in this study. To detect Taq DNA polymerase inhibitors, we used the TRAP assay described by Kim and Wu (17) , which included the amplification of an internal control of 36 bp in each assay, and a false-negative result was concluded when the 36-bp amplified product was not observed. To detect telomerase inhibitors, we examined protein extracts from each specimen before and after spiking with telomerase-positive SW480 colorectal cancer cells. Absence of detectable TA in the spiked extract was taken to indicate the presence of telomerase inhibitors. No inhibitors could be detected using any of these methods.

A strong correlation between hTERT mRNA expression and TA was observed in 36 of 43 (84%) STS (P < 0.0001), hence supporting the close relationship between both parameters and the crucial role of the hTERT gene up-regulation in telomerase activation (18 , 23, 24, 25, 26) . Four hTERT-positive STSs failed to express detectable enzyme activity, and we showed that this was not due to the presence of telomerase/PCR inhibitors. The presence of alternately spliced hTERT transcripts deleted in critical regions of the reverse transcriptase (25 , 26) , abnormalities in the RNA template component of telomerase, or unbalanced levels of expression and/or posttranscriptionnal modifications of the different telomerase subunits (hTR, TLP1, and hTERT; Refs. 23 and 26 ) could account for enzyme inactivity and, thus, explain those discrepancies. TA was observed in the absence of detectable hTERT mRNA in three cases. In the latter situation, an increased degradation rate of the hTERT mRNA compared to that of the enzyme may explain this type of discordance.

A recurrent and crucial problem in diagnostic pathology is to differentiate true sarcomas from clinically benign but morphologically malignant-looking soft tissue lesions. Nodular fasciitis and myositis ossificans are prototypical examples of such lesions. All benign lesions of our series (including a case of myositis ossificans) were negative for telomerase. Because 57% of sarcomas were also telomerase-negative lesions (including malignant fibrous histiocytomas and leiomyosarcomas), we, therefore, conclude that TA cannot be used to distinguish a benign lesion from a malignant one. This observation is of particular importance for the pathologist because malignant fibrous histiocytoma and leiomyosarcoma are those sarcomas that are most likely to be confused with pseudosarcomatous lesions. Along the same line, TA cannot be used to separate a well-differentiated fibrosarcoma from a desmoid tumor or a well-differentiated "lipoma-like" liposarcoma from a conventional lipoma, all four lesions being telomerase negative.

In ~50% of the intermediate- and high-grade STSs, TA was undetectable. In addition, several locally recurring tumors and metastases did not express TA. This indicates that TA is not a reliable marker of aggressiveness in STS and cannot be used as a prognostic indicator in this tumor category, contrasting with what has been reported for epithelial neoplasms (9 , 19) , neuroblastoma (8) , meningioma (7) , and several other tumor types (5) . Although our series included a limited number of cases per histological category, which precludes definitive conclusions, telomerase activation might be histology related in being predominantly negative in leiomyosarcomas and malignant fibrous histiocytomas.

Neither TA nor hTERT mRNA was found in 21 of 43 (49%) STSs, including 2 recurrences and 4 metastases. This suggests that TA is not an essential prerequisite for sarcoma development nor for its metastatic dissemination. Similar observations were made for transplantable osteosarcomas (27) as well as for renal cell carcinoma (28) and retinoblastoma (6) . To explain the absence of TA in some tumors, despite optimal tissue preservation and absence of telomerase inhibitors, the existence of an alternative telomerase-independent mechanism for cell immortality via telomere lengthening has been suggested (10 , 29 , 30) . Although such a mechanism might be operative in some sarcomas and sarcoma cell lines (29 , 30) , it is unlikely to be universal. Indeed, a recent study (31) showed that about half of STS have short telomeres, whereas in only 17% of the tumors examined the chromosomes had elongated telomere repeats. In addition, this study also showed differences in telomere length patterns between primary tumors and recurrences. On the basis of the latter findings, telomere lengthening, whether it occurs through telomerase activation or not, is unlikely to play a dominant role in sarcoma development and maintenance. Additional studies focusing on the relationship between telomere length and TA in STS are needed to clarify this issue. Another explanation that would account for the lack of TA in a significant number of STSs is the fact that telomerase expression might be a field- and/or a time-dependent phenomenon. Indeed, intratumoral variations in telomerase expression have recently been documented in high-grade astrocytomas (12) , and it is conceivable that such a phenomenon occurs also in STSs. TA and hTR expression generally correlates with growth rate (11, 12, 13) , and one might suppose that the lack of TA in some STS is associated with low proliferative activity. Indeed, most of our telomerase-negative STS showed a low proliferation rate, as assessed by Mib-1 staining, suggesting a relationship between both parameters. However, this is hampered by the fact that 76.5% of telomerase-positive STS showed also low labeling indices (i.e., Mib-1-positive nuclei ratio of <=15%), hence precluding any conclusions.

In conclusion, we showed that TA and hTERT mRNA are not expressed in benign mesenchymal lesions. In STSs, TA is restricted to a subset of intermediate- and high-grade sarcomas, might be histology dependent, and, as yet, cannot be used as a diagnostic or prognostic tool.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed, at Institut Universitaire de Pathologie, 25 rue du Bugnon, CH-1011 Lausanne, Switzerland. Phone: 41 21 314 7111; Fax: 41 21 314 7115; E-mail: louis.guillou{at}chuv.hospvd.ch Back

2 The abbreviations used are: hTERT, human telomerase reverse transcriptase; TA, telomerase activity; TRAP, telomerase repeat amplification protocol; hTR, human telomerase RNA; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; STS, soft tissue sarcoma. Back

Received 12/ 2/98. Accepted 5/ 3/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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