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Molecular Oncology, Markers, Clinical Correlates |
Departments of Pathology [T. G., R. S-S., C. H., A. R.] and General Surgery [M. P., H. U. S., H. L.], Otto-von-Guericke University, 39120 Magdeburg, Germany
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Because of the biological behavior of GISTs, both pathologists and surgeons are faced with problems related to diagnostics and therapy. Golden and Stout (1) were the first to describe mesenchymal tumors of the gut. Because of the cellular characteristics of these tumors Stout (2) interpreted them as myogenic neoplasms and introduced the term "leiomyoblastomas" for those tumors composed of epithelioid cells (3) . In 1969, electron microscopy revealed that not all tumors described by Stout (2) showed myogenic characteristics (4) . By contrast, there were indications of Schwann cells, which are suggestive of a neurogenic origin (5) . About 20 years later, with the help of immunohistology, neurogenic (6) as well as myogenic (7) markers could be found.
Partially, the marker profile depends on the localization of these tumors in the gut (8 , 9) . The frequent observation of a positive immunohistological reaction with the CD34 antibody, with the majority of GISTs being positive for this hematopoetic progenitor cell antigen, allowed the conclusion that these tumors are neoplasms originating from primitive mesenchymal cells (10) . Recent investigations, which meanwhile have been corroborated by molecular genetic investigations, show that GISTs have common features with the interstitial cell of Cajal, the so-called pacemaker cells of the gut (11 , 12) . In these cells, CD117 is expressed as the product of the c-kit proto-oncogene and plays a role in the development of this cell type (13) . Sarlomo-Rikala et al. (14) pointed out that the CD117 antibody is a useful marker for diagnosing GISTs and for separating them from true leiomyomas and neurogenic tumors. To date, only a few studies have investigated molecular genetic markers for the characterization of the biological behavior of GISTs.
The importance of telomerase has already been validated concerning the expression and progression of numerous malignant epithelial tumors (15, 16, 17, 18) . This enzyme regulates the de novo synthesis of the telomeres, the ends of eukaryotic chromosomes, protecting them from loss, fusion, and degradation (19 , 20) . Telomeres consist of thousands of copies of the single hexanucleotide repeat TTAGGG.
With each cell division, the chromosomal ends undergo shortening as long as they have reached a critical length, leading to apoptosis (21) . Hence, telomeres virtually constitute one of the life-maintaining mechanisms of the cell. If the chromosomal ends are renewed by activating telomerase, the cell will be immortal. This is thought to be one mechanism that guarantees the survival of particularly strongly proliferating tissue, such as malignant tumors (19) .
The aim of our study was to investigate telomerase activity and the expression of the telomerase subunits: hTR and hTRT, making a comparison with extragastrointestinal, myogenic, or neurogenic sarcomas. Moreover, we addressed the issue of whether histologically, the biological behavior of the GISTs correlates with telomerase activity, and which clinicopathological factors show an association with the activation of this enzyme.
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PATIENTS AND METHODS |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Methods.
The tumor specimens were routinely processed at the Department of
Pathology, Otto-von-Guericke University. The tumors were classified
according to the grading system proposed by Lewin (22)
.
According to Amin et al. (23)
, the "cutoff
point" of the mitotic activity between benign and malignant was
determined as 5 mitoses per 50 high power field. The
extragastrointestinal sarcomas were graded according to the system
proposed by Trojani et al. (24)
. In addition to
routine preparation, further representative tumor materials from GISTs
and the extragastrointestinal sarcomas were prepared, snap-frozen
in liquid nitrogen, and stored at -70°C. To estimate the percentage
of tumor cells in H&E slides, tumor tissue corresponding to all
snap-frozen tumor samples was taken. To prevent false-negative results,
we used only those cases in which at least 80% of tumor cells were
seen. A dilutional effect on the DNA genetic analysis could thus be
excluded. Because the small GISTs could be enucleated very easily from
the adjacent tissue, there was no need for a microscopic dissection. In
the case of large tumors, tissue without necroses was obtained from the
center of the tumor.
Immunohistology.
The immunohistochemical reactions were performed on formalin-fixed and
paraffin-embedded tissue sections using the ABC (avidin-biotin-complex)
method with either peroxidase or alkaline phosphatase. Paraffin
sections were deparaffinized, rehydrated, and exposed to the primary
antibodies. The antibodies for NSE (Vector, Burlingame, CA; dilution,
1:9) and CD117 (Immunotech, Marseille, France; dilution, 1:80) were
diluted in a buffer containing 50 ml of RPMI 1640 (Life Technologies,
Inc., Eggenstein, Germany), 450 ml of distilled water, 50 ml of fetal
bovine serum (Life Technologies, Inc., Eggenstein,
Germany), and 0.5 g of sodium azide (Sigma Chemical Co.,
Deisenhofen, Germany; pH, 7.4–7.6). The other antibodies were diluted
in a commercially available dilution buffer (Ventana Medical Systems,
Tucson, AZ).
Stainings for Desmin (Immunotech; dilution, 1:150), SM-Actin (Immunotech; dilution, 1:150), S-100 (Dako, Hamburg, Germany; dilution, 1:500) and CD 34 (Biogenex, Hamburg, Germany; dilution, 1:20) were done automatically on the Ventana Nexes Immunostainer (Ventana Medical Systems) with peroxidase as detection system, whereas NSE and CD117 were stained manually at 37°C. Detection was done either by Ultratech-peroxidase (CD117) or Vectastain-alkaline phosphatase (NSE). For alkaline phosphatase, the following substrate solution was used and incubated for 25 min with gentle agitation: 44 ml of Tris buffer, 16 ml of propandiol, 0.025 g of levamisole, 0.125 g of newfuchsin, 0.3 ml of sodium nitrite (4%), 0.035 g of naphtol-as-bi, and 0.4 ml of dimethylformamide (all Sigma) at pH 8.7. For peroxidase, a commercially available diaminobenzidine solution was used (Immunotech), following the manufacturer‘s instructions. All substrate reactions were performed at room temperature. The sections were counterstained with Mayer’s hematoxylin and embedded in gelatin.
Determination of Telomerase Activity.
Frozen tumor tissue was homogenized in 200 µl of
3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate lysis buffer
(telomerase extraction buffer) on ice (25)
. After 30 min
of incubation on ice, the lysate was centrifuged at 16,000 x
g, and the supernatant was snap frozen in liquid nitrogen
and stored at -80°C. Protein concentration was measured by the
Bradford assay (Bio-Rad Laboratories, Richmond, CA). To exclude
variations in telomerase activity resulting from Taq polymerase
inhibitors present in the tissue extract, we tested a series of
dilutions (4, 0.4, and 0.2 µg/µl).
Telomerase activity was determined by using the ONCOR TRAP-eze telomerase detection kit (Oncor; Ref. 26 ). The following control reactions were done: (a) amplification of a 36-bp internal standard (provided in the kit) to identify false-negative samples containing Taq polymerase inhibitors; (b) a heat-inactivated control as a negative control; (c) a negative control without addition of a protein extract to exclude PCR-contamination; and (d) a TSR8-control, which should yield five to six signals (provided in the kit).
The TRAP assay was considered valid when all of the controls showed the expected values. To minimize the possibility of false-negative results because of tissue degradation, the suitability of the RNA in the protein extract was tested by RT-PCR amplification of a 495-bp fragment of the human ß-actin gene with the primers 5'-CATGCCATCCTGCGTCTGGAC-3' and 5'-CACGGAGTACTTGCGCTCAGGAGG-3'.
Cy5-labeled PCR products were electrophoresed on 0.5-mm thick polyacrylamide gels containing 7 M urea (Ready Mix; Pharmacia Biotech) on an automated sequencer (ALF Express; Pharmacia Biotech) and detected as a ladder of 6-base increments starting at 50 nucleotides: 50, 56, 62, 68, and so on. PCR and amplification conditions were described in detail by Schneider-Stock et al. (27) .
Interpretation of Data.
The definition of a genuine telomerase signal was any measurable signal
that was greater than the signal level measured from the same area in
the corresponding heat-inactivated control reaction. A value was
considered too low if the signal strength of telomerase-positive
reaction was equal or less than the signal strength of its
corresponding heat reaction. A value was considered positive if the
signal strength was at least twice the SD of its corresponding
heat-treated reaction. The TRAPeze telomerase kit uses a modified
reverse primer sequence that allows the estimation of telomerase
processivity. An extract was considered negative for telomerase
activity when the 36-bp band was revealed and no discernable ladder
pattern was found. Telomerase activity was interpreted as weak when
only the 50-bp band was seen. Telomerase activity was considered highly
positive when the 36-bp internal control band and a ladder of PCR
products with 6-base increments were visible.
Determination of hTRT and hTR mRNA Expression.
Total RNA was isolated by using TRIzol reagent (Life Technologies,
Inc., Gaithersburg, MD) and treated with DNase (Life Technologies,
Inc., Gaithersburg, MD). Reverse transcription was made according to
the manufacturer’s instructions (Promega Corp., Madison, WI). RT-PCR
conditions of hTRT and hTR were described by Schneider-Stock et
al. (28)
.
HTRT primers were: sense, 5'-CGGAAGAGTGTCTGGAGCAA-3'; and antisense, 5'-GGATGAAGCGGAGTCTGGA-3'. hTR primers were: sense, 5'-TCTAACCCTAACTGAGAAGGGCGTAG-3'; and antisense, 5'-GTTTGCTCTAGAATGAACGGTGGAAG-3'.
Amplification was performed in an automated thermal cycler (Multicycler PTC 200; MJ Research, Watertown, MA) for 31 cycles (hTRT) and 28 cycles (hTR), respectively. Cycling conditions were: 94°C for 45 s, 60°C for 45 s, and 72°C for 90 s. HTRT and hTR mRNA expressions were determined using a semiquantitative RT-PCR assay.
ß2-M mRNA was amplified to normalize equal amounts of RNA template using the primers and PCR conditions described in one of our previous studies (29) . A negative control reaction without cDNA was included in each experiment. PCR products were resolved on ultrathin native polyacrylamide gels cross-linked with piperazine diacrylamide, baked on GelBond Pag (FMC, Rockland, ME), and visualized by silver staining (30) . Bands were directly quantified with the use of laser densitometry (VDS; Pharmacia Biotech). Expression was evaluated by estimating the intensity ratio between the hTRT or hTR signal and the corresponding ß2-M signal. Normal placenta tissue of an adult woman was used as a negative control. The expression level was defined as normal in the telomerase-negative placenta tissue. In comparison with the placenta, 3-fold or more intensified signals were assessed as increased expression. The expressions of hTRT and hTR were reported as absent or present for the purpose of this study. Different expression levels were not classified. RNA was reverse transcribed twice, and each RT-PCR was done at least twice to ensure consistency. The NCI-H23 lung carcinoma cell line and human 293 cells were used as positive controls with known elevated hTRT mRNA expression and hTR mRNA expression (31) . All cell lines were obtained from the American Type Culture Collection (Rockville, MD).
Statistical Evaluation.
The significance of associations between genetic and immunohistological
findings and clinicopathological factors was evaluated statistically by
Fisher’s exact test (two tailed) and the 
2
test according to Pearson using the SAS statistical software system
(SAS Institute, Inc., Cary, NC). A probability of P <
0.05 was considered significant.
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RESULTS |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. In 13 cases, the primary
GISTs were localized in the stomach, and in the small intestine in
three. The mean age of the patients was 59.3 years (9–85 years).
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Almost all GISTs, independent of their localization, showed a strong immunoreaction with the CD34 and CD117 antibodies. Only two gastric stromal tumors (15.4%) expressed myogenic markers (sm-actin and/or desmin), whereas nine tumors (69.2%) showed a neurogenic marker profile (S100-protein and/or NSE) at the same time.
In all three intestinal stromal tumors, neurogenic as well as myogenic markers were expressed. The CD34 and CD117 antibody reactions were negative in these tumors.
Telomerase Activity in GISTs Compared with Extragastrointestinal
Sarcomas.
In 81% (17 of 21) of the malignant GISTs, in their recurrences and
liver metastases, telomerase activity was found. Sixty-seven % showed
high activity (Fig. 1)
. The gastric
stromal tumor with an uncertain malignant potential (case 2) showed
only low telomerase activity. There was no activity in two benign and
two malignant tumors. In one malignant stromal tumor of the small
intestine, the primary tumor showed no activity at first but displayed
a marked activity in its recurrence (case 12).
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; however, high activity was found in
only 18% of leiomyosarcomas (4 of 22) and in 48% of MPNSTs (10 of
21). Although there was no significant difference in the enzyme
activation in all tumor groups investigated in this study
(P = 0.683), GISTs showed a remarkable higher level of
telomerase activity (P = 0.024). There was no
association between telomerase activity and tumor grade and
localization of the extragastrointestinal sarcomas. Furthermore, an
association of telomerase activity with gender and the age of the
patients with GIST or extragastrointestinal sarcomas was not observed.
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On the other hand, hTRT was expressed in all GISTs that showed an
activation of telomerase (Fig. 3
; Table 2
). Compared with the other sarcomas,
there was a strong association between hTRT expression and activation
of the telomerase in GISTs (P < 0.0001). This
association could not be observed in leiomyosarcomas (P = 0.091) and MPNSTs (P = 0.231). In leiomyosarcomas,
the hTRT subunit was expressed only in those cases with high levels of
telomerase activity (Fig. 4)
. Eleven
leiomyosarcomas with weak telomerase activity did not express hTRT.
With some exceptions, the same situation was given in MPNSTs. In
six cases, telomerase was activated without hTRT expression. In only
one case of MPNST hTRT was expressed, but there was no telomerase
activity.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A further aim of our study was to check the expression of the telomerase subunits hTR and hTRT. Because it has been shown that the telomerase-associated protein TP1, the third subunit of telomerase, does not influence the enzyme activity (38) , and that only a posttranscriptional modification of the protein may affect telomerase activity (39) , we did not investigate our series of tumors for the expression of TP1.
hTR, the RNA component of telomerase, was always positive in all GISTs, independent of its biological behavior and the clinicopathological data, as well as in all MPNSTs and leiomyosarcomas. There was no correlation between the expression of hTR and telomerase activity. Although several investigators could almost always show an increased expression of hTR in malignant tumors (40, 41, 42) , there is obviously no correlation of hTR and the level of telomerase activity in each case (43) .
A certain correlation exists between telomerase activity and the expression of the catalytic subunit hTRT (44, 45, 46) . That hTRT is in fact a critical determinant in the activation of telomerase is reflected in the fact that there is a strong association between the expression of hTRT and telomerase activity in all GISTs. Both in leiomyosarcomas and MPNSTs, the relationship between hTRT expression and activation of telomerase was not seen in all cases. The leiomyosarcomas showed an expression of hTRT in those tumors having high telomerase activity. Telomerase activity was found in six MPNSTs not showing expression of hTRT. This discordance was explained by the theory that other factors may substitute for hTRT in conferring full telomerase activity (47) . Only one MPNST expressed hTRT without activation of telomerase. In this case, two possibilities were suggested: (a) posttranscriptional modification of the subunits could play a role in the regulation of telomerase activity; (b) different expression levels of each subunit and/or the balance of the expression levels between the subunits might play a critical role in determining enzymatic activity (47) .
In conclusion, compared with extragastrointestinal myogenic or neurogenic sarcomas, malignant GISTs express telomerase at a significantly higher level. Benign GISTs show no enzymatic activity. However, a close correlation between telomerase activity and hTRT does exist. That telomerase is reactivated only in a recurrence suggests that the activation of this enzyme is a late event and possibly related to tumor progression.
Although all soft tissue tumor entities investigated in this study resemble each other by conventional histology, there are obviously remarkable differences in the telomerase activity and in the expression of the telomerase subunits. In contrast to other soft tissue tumors, a final consensus on grading GISTs has not been achieved, and the biological behavior is often uncertain, leading to problems in the management of these tumors. Therefore, it is necessary to find new markers that are able to shed light on this matter. Telomerase activity and the expression of the telomerase subunit hTRT can give a hint to the tumor behavior. Further investigations are necessary to validate these results as a prognostic factor.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 The research work was supported by the Grant
10-1501-Schn2 from the Dr. Mildred-Scheel-Stiftung, Bonn, Germany, and
by Grant 97.011.2 from the Wilhelm-Sander-Stiftung, Munich, Germany. 
2 To whom requests for reprints should be
addressed, at Department of Pathology, Otto-von-Guericke University,
Leipziger Strasse 44, 39120 Magdeburg, Germany. Phone: 49-391-67-13182;
Fax: 49-391-67-15818; E-mail: Thomas.Guenther{at}Medizin.Uni-Magdeburg.de
3 The abbreviations used are: GIST,
gastrointestinal stromal tumor; hTR, human telomerase RNA; hTRT,
human telomerase reverse transcriptase; MPNST, malignant peripheral
nerve sheath tumor; NSE, neuron-specific enolase; TRAP,
telomeric repeat amplification protocol; RT-PCR, reverse
transcription-PCR; ß2-M, ß2-microglobulin.
Received 9/22/99; revised 1/28/00; accepted 2/ 1/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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  R. Schneider-Stock, C. Boltze, J. Lasota, B. Peters, C. L. Corless, P. Ruemmele, L. Terracciano, M. Pross, L. Insabato, D. Di Vizio, et al. Loss of p16 Protein Defines High-Risk Patients with Gastrointestinal Stromal Tumors: A Tissue Microarray Study Clin. Cancer Res., January 15, 2005; 11(2): 638 - 645. [Abstract] [Full Text] [PDF]
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