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Original Studies |
Laboratoire d’Anatomie Pathologique and CNRS (P.B., N.C., R.C.Z., G.D.), UPR8291, Centre Hospitalier Universitaire de Purpan, 31059 Toulouse Cédex; Service d’Endocrinologie (F.L.), Hôpital Haut Lévèque, Bordeaux; Service d’Endocrinologie (F.B.-B.), Centre Hospitalier Universitaire de Rangueil, Toulouse; and Laboratoire d’Anatomie Pathologique (H.T., J.-P.M.), CHU de Bordeaux, France
Address all correspondence and requests for reprints to: Pierre Brousset, M.D., Ph.D., Laboratoire d’Anatomie Pathologique, Centre Hospitalier Universitaire de Purpan, Place du Dr Baylac, 31059 Toulouse Cédex, France.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Thyroid carcinomas originating from the follicular epithelium are composed of three main groups with different histological and clinical features (10). Among them, follicular and undifferentiated carcinomas are more agressive than papillary carcinomas. Because all these tumors have different pathogenesis and clinical behavior, we sought to determine the patterns of telomerase activity in a series of thyroid carcinomas.
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Materials and Methods |
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T cell leukemia CEM and Ishikawa cell lines (8) were used as
positive controls for telomerase activity. Twenty samples of normal
thyroid tissues and 12 adenomas, 15 papillary carcinomas, 6 follicular
carcinomas, and 3 undifferentiated carcinomas were evaluated in this
study. Tumors were classified according to the World Health
Organization classification (10). Adenomas were classified as follows:
6 cases of microfollicular, 4 cases of macrofollicular, and 2 cases of
trabecular pattern. All cases of papillary carcinomas were thicker than
1 cm and 2 cases corresponded to a Hürthle cell variant (10). One
of the 3 cases of undifferentiated carcinomas was an insular variant.
All results are summarized in Table 1
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Protein concentrations were determined by the Bradford assay (Bio-Rad) by using BSA as internal control.
Telomerase (TRAP) assay
The procedure was similar to that described by Kim et al. (4) and Piatyszek et al. (11), slightly modified as already published (8). Briefly, step 1 consisted of extension of a telomerase substrate (TS) oligomer (5'AATCCGTCGAGCAGAGTT-3'), and step 2 consisted of a hot-start PCR amplification of the product using a reverse CX primer [5'-(CCCTTA)3CCCTAA-3'].
Two to 4 µL of CHAPS extracts (6 µg of protein) were tested in a
PCR tube in 50 µL of a reaction buffer containing 68 mmol/L Kcl, 1.5
mmol/L MgCl2, 20 mmol/L Tris-HCl (pH 8.3), 1 mmol/L EGTA, 0.05% Tween
20, 0.5 µmol/L T4 gene 32 protein (Boehringer, Mannheim, France), 50
µmol/L of each deoxynucleotide triphosphate (dATP, dTTP, dGTP, dCTP),
0.25 µL (
-32P)dCTP, 0.25 µL
(-32P)dTTP (10 µCi/µL each), 2.5 U Taq
DNA polymerase, 0.1 µg of primer TS, and 0.1 µg of primer CX.
The tube was incubated 30 min at 23 C to allow for the extension of the TS primer by the telomerase. After elongation, the sample was heated at 94 C for 3 min. The PCR assay consisted of 30 cycles of 94 C for 30 sec, 50 C for 30 sec, and 72 C for 45 sec. Controls were included in each assay, as already described (8); positive control (1 lymphoma cell line) and negative controls: 1) predigestion of protein extracts from a positive control with RNase A (0.5 µg for 10 µL extract, 15 min at room temperature; 2) no protein; and 3) no CX primer. To assess sensitivity of the TRAP assay and to rule out the presence of telomerase or Taq polymerase inhibitors, protein extracts from a positive control (Ishikawa lymphoma cell line) were diluted at 1:10, 1:100, 1:250, 1:500, 1:750, and 1:1000 in protein extracts from negative cases. A signal at 1:1000 corresponded to the detection of the activity of 10 positive cells in a background of 104 negative cells.
PCR amplification products were analyzed on 10% polyacrylamide gel exposed directly to an autoradiographic film (Kodak, X-OMAT, Rochester, NJ) using intensifying screens.
Alkaline phosphatase activity
The stability of alkaline phosphatase seems to be similar to that of telomerase (11). Thus, this enzyme is a useful independent marker to determine the integrity of telomerase extracts. We investigated the activity of alkaline phosphatase on frozen sections obtained from TRAP-positive and TRAP-negative tumors. Frozen sections were briefly fixed in acetone for 5 min and then incubated with a solution of New fuschin chromogen (Sigma, France) to reveal the activity of the enzyme. A red precipitate was seen in endothelial cells of virtually all blood vessels, and this signal could be abolished by a 15-min incubation with a solution of levamisole (1 mg in 0.05 N Tris-HCl, pH 8.7).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and shown in Fig. 1. Telomerase activity was not detected
in normal thyroid tissues (0/20). Three of 15 cases (20%) of papillary
carcinomas were positive for the TRAP assay. None of the two cases of
papillary carcinomas with Hürthle cells were positive. There was
no significant difference regarding the size or the differentiation of
the tumors, when considering the results of the TRAP assay. Similarly,
the presence of metastases could not be correlated to the detection of
telomerase activity, because only 1 of the 4 papillary carcinomas with
cervical lymph node involvement was TRAP-positive. Four of 6 cases of
follicular carcinoma (66%) displayed detectable levels of telomerase
activity. The intensity of the signal varied from one case to another.
Two of 3 cases (66%) of undifferentiated carcinomas were positive for
telomerase activity. The one case of the insular type was TRAP
positive.
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), whereas all other cases, irrespective of their
subtype, remained completely negative. The positive case had no
particular morphological and clinical aspect. It was difficult to establish a grade in telomerase activity, but this activity varied from case to case. For example, all cases were checked morphologically, and we observed slight differences in the density of cells, from one tumor to another, that may explain variations in telomerase activity. However, the density of tumor cells was similar, overall, in TRAP-positive and TRAP-negative cases within the same histological subtype.
We checked to verify that there was no display of telomerase inhibitors
in TRAP-negative cases. It was possible to detect the activity from 10
cells of the TRAP-positive lymphoma cell line diluted in a background
of 9990 cells from TRAP-negative thyroid tumors (dilution 1:1000; Fig. 2). There was no difference in the
activity of alkaline phosphatase on frozen sections from TRAP-positive
and TRAP-negative tumors.
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Discussion |
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The results provide some evidence that telomerase is activated during thyroid carcinogenesis. This is not surprising, because the vast majority of cancers exhibit telomerase activity (4). Our study, however, suggests that telomerase activation is much less frequent in thyroid tumors, overall, compared with other types of cancers, and that telomerase activity is rather infrequent in the papillary subtype. Telomerase activation was frequently noticed in follicular and undifferentiated carcinomas, which are commonly more aggressive than papillary carcinomas (10). However, the numbers of the tumors tested here are small, and statements about the prevalence of telomerase activity in these cancers are difficult to make. Our data do not allow us to draw any definite conclusions, but we think that telomerase activity should be tested as a prognostic factor in thyroid cancers.
We think that the detection of telomerase activity may be helpful in differentiating dystrophic thyroid lesions from carcinomas and may be extended to cytological smears obtained after fine-needle aspirates of thyroid nodules. So far, cytodiagnosis seems poorly reliable and may be enhanced by the investigation of telomerase activity in cell suspensions. Such a combination has been validated for detecting cancer cells in breast (9) and in bladder washings and urine (12).
The finding of telomerase activity in one case of microfollicular adenoma is intriguing and raises important questions. First, this case was classified as microfollicular adenoma because it lacked morphological criteria of follicular carcinoma (10). The activation of telomerase in this lesion may indicate that the tumor contains cells undergoing a malignant transformation. Further experiments based on tissue microdissection may give some clues for understanding thyroid carcinogenesis. A strategy of selective cell analysis, from different areas of a given tumor, looking for a heterogeneous pattern of telomerase activity may yield interesting information.
In conclusion, we found that telomerase is not activated in normal thyroid cells, compared with other normal tissues such as breast (13). We observed that telomerase activity was more frequently associated with agressive thyroid tumors of follicular and undifferentiated subtypes than with papillary carcinomas. The finding of telomerase activity in one case of microfollicular adenoma opens the question as to whether some adenomas may contain dysplastic or malignant cell subpopulation.
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Footnotes |
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Received June 12, 1997.
Revised August 13, 1997.
Accepted August 22, 1997.
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