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Histology (A.-C.G., C.M., S.G., C.d.B., J.-F.D., I.M.C., M.-C.M.), Endocrinology (C.D.), and Pathology Units (M.-C.N., J.R.), Université Catholique de Louvain Medical School, and Institut de Recherche Interdisciplinaire, Université Libre de Bruxelles (S.C., F.M.), B-1070 Brussels, Belgium; Service d’Anatomie et de Cytologie Pathologiques, Hôpital Ambroise Paré (B.F.), F-912100 Boulogne, France; and Academic Medical Center, University of Amsterdam (J.J.M.D.V.), NL-1012 Amsterdam, The Netherlands
Address all correspondence and requests for reprints to: Dr. M.-C. Many, Histology Unit, Université Catholique de Louvain, Medical School, UCL-5229, 52 avenue Mounier, B-1200 Brussels, Belgium. E-mail: many{at}isto.ucl.ac.be.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cancers are generally detected as cold nodules on thyroid scintiscans, suggesting alterations in Tg iodination besides other possible defects in iodine metabolism. Previous studies suggested that expression anomalies of some of the thyroid-specific proteins could occur in cancers, e.g. TPO (12), NIS (13), and pendrin (9), whereas TSH receptor (TSHr) expression remained unchanged (14).
The aim of the present study was to correlate the loss of Tg-iodinating properties in thyroid cancers with the expression of the various proteins involved in intrathyroidal iodine metabolism to characterize the functional status of malignant thyroid disorders compared with benign conditions. This was performed by analyzing variations in the expression of Tg, T4-containing Tg (Tg-I), NIS, pendrin, TPO, ThOXs, and TSHr.
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Materials and Methods |
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Ninety-five samples of normal human thyroids and the same number of pathological cases were examined. They included 13 hot nodules (divided into two groups, five from patients receiving an antithyroid treatment and eight from patients without treatment), 11 multinodular goiters (MNG), eight adenomatous goiters, five follicular adenomas, seven insular carcinomas, 13 follicular carcinomas, two metastases of follicular carcinomas, nine follicular variants of papillary carcinomas, 16 papillary carcinomas, one metastasis of a papillary carcinoma, eight Hurthle cell adenomas, and two Hurthle cell carcinomas. All samples were selected from a tissue bank of the pathology department. They were fixed in Bouin’s fluid or formaldehyde and embedded in paraffin. The type of fixative did not modify the immunohistochemical results. Ethical rules were respected according to guidelines of the ethical committee of Université Catholique de Louvain.
Immunohistochemical analysis of thyroid-specific proteins
Sections (5 µm) were laid on SuperFrost glass slides (Menzel-Glaser, Brauschweig, Germany) and used for the detection of thyroid-specific proteins (Table 1
): Tg (polyclonal antibody, DAKO, Glostrup, Denmark), Tg-I (B1, monoclonal antibody) (15), NIS (monoclonal antibody) (16), pendrin (polyclonal antibody, gift from I. Royaux), ThOXs (polyclonal antibody raised against ThOX1 and ThOX2) (10), TPO (monoclonal antibody mAb47, gift from J. Ruf), and TSHr (polyclonal antibody, Novocastra, Newcastle, UK). Sections were dewaxed and rehydrated. Those used for NIS detection were pretreated in a microwave oven in Tris-citrate buffer (pH 9.5) for one cycle of 3 min at 750 watts and three cycles of 3.5 min at 350 watts.
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for incubating times and dilutions). After two washes in PBS-BSA, binding of antibodies was detected using a second antibody conjugated to a peroxidase-labeled polymer (EnVision detection, DAKO) for 30 min at room temperature. After two washes in PBS-BSA, peroxidase activity was revealed with the 3-amino-9-ethylcarbazole substrate (DAKO). Sections were counterstained with Mayer’s hematoxylin, rinsed, and mounted in Paramount (DAKO). The binding of anti-Tg antibody was detected with a second polyclonal antibody, whereas that of anti-Tg-I antibody was detected with a second monoclonal antibody. The peroxidase activity was here revealed with 3,3'-diaminobenzidine tetrahydrochloride (Aldrich, Bornem, Belgium). Sections were dehydrated and mounted in DPX (BDH, Poole, UK). Several negative controls were performed: absence of the first antibody or replacement of the first antibody with preimmune serum. Controls of the Tg-I immunostaining were performed by preincubating the first antibody with iodinated Tg, noniodinated Tg, or T4. Different dilutions (from 1:4000 to 1:250) of anti-Tg-I antibody were also tested to ascertain the absence of labeling in thyroid cancers. Controls were also performed before and after inhibition of endogenous peroxidase activity with H2O2.
For NIS, pendrin, and TPO immunostainings, the number of positive cells was counted in 10 microscopic fields at a magnification of x250 in five cases of normal thyroids and of follicular variants of papillary, papillary, follicular, and insular carcinomas. In these cases, immunostaining was indeed heterogeneous. The percentage of positive cells was then calculated. Statistical analysis was performed using ANOVA.
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Results |
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In all normal and pathological cases, the follicular lumina were stained with anti-Tg polyclonal antibody (Fig. 1, I. A–D), indicating that Tg was present in all types of carcinomas. It was also detected in metastatic thyroid tissues (Table 2).
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, I. G and H). Metastases of follicular carcinomas and papillary carcinomas were also negative for Tg-I. In normal glands (Fig. 1, I. E), MNG, adenomatous goiters, and follicular adenomas (Table 2), all follicular lumina contained Tg-I. They were also strongly stained in eight of eight hot nodules obtained from patients without antithyroid medication (Fig. 1, I. F), whereas Tg-I was only detected in a few follicles in three of five cases of patients with hot nodules treated with strumazol. Hurthle cells adenomas were the sole noncancerous tissues to be entirely negative for Tg-I. Tg-I immunostaining may appear to be useful in the diagnosis of carcinomas.
Tg-I staining was inhibited when the first antibody was incubated with iodinated Tg or T4, but not with noniodinated Tg, confirming the specificity of this antibody (Fig. 1, I. I and J). In hot nodules, Tg-I labeling was not only intraluminal, but was also located in the cytoplasm of follicular cells. This cytoplasmic staining was inhibited by preincubation with iodinated Tg or T4 (Fig. 1, I. K and L).
Immunostaining of TSHr
In all normal (Fig. 1, I. M) and pathological tissues (Fig. 1, I. N and O), TSHr was detected in the thyrocytes. In follicular (Fig. 1, I. N) and papillary (Fig. 1, I. O) carcinomas, the intensity of staining was variable from one region to another in a given sample and between cases, but strong expression was often observed, for example in some papillae of papillary carcinomas.
Immunostaining of iodide transport markers: NIS and pendrin
In normal thyroids (Fig. 1, II. A) and multinodular goiters, NIS was observed at the basolateral pole of the cells, but the staining was highly heterogeneous between follicles and cells, among which 35% were stained (Table 3). In hot nodules (Fig. 1, II. B), NIS was also detected at the basolateral pole (Table 4), but its expression was strongly increased, and the majority of cells were positive. NIS was detected in eight of eight cases of adenomatous goiters and in three of three cases of follicular adenomas (Table 2). It was absent in carcinomas (Fig. 1, II. C and D), except in very rare cells (Table 3) in five of 10 follicular carcinomas, three of seven follicular variants of papillary carcinomas, and two of six insular carcinomas (Table 2). In our samples, NIS has never been detected in papillary carcinomas or in metastatic tissues. When NIS was expressed in carcinomas, it was often diffusely distributed in the cytoplasm (Table 4).
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) of follicular cells in normal thyroids (Fig. 1, II. E) and in MNG (Table 2). Its expression was slightly increased in hot nodules without treatment (Fig. 1, II. F), but only two of five treated hot nodules were positively stained (Table 2). In adenomatous goiters and follicular adenomas, pendrin was always strongly expressed. Pendrin remained weakly expressed at the apical pole of 64% of cells (Table 3) in six of six follicular carcinomas (Fig. 1, II. G), whereas it was absent in the other types of carcinomas (Fig. 1, II. H), except in a few cells of two of five follicular variants of papillary carcinomas, two of nine papillary carcinomas, and one of five insular carcinomas (Tables 2 and 3). Pendrin was never detected in Hurthle cell carcinomas and was seen in rare cells of one of two Hurthle cell adenomas. The basal transporter of iodide, NIS, is thus absent in all types of carcinomas, whereas the putative apical transporter, pendrin, is a marker that remains weakly present in carcinomas of follicular type.
Immunostaining of Tg iodination markers: TPO and ThOXs
TPO was detected at the apical pole (Table 4) of 92% of cells (Table 3) from normal thyroids (Fig. 1, II. I), but also in MNG and follicular adenomas. Its expression was strongly increased in hot nodules (Fig. 1, II. J) and in adenomatous goiters. In carcinomas, a strong staining was detected in 55% of cells of seven of seven insular carcinomas and in 13 of 13 follicular carcinomas where 26% of the cells were positive (Fig. 1, II. K, and Tables 2 and 3). However, the labeling was diffuse in the cytoplasm (Table 4). Two of 2 metastases from follicular carcinomas also expressed TPO. In follicular variants of papillary carcinomas, a weak staining for TPO was observed in 17% of the cells, in nine of nine cases (Tables 2 and 3). The localization was also cytoplasmic (Table 4). On the other hand, 16 of 16 papillary carcinomas (Fig. 1, II. L) remained negative for TPO, whereas staining could only be detected in some cells of Hurthle cell adenomas and carcinomas.
ThOX1 and ThOX2 were detected in normal thyroids (Fig. 1, II. M), in 11 of 11 MNG, in eight of eight adenomatous goiters, and in three of three follicular adenomas (Table 2). Their expression was slightly increased in five of five hot nodules without treatment (Fig. 1, II. N) and in three of five treated hot nodules. Its localization was mainly apical (Table 4). ThOXs immunostaining was weak in seven of seven insular carcinomas, in 11 of 11 follicular carcinomas (Fig. 1, II. O), and in seven of eight follicular variants of papillary carcinomas, whereas 13 of 13 papillary carcinomas (Fig. 1, II. P) showed strong staining, but diffusely located throughout the cytoplasm (Table 4). ThOXs were also detected in some cells of seven of eight Hurthle cell adenomas and in two of two Hurthle cell carcinomas (Table 2).
Papillary carcinomas thus appear to lose TPO expression, but to keep a strong expression of ThOXs in cytoplasm. They differ therefore from other carcinomas in which both TPO and ThOX expressions are maintained.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The major observation of our work is that all types of carcinomas lose their capacity to iodinate Tg. This has been demonstrated by the absence of specific immunodetection of Tg-I using B1 monoclonal antibody, whose specificity to recognize a high T4 content within the N-terminal Tg fragment was demonstrated by ELISA (15). This specificity was confirmed by the inhibition of immunostaining after prior incubation of the antibody with iodinated Tg or T4.
Another common modification occurring in all types of carcinomas that we have analyzed is the defect in iodide transport ascertained by the reduction of NIS expression. This is in agreement with previous studies that showed a decrease in or a loss of NIS protein expression in thyroid cancers (29, 30, 31, 32) and in NIS gene expression in papillary thyroid cancers (33). This last observation is in agreement with the absence of NIS immunostaining in our cases of papillary carcinomas. However, there are also opposite observations. There is one paper reporting an increased NIS expression in papillary carcinomas (34). In addition, recent immunohistochemical studies (35, 36) indicated that NIS protein is present mostly in the intracellular compartment in a high proportion (80%) of both follicular and papillary thyroid carcinomas. This raises a question about NIS activity when it is located in the cytoplasm. In the work by Dohan et al. (35) and Wapnir et al. (36), the percentage of differentiated carcinomas expressing NIS was higher than in our study (25%). This could be explained by the use of different antibodies. With our antibody, the intensity of the labeling seems to be weaker in a lower proportion of cells (35% in normal thyroids). Of note, staining for NIS was found in normal thyroid tissue adjacent to the tumor analyzed, excluding technical pitfalls during the immunostaining procedure. The pathological zone was also very heterogeneous, justifying the usefulness of counting the percentage of labeled cells in a large number of microscopic fields. It is crucial to determine the presence and function of NIS, because it has been shown (37) that NIS expression is associated with a lower risk of recurrence of thyroid carcinomas in children and adolescents.
Previous studies (9, 32) have shown a decrease in pendrin mRNA in thyroid cancers. However, our results have demonstrated labeling differences among carcinomas. Indeed, pendrin was expressed in 64% of cells of follicular carcinomas, although at a lower level than in normal thyroids, and was undetectable in papillary carcinomas. Similarly, TPO expression was maintained in follicular carcinomas and in follicular variants of papillary carcinomas, although in a scattered few cells (26 and 17%, respectively). It was considered absent in papillary carcinomas, with occasional labeling in 1.5% of cells. Differentiated carcinomas have been shown to express TPO mRNA (14). In about two thirds of them, TPO protein is expressed, but to a more variable extent than in normal tissue (38). When studying TPO expression by immunohistochemistry, results are influenced by the type of antibody used. As reported by de Micco et al. (12, 39), immunostaining with polyclonal antibody was positive in all benign tumors and in 50% of carcinomas. On the contrary, with the monoclonal antibody mAb47, there was a reduction or an absence of TPO in a majority of differentiated carcinomas. Using the same antibody, we confirmed these observations, but we also noted differences between follicular and papillary carcinomas.
As TPO activity requires H2O2, we analyzed the expression of ThOX1 and ThOX2, which are known to be involved in H2O2 generation (10). ThOXs were detected in all types of carcinomas, and their expression was increased in papillary carcinomas. However, our results did not provide information about their biochemical activity and, hence, about H2O2 production. Indeed, ThOXs staining in carcinomas was mainly cytoplasmic. As recently suggested, this abnormal cytoplasmic location could be associated with the absence of H2O2 production (40).
The TSHr was expressed in nearly all normal and pathological samples. This is in agreement with previous reports that showed the persistence of TSHr in papillary carcinomas and with studies stating that TSHr is always present in thyroid epithelial cells even if they are dedifferentiated (41, 42). The persistence of TSHr expression indicates that tumor cells keep the ability to respond to TSH. This has therapeutic implications, as TSH administration could therefore stimulate iodide transport and improve radioiodine therapy of carcinomas (43).
In conclusion, differentiation properties of thyroid cells, namely the expression of proteins involved in the iodine metabolism, may be useful for the diagnosis of thyroid cancers. In contrast to adenomas, T4-rich iodinated Tg detected by B1 monoclonal antibody was absent in the follicular lumina of all types of carcinomas. The use of this antibody could therefore be of great interest in the distinction between follicular adenomas and carcinomas. To prove it, the number of cases should be obviously increased. However, the difference in T4-rich Tg expression between follicular adenomas and carcinomas is associated with modifications in the expression of other proteins involved in iodine metabolism. In follicular adenomas, as in other benign thyroid lesions, NIS, pendrin, ThOXs, and TPO are expressed in all cases at the right subcellular position. On the contrary, in follicular carcinomas there is a strong reduction in both NIS and TPO expression. Moreover, TPO and ThOXs proteins are predominantly located in the cytoplasm rather than at the apical pole. The loss of the mechanism leading to Tg iodination seems to be different in carcinomas and in hypofunctioning untransformed follicles. In cold benign follicles, TSHr and NIS expression persisted, whereas the apical markers, pendrin, TPO, and ThOXs, vanished (44). In carcinomas, NIS expression consistently disappeared. The expression of apical markers depended on the type of tumor, suggesting that defects in Tg iodination may have different causes. In follicular carcinomas, this is mainly due to the loss of iodide transport from blood across the basolateral membrane, as the expressions of pendrin, TPO, and ThOXs are maintained, although in a reduced number of cells for TPO and NIS and with an odd cellular targeting for both TPO and ThOXs. By contrast, pendrin and TPO are undetectable in papillary carcinomas, and the localization of ThOXs is also modified, with the protein being diffusely located in the cytoplasm. The immunohistochemical detection of pendrin, TPO, and ThOXs could therefore aid in the diagnosis of thyroid carcinomas and in distinguishing between follicular and papillary types.
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Footnotes |
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Part of this work was presented at the 26th Annual Meeting of the European Thyroid Association, Milan, Italy, 1999, and at the 12th International Thyroid Congress, Kyoto, Japan, 2000.
Abbreviations: I-, Iodide; MNG, multinodular goiter; NIS, Na+/I- symporter; Tg, thyroglobulin; Tg-I, T4-containing thyroglobulin; ThOX, thyroid oxidase; TPO, thyroperoxidase; TSHr, TSH receptor.
Received April 3, 2002.
Accepted June 23, 2003.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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