Evaluation of insulin-like growth factor II, cyclooxygenase-2, ets-1 and thyroid-specific thyroglobulin mRNA expression in benign and malignant thyroid tumours

OBJECTIVE: We evaluated three markers (insulin-like growth factor II (IGF-II), cyclooxygenase-2 (COX-2) and ets-1) of thyroid growth stimulation and cell transformation together with a thyroid-specific marker (thyroglobulin (Tg)) for their potential to differentiate benign and malignant follicular thyroid neoplasia (FN). DESIGN AND METHODS: mRNA expression levels were determined by real-time PCR in 100 snap-frozen thyroid samples: 36 benign thyroid nodules with different histology and function (19 cold (CTN) and 17 toxic thyroid nodules (TTN)), 36 corresponding normal thyroid tissues of the same patients, eight Graves' disease (GD) thyroids, 10 follicular thyroid carcinomas (FTC) and 10 papillary thyroid carcinomas (PTC). RESULTS: Mean IGF-II and COX-2 levels were not significantly altered between benign and malignant thyroid nodules (IGF-II) or nodular (FTC, TTN, CTN) and normal thyroid tissues (COX-2). In contrast, eight- to tenfold upregulation of ets-1 was observed in PTC and three- to fourfold upregulation of ets-1 was observed in FTC (and GD) compared with benign thyroid nodules and normal thyroid tissues. In addition, thyroglobulin mRNA expression was markedly downregulated (50- to 100-fold) in FTC, PTC and GD samples compared with benign nodular and normal thyroid tissues. Hence an ets-1/Tg ratio >20 distinguished differentiated thyroid cancer from benign nodular or normal thyroid tissue. We then studied ets1- and Tg mRNA expression levels in fine needle aspiration cytology (FNAC) samples. However, in a consecutive series of 40 FNAC samples only equivocal results were obtained on 38 benign and two malignant (FTC) thyroid tumour samples. CONCLUSIONS: Upregulation of ets-1 and downregulation of Tg mRNA expression occur in differentiated thyroid cancer and may facilitate pre-operative identification of thyroid malignancy depending on further evaluation of these potentially promising markers in a larger series of benign and malignant thyroid tumours and their FNAC samples.     

Characterisation of DEHAL1 expression in thyroid pathologies

Iodotyrosine dehalogenase 1 (DEHAL1) is a transmembrane protein involved in the recycling of iodide in the human thyroid. The aim of the present study was (I) to investigate whether DEHAL1 expression is different in differentially functioning thyroid pathologies and (II) to evaluate DEHAL1 as a possible marker of thyroid cell differentiation. DESIGN AND METHODS: Real-time PCR for DEHAL1 and its isoform DEHAL1B was performed in a series of 105 thyroid specimens, including toxic thyroid nodules (TTN), Graves' disease (GD) thyroids, benign cold thyroid nodules (CTN), normal thyroid tissues and thyroid cancers (follicular thyroid carcinomas (FTC), papillary thyroid carcinomas (PTC), partially differentiated thyroid cancers (PDTC) and anaplastic thyroid carcinomas (ATC)). In addition, DEHAL1 protein expression was studied by immunohistochemistry in 163 benign and malignant thyroid pathologies and normal thyroids. RESULTS: (I) The highest DEHAL1 mRNA levels were found in GD thyroids, while downregulation of DEHAL1 and DEHAL1B mRNA occurred in PTC and ATC (P<0.001 and <0.05 respectively); (II) DEHAL1 protein was overexpressed in TTNs and GD thyroids with predominant apical staining in all samples; (III) a weaker and patchy staining pattern was found in CTNs and normal thyroids; (IV) in differentiated thyroid cancers (FTC and PTC), a diffuse cytoplasmic DEHAL1 expression was found; and (V) in PDTC and ATC, DEHAL1 expression was faint or absent. CONCLUSION: Upregulation of DEHAL1 protein expression and sublocalisation of DEHAL1 at the apical cell pole in TTNs and GD thyroids is consistent with increased thyroid hormone turnover during thyrotoxicosis. Diffuse cytoplasmatic localisation or downregulation of DEHAL1 expression in thyroid cancers suggests alteration or loss of DEHAL1 function during thyroid cell dedifferentiation.     

Galectin-3 messenger ribonucleic acid and protein are expressed in benign thyroid tumors

Galectin-3 is a protein of the lectin family that has been associated with neoplastic processes in various tissues. In the thyroid, expression of this protein has been described in differentiated follicular cancer, suggesting that the immunohistochemical study of galectin-3 may be a potential marker of malignancy in thyroid neoplasms. The confirmation of these results may represent an extremely useful tool for presurgical diagnosis and medical conduct. In this study, galectin-3 protein and mRNA expression were analyzed in the thyroid tissues from 87 patients with histomorphological diagnosis of multinodular goiter (MNG) (n = 24), follicular adenoma (n = 31), follicular carcinoma (n = 20), papillary carcinoma (n = 12), and five normal tissues. Galectin-3 protein expression was detected by immunohistochemical method in light, fluorescence, and confocal microscopy, using monoclonal antibody. Galectin-3 mRNA expression was detected by the RT-PCR method. Our results showed that the majority of carcinomas expressed galectin-3 protein (follicular, 90%; papillary, 100%). However, in contrast to the previously published data, benign lesions also expressed galectin-3 (adenoma, 45%; MNG, 17%). We further demonstrated by RT-PCR that thyroid tissues with diagnosis of adenoma and MNG-expressed galectin-3 mRNA. Although the galectin-3 immunostaining demonstrated a sensitivity of 93.8% in the identification of cancer, the accuracy in the distinction between benign and malignant tissues was 77.0%. This accuracy was even lower (68.6%) when the galectin-3 expression in follicular adenoma was compared with follicular carcinoma. Thus, the use of galectin-3 immunodetection as a molecular marker for thyroid carcinoma must be interpreted with caution, particularly in the differentiation between thyroid follicular carcinoma and follicular adenoma.     

Differential expression of IgG Fc binding protein (FcgammaBP) in human normal thyroid tissue,thyroid adenomas and thyroid carcinomas

The genetic events involved in thyroid carcinogenesis are still incompletely understood. Several rearrangements and mutations of oncogenes have been implicated in the development of thyroid papillary carcinomas, follicular adenomas and carcinomas. However, none of these molecular alterations is suitable either as a general marker for the diagnosis of thyroid carcinomas or to differentiate between thyroid follicular adenomas and carcinomas. In order to identify new genes with altered expression which could serve as such markers, we analyzed RNA from thyroid tumor and normal tissue using a novel technique called restriction-mediated differential display. Several differentially expressed genes were identified, including the gene for IgG Fc binding protein (FcgammaBP). Differential expression of FcgammaBP was confirmed by quantitative real-time RT-PCR. Our experiments showed that IgG Fc binding protein (FcgammaBP) is differentially expressed in normal thyroid tissue, thyroid adenomas and thyroid carcinomas. While the FcgammaBP gene is constitutively expressed in normal thyroid tissue, its expression is significantly increased in follicular thyroid adenomas and significantly decreased in papillary and follicular thyroid carcinomas. Thus, measurement of the expression levels of FcgammaBP in thyroid biopsies might help to make the otherwise difficult distinction between a thyroid follicular adenoma and a follicular carcinoma.     

Sodium/iodide symporter (NIS) and pendrin are expressed differently in hot and cold nodules of thyroid toxic multinodular goiter.

OBJECTIVE: The expression of two iodide transporters, the sodium/iodide symporter (NIS) and pendrin, was analyzed in thyroid tissues of patients with toxic multinodular goiter (TMNG) and non-toxic multinodular goiter (MNG). METHODS: The levels of NIS and pendrin proteins were analyzed in total protein extracts from nodular and non-nodular tissues by Western blot. RESULTS: In tissue samples from TMNG, we found an increased expression of NIS (2.5-fold) in the hot nodules, and similar levels between cold nodules and non-nodular tissues. In contrast, the levels of pendrin were slightly increased in both hot and cold nodules from TMNG, and decreased (about twofold) in cold nodules from MNG. We also noticed that there was no relationship between NIS and pendrin expression. CONCLUSIONS: Our data demonstrate that hot nodules from TMNG express a higher number of iodide transporters (mainly NIS), whereas cold nodules from TMNG, but not from MNG, show levels of the two proteins comparable with normal tissue, suggesting a role in vivo of TSH in maintaining the expression of NIS and pendrin protein in normal thyroid tissue. Finally, different mechanisms are involved in the regulation of NIS and pendrin expression.     

Molecular profiling distinguishes papillary carcinoma from benign thyroid nodules

Recently we identified a molecular basis for differentiating benign and malignant follicular thyroid tumors. The purpose of these studies was to determine whether molecular analysis can be used to differentiate papillary thyroid carcinomas from benign thyroid nodules. Gene expression patterns of 14 papillary thyroid carcinomas and 21 benign tumors were analyzed by oligonucleotide array analysis. The carcinomas included seven classical papillary thyroid carcinomas (PTC) and seven follicular variant of PTC (FVPTC), and the benign tumors included 14 follicular adenomas and seven hyperplastic nodules. A hierarchical clustering analysis was performed to examine the groups for potential differences. The combined PTC and FVPTC groups had a distinct gene expression profile compared with the benign lesions. The sensitivity for a diagnosis of carcinoma was 93%, with a 100% specificity (one FVPTC clustered with the benign nodules). Cancer gene profiles contained both known (Met and galectin-3) and previously unidentified genes. Gene profiling is a reliable means of distinguishing PTC, FVPTC, and benign tumors of the thyroid. These gene profiles may provide insight into the pathogenesis of papillary thyroid carcinoma and may ultimately enhance the preoperative diagnosis of thyroid nodules on a molecular basis.     

Determination of galectin-3 messenger ribonucleic Acid overexpression in papillary thyroid cancer by quantitative reverse transcription-polymerase chain reaction

Galectin-3, a lectin-family protein that appears to be involved in malignant transformation, has been reported to be an accurate immunohistochemical marker for thyroid cancer. However, immunohistochemistry is a subjective method that can be difficult to apply to cytologic specimens. Therefore, we sought to develop an objective and quantitative assay to measure galectin-3 mRNA in thyroid tissue to enhance potential clinical use of galectin-3 in the molecular analysis of thyroid nodules. In this study, total RNA from 37 snap-frozen thyroid tissue specimens was isolated from eight papillary and nine follicular thyroid cancers, six follicular adenomas, seven adenomatoid nodules, and seven normal thyroid lobes from patients undergoing thyroidectomy. Normalized levels of galectin-3 mRNA, expressed as picograms per nanogram GAPDH mRNA, were higher in papillary carcinomas (3327 pg/ng) and follicular adenomas (1314 pg/ng) than in thyroid normal tissue (426 pg/ng; P = 0.0012 and 0.032, respectively). Galectin-3 mRNA levels were also higher in papillary cancers than in adenomatoid nodules (P = 0.0012). However, galectin-3 mRNA levels were not statistically greater in follicular carcinomas than either normal tissue or follicular adenomas (P = 0.068 and 0.12, respectively). In summary, in comparison to galectin-3 immunohistochemistry, quantitative measurement of galectin-3 mRNA appears useful in the identification of papillary thyroid cancers (PTCs) but does not appear to be useful in distinguishing follicular carcinomas from follicular adenomas.     

Negative correlation between thyroperoxidase and dual oxidase H2O2-generating activities in thyroid nodular lesions

Iodine incorporation into thyroglobulin is dependent on the activities of both thyroperoxidase (TPO) and thyroid dual oxidase 2 (DuOx2). Although TPO expression is decreased in some thyroid nodular lesions, DuOx1 and 2 mRNA expressions are maintained, but DuOx H2O2-generating activity has never been evaluated in such tumors. Our goal was to determine DuOx activity in hypofunctioning lesions of the thyroid. We evaluated H2O2 generation by DuOx in 12 paranodular to cold nodule samples, 17 non-toxic multinodular goiters (MNG; 33 samples), 3 papillary carcinomas (PC; 4 samples), 3 follicular carcinomas (FC; 4 samples), and 10 follicular adenomas. DuOx activity was detected in all paranodular tissues (121+/-23 nmol H2O2/h per mg protein), but was undetectable (<1 nmol H2O2 generated) in all PC, two out of four FC samples and seven out of ten adenomas. In 11 MNG at least two different areas of the goiter have been evaluated, and in 5 of these goiters one of the samples had DuOx activity below the limit of detection. The coefficient of variation in MNG samples ranged from 11.3 to 57.2%. Interestingly, in all the adenomas studied, TPO activity (486+/-142 U/g protein, n=8) was well within the range found in paranodular tissues (414+/-116 U/g protein, n=3). We found a significant negative correlation between DuOx and TPO activities, suggesting that these enzymes are regulated in opposite directions, at least in thyroid tumors.     

Expression of tpo mRNA in thyroid tumors: quantitative PCR analysis and correlation with alterations of ret, Braf , ras and pax8 genes

Immunocytochemistry (ICC) of thyroid peroxidase (TPO) using the monoclonal antibody MoAb47 has been used as malignancy marker on thyroid fine needle aspiration. However, little is known about the fate of TPO in thyroid carcinoma. We performed a qualitative PCR (Q-PCR) analysis to measure the expression of variants of tpo mRNA in 13 normal tissue samples, 30 benign tumors (BT), 21 follicular carcinomas (FC), 20 classical papillary carcinomas (PCc), 12 follicular variants of papillary carcinomas (PCfv) and nine oncocytic carcinomas (OC). We also studied mutations involving the ras, Braf, ret or pax8 genes. Results of Q-PCR were closely correlated with those of ICC (P < 0.0001; R = 0.59) and showed that overall tpo expression was lower in all carcinomas than in normal and BT (P < 0.05). The ratio tpo2 or tpo3 to tpo1 was inversed in follicular tumors. Genetic mutations were observed in 90% of PCc, 61.9% of FC, 41.7% of PCfv, 0% of OC and 10% in BT. pax8-ppar gamma1 rearrangement was correlated with qualitative changes in tpo mRNA (P < 0.01). These results confirmed the decrease of TPO expression in 97% of thyroid carcinomas regardless of histological type and the overexpression of shorter splice variants in follicular tumors. Both reduction in quantity of TPO and impairment of its maturation process could account for the atypical immunohistochemical reaction of MoAb47 with TPO.     

Gangliosides and phospholipids in human thyroids responsive and unresponsive to thyrotropin

The patterns of gangliosides and phospholipids and their relation to TSH response were examined in twenty-six malignant thyroid tumors (4 follicular, 6 papillary, 5 medullary, 11 anaplastic carcinomas) and thirty-six hyperplastic goiters. Thirteen thyroid tissues adjacent to benign tumors with no evidence of macroscopic or microscopic abnormalities were used as normal tissue. In normal thyroids the major ganglioside was GD3 (44%) and GM3 was the second ganglioside (20%). In minor amounts GD1a (8.6%), GD1b (6.2%), GT1b (5.7%) and GM1 (5.6%) were present. In hyperplastic goiters and in follicular carcinomas the patterns of gangliosides were similar to that of normal tissue except for GM3 which, in the last tissue, was higher (34%). In papillary carcinomas low levels of GM3 (11%) and GT1b (0.8%) with a high level of GD1b (12.6%) were found. In anaplastic carcinomas GM3 was very high (47%) whereas GD3 was low (18%). In these tumors also a high percentage (14.0%) of GD1a was found. In medullary carcinomas the lowest levels of GM3 (4%) with the highest level of GD3 (64%) were found. Although large differences of the gangliosides distribution were clearly encountered in the various pathological human thyroid, no correlation between lack of TSH response and some individual ganglioside could be made. No differences in the individual phospholipid in the various tissues studied were found.     

Iodine and cancer.

Thyroid carcinomas are the most frequent endocrine malignancies. Among thyroid carcinomas the most frequent types are the differentiated forms (follicular, papillary or mixed papillary-follicular), whereas anaplastic thyroid carcinoma and medullary thyroid carcinomas are rare. Animal experiments have demonstrated a clear increase in incidence of thyroid epithelial cell carcinomas after prolonged iodine deficiency leading to a situation of the thyroid gland by thyrotropin and possibly other growth factors. However, the overall incidence of differentiated thyroid carcinoma is generally not considered to be influenced by the iodine intake of a population, whereas the distribution of the types of thyroid carcinoma seems to be related to the intake of iodine, with fewer of the more aggressive follicular and anaplastic carcinomas and more papillary carcinomas in iodine rich areas. Populations starting iodine prophylaxis demonstrate an increase in the ratio of papillary to follicular carcinoma. Because a population with higher iodine intake usually has fewer benign nodules in the thyroid gland and the incidence of thyroid carcinomas is similar to an iodine-deficient region, the diagnostic work-up of nodules in the thyroid gland may become affected. The incidence of other cancers, such as breast cancer, may be influenced by the iodine intake, but too few studies are available at present. The present article summarizes available data from both epidemiological studies, animal experiments, and basic gene transfection studies. The overall incidence for a relationship between iodine and cancer is poor and future studies are warranted.     

Immunohistochemical detection and localization of a 72-kilodalton heat shock protein in autoimmune thyroid disease.

Recently described immunological functions for heat shock proteins (HSPs) and our previous demonstration of site-selective HSP-72 expression in cultured fibroblasts derived from extrathyroidal manifestations of Graves' disease (GD) prompted us to determine whether expression of the inducible 72-kilodalton HSP can be detected in human thyroid tissues. Immunohistochemistry was performed on formalin-fixed paraffin-embedded thyroid tissue specimens from patients with GD, Hashimoto's thyroiditis (HD), and multinodular goiter (MNG) as well as on normal thyroid tissue. A mouse monoclonal anti-HSP-72 antibody and an ultrasensitive avidin-biotin-peroxidase complex detection system were used for these studies. Striking differences in HSP-72 immunoreactivity were detected both between tissues from GD and HD compared with MNG and normal thyroid and between GD thyroid glands treated preoperatively with antithyroid medication and untreated GD glands. Strong HSP-72 reactivity in GD and HD tissues was detected in thyroid follicles as well lymphocytic infiltrates. No HSP-72 reactivity was detected in MNG or normal thyroid tissue. HSP-72 immunoreactivity was markedly reduced in GD glands that received preoperative antithyroid drug treatment. In conclusion, high levels of HSP-72 expression in autoimmune thyroid disease may reflect a state of chronic cellular stress, but could also represent an immunomodulatory factor of relevance in the autoimmune process in GD.     

Transforming events in thyroid tumorigenesis and their association with follicular lesions

Thyroid tumors comprise a broad spectrum of neoplastic phenotypes, and distinct molecular events have been implicated in their pathogenesis. Pituitary tumor transforming gene, originally isolated from GH(4) pituitary cells, is tumorigenic in vivo, regulates basic fibroblast growth factor secretion, and is homologous to a securin inhibitor of chromatid separation. Pituitary tumor transforming gene 1 is expressed at low levels in several normal human tissues and is abundantly expressed in neoplasms, including colorectal carcinoma, where pituitary tumor transforming gene expression correlated highly with tumor invasiveness. As pituitary tumor transforming gene is regulated by E and as thyroid cancer shows a strong female preponderance, we examined pituitary tumor transforming gene 1 expression and action in human thyroid tumors and in normal human and rat thyroid cells. Increased pituitary tumor transforming gene 1 expression was evident early in thyroid tumors and was most abundantly expressed in a subset of thyroid hyperplasia, follicular adenomas, and follicular carcinomas (1.8-fold; P < 0.0001). Pituitary tumor transforming gene 1 overexpression in rat FRTL5 thyroid cells and in primary human thyroid cell cultures causes in vitro transformation and produces a dedifferentiated neoplastic phenotype. As pituitary tumor transforming gene 1 was abundantly overexpressed in follicular adenoma and follicular carcinoma, we propose that pituitary tumor transforming gene overexpression may play a role in the early molecular events leading to divergent development of follicular and papillary carcinoma.     

Estrogen receptor (ER)-beta, but not ER-alpha, is present in thyroid vessels: immunohistochemical evaluations in multinodular goiter and papillary thyroid carcinoma.

OBJECTIVE: Estrogen receptors (ERs) have been demostrated in the vessel structures of several systems. Little is known on the presence of ERs in the thyroid vessels. DESIGN: We immunohistochemically evaluated both ER-alpha and ER-beta immunoreactivity (IR) in both vascular and follicular thyroid cells in tissue samples from 17 cases of multinodular goiter (MNG) and 17 cases of papillary thyroid carcinoma (PTC). MAIN OUTCOME: ER-alpha IR was undetectable in either tissue examined. In 100% of MNG samples, nuclear ER-beta IR was detected in both endothelial and follicular cells. In PTC samples, endothelial nuclear ER-beta IR was found in 100% of cases, whereas the nuclear staining of follicular cells was found in 83% of cases. The intensity of staining of the endothelial ER-beta IR was comparable between MNG and PTC. However, when follicular cells were considered, a tendency toward a decrease in nuclear staining and a significant increase in cytoplasmic staining were found in PTC lesions as compared to MNG. CONCLUSIONS: This study demonstrated that ER-beta, but not ER-alpha, IR is present in the endothelium of thyroid vessels. Furthermore, although data need to be confirmed in larger observations, these results suggest the lack of differences in the pattern of vascular ER-beta IR between MNG and PTC.     

Correlation of cytoplasmic beta-catenin and cyclin D1 overexpression during thyroid carcinogenesis around Semipalatinsk nuclear test site.

The Semipalatinsk nuclear test site (SNTS), the Republic of Kazakhstan, has been contaminated by radioactive fallout. The alteration of oncogenic molecules in thyroid cancer around the SNTS was considered worthy of analysis because it presented the potential to elucidate the relationship between radiation exposure and thyroid cancer. This study aimed to analyze both beta-catenin and cyclin D1 expressions in thyroid carcinomas around the SNTS. We examined nine cases of chronic thyroiditis, eight cases of follicular adenomas, and 23 cases of papillary carcinomas. Immunohistochemically, all carcinomas displayed a strong cytosolic beta-catenin expression, while both chronic thyroiditis and follicular adenomas showed a significantly lower cytoplasmic beta-catenin (22.2% and 37.5%, respectively). No cyclin D1 immunoreactivity was evident in chronic thyroiditis. In contrast, 62.5% of follicular adenomas and 87.0% of papillary carcinoma showed cyclin D1 overexpression. Additionally, a strong correlation between cytoplasmic beta-catenin and cyclin D1 expression was suggested in thyroid tumors. This study revealed a higher prevalence of both aberrant beta-catenin expression and cyclin D1 overexpression in papillary thyroid cancers around the SNTS than sporadic cases. The analysis of the alteration of the Wnt signaling-related molecules in thyroid cancer around the SNTS may be important to gain an insight into radiation-induced thyroid tumorigenesis.