Characterization of autonomous thyroid adenoma: metabolism, gene expression, and pathology.

Fifty-one in vivo characterized autonomous single adenomas have been studied for functional parameters in vitro, for gene and protein expression and for pathology, and have been systematically compared to the corresponding extratumoral quiescent tissue. The adenomas were characterized by a high level of iodide trapping that corresponds to a high level of Na+ /iodide symporter gene expression, a high thyroperoxidase mRNA and protein content, and a low H2O2 generation. This explains the iodide metabolism characteristics demonstrated before, ie, the main cause of the "hot" character of the adenomas is their increased iodide transport. The adenomas spontaneously secreted higher amounts of thyroid hormone than the quiescent tissue and in agreement with previous in vivo data, this secretion could be further enhanced by thyrotropin (TSH). Inositol uptake was also increased but there was no spontaneous increase of the generation of inositol phosphates and this metabolism could be further activated by TSH. These positive responses to TSH are in agreement with the properties of TSH-stimulated thyroid cells in vitro and in vivo. They are compatible with the characteristics of mutated TSH receptors whose constitutive activation accounts for the majority of autonomous thyroid adenomas in Europe. The number of cycling cells, as evaluated by MIB-1 immunolabeling was low but increased in comparison with the corresponding quiescent tissue or normal tissue. The cycling cells are observed mainly at the periphery; there was very little apoptosis. Both findings account for the slow growth of these established adenomas. On the other hand, by thyroperoxidase immunohistochemistry, the whole lesion appeared hyperfunctional, which demonstrates a dissociation of mitogenic and functional stimulations. Thyroglobulin, TSH receptor, and E-cadherin mRNA accumulations were not modified in a consistent way, which confirms the near-constitutive expression of the corresponding genes in normal differentiated tissue. On the contrary, early immediate genes expressions (c-myc, NGF1B, egr 1, genes of the fos and jun families) were decreased. This may be explained by the proliferative heterogeneity of the lesion and the previously described short, biphasic expression of these genes when induced by mitogenic agents. All the characteristics of the autonomous adenomas can therefore be explained by the effect of the known activating mutations of genes coding for proteins of the TSH cyclic adenosine monophosphate (cAMP) cascade, all cells being functionally activated while only those at the periphery multiply. The reason of this heterogeneity is unknown.

Study of gene expression in thyrotropin-stimulated thyroid cells by cDNA expression array: ID3 transcription modulating factor as an early response protein and tumor marker in thyroid carcinomas.

Induction of cell proliferation by mitogen or growth factor stimulation leads to the specific induction or repression of a large number of genes. To identify genes differentially regulated by the cAMP-dependent transduction pathway, which is poorly characterized so far, we used the cDNA expression array technology. Hybridizations of Atlas human cDNA expression arrays with (32)P-labeled cDNA probes derived from control or thyrotropin (TSH)-stimulated dog thyrocytes in primary culture generated expression profiles of hundreds of genes simultaneously. Among the genes that displayed modified expression, we selected the transcription factor ID3, whose expression was increased by a cAMP-dependent stimulus. ID3 overexpression after TSH stimulation was first verified by Northern blotting analysis, and its mRNA regulation was then investigated in response to a variety of agents acting on thyrocyte proliferation and/or differentiation. We show that: (1) ID3 mRNA induction was stronger after stimulation of the cAMP cascade, but was not restricted to this signaling pathway, as phorbol myristate ester (TPA) and insulin also stimulated mRNA accumulation; (2) in contrast, powerful mitogens for thyroid cells, epidermal growth factor and hepatocyte growth factor, did not significantly modify ID3 mRNA levels; (3) ID3 protein levels closely parallelled mRNA levels, as revealed by immunofluorescence experiments showing a nuclear signal regulated by TSH; (4) in papillary thyroid carcinomas, ID3 mRNA was downregulated. Our results suggest that ID3 expression might be more related to the differentiating process induced by TSH than to the proliferative action of this hormone.

The expression profiles of the galectin gene family in primary and metastatic papillary thyroid carcinoma with particular emphasis on galectin-1 and galectin-3 expression

INTRODUCTION: Galectin family members have been demonstrated to be abnormally expressed in cancer at the protein and mRNA level. This study investigated the levels of galectin proteins and mRNA expression in a large cohort of patients with papillary thyroid carcinoma and matched lymph node metastases with particular emphasis on galectin-1 and galectin-3. METHODS: mRNA expression of galectin family members (1, 2, 3, 4, 7, 8, 9, 10 and 12) were analysed by real-time polymerase chain reaction in 65 papillary thyroid carcinomas, 30 matched lymph nodes with metastatic papillary thyroid carcinoma and 5 non-cancer thyroid tissues. Galectin-1 and 3 protein expression was determined by immunohistochemistry in these samples. RESULTS: Significant expression differences in all tested galectin family members (1, 2, 3, 4, 7, 8, 9, 10 and 12) were noted for mRNA in papillary thyroid carcinomas, with and without lymph node metastasis. Galectin-1 protein was more strongly expressed than galectin-3 protein in papillary thyroid carcinoma. Galectin-1 protein was found to be overexpressed in 32% of primary papillary thyroid carcinomas. A majority of lymph nodes with metastatic papillary thyroid carcinoma (53%) had significantly increased expression of galectin-1 protein, as did 47% of primaries with metastases. Galectin-1 mRNA levels were decreased in the vast majority (94%) of primary thyroid carcinomas that did not have metastases present. Galectin-3 protein levels were noted to be overexpressed in 15% of primary papillary thyroid carcinomas. In primary papillary thyroid carcinoma with lymph node metastases, 32% had over expression of galectin-3 protein. Overexpression of galectin-3 mRNA was noted in 58% of papillary thyroid carcinomas and 64% of lymph nodes bearing metastatic papillary thyroid carcinoma. Also, primary papillary thyroid carcinoma with lymph node metastases had significantly higher expression of galectin-3 mRNA compared to those without lymph node metastases. CONCLUSION: Galectin family members show altered expression at the mRNA level in papillary thyroid cancers. Overexpression of galectin-1 and 3 proteins were noted in papillary thyroid carcinoma with lymph node metastases. The results presented here demonstrated that galectin-1 and galectin-3 expression have important roles in clinical progression of papillary thyroid carcinoma.

Co-regulatory potential of vascular endothelial growth factor–A and vascular endothelial growth factor–C in thyroid carcinoma

Vascular endothelial growth factor (VEGF) promotes growth of blood or lymphatic vessels. The aim of the current study is to identify relationships between VEGF-A and VEGF-C, and their impact in angiogenesis and metastases in thyroid cancers. VEGF-A and VEGF-C mRNA and protein expression was investigated in 136 thyroid cancers (123 papillary thyroid carcinomas and 13 undifferentiated thyroid carcinomas) and 40 matched lymph node metastases with papillary thyroid carcinoma using reverse transcription polymerase chain reaction and immunohistochemistry. VEGF-A and VEGF-C mRNA expression was significantly different between conventional papillary thyroid carcinoma, follicular variant of papillary thyroid carcinoma, and undifferentiated thyroid carcinomas (P = 1 x 10(-6) and 1 x 10(-5), respectively). In undifferentiated carcinoma, VEGF-A and VEGF-C protein overexpression was noted in all cases. VEGF-A and VEGF-C mRNA overexpression was noted in 51% (n = 62) and 27% (n = 33) of the papillary thyroid carcinomas, whereas VEGF-A and VEGF-C protein overexpression was also identified in 70% (n = 86) and 62% (n = 76) of the carcinomas. VEGF-A mRNA was significantly higher in cancers with lymph node metastases compared with nonmetastatic cancers (P = .001), whereas most metastatic cancers underexpressed VEGF-C (P = .0002), with a similar trend for protein. The expression of VEGF-A and VEGF-C correlated with each other at both mRNA and protein levels (P = .00004 and .003, respectively). In summary, VEGF-A and -C expressions correlate with the pathological parameters and metastatic status of thyroid carcinomas. The significant correlations between the expressions of these genes add weight to hypotheses concerning VEGF-A and -C interaction in cancer progression.

Single nucleotide polymorphisms and mRNA expression of VEGF-A in papillary thyroid carcinoma: Potential markers for aggressive phenotypes

BACKGROUND AND OBJECTIVES Polymorphisms of the VEGF gene are known to affect the biological behaviour of cancers but have seldom been studied in thyroid cancer. The aim of the current study is to evaluate the prevalence and relevance of VEGF-A polymorphisms and mRNA expression in papillary thyroid carcinoma (PTC). MATERIALS AND METHODS Genomic DNA and total RNA were isolated from paraffin-embedded tissue from 91 PTC (51 conventional PTC and 40 follicular variant) and 78 control thyroid tissues. Three DNA polymorphisms (+936C > T, +405C > G and -141A > C) in the 3' and 5' untranslated region (3'-UTR, 5'-UTR) of VEGF-A were studied using PCR and RFLP. Also, the mRNA expression of VEGF-A in these tissues was studied by real-time PCR. RESULTS Distribution of polymorphisms in the 5'-UTR (VEGF-VEGF -141A > C and +405C > G) and 3'-UTR (VEGF +936C > T) were all significantly different in PTC and benign thyroid tissue (p = 0.0001, 0.001 and 0.028 respectively). The VEGF -141 C allele was more common in PTC with lymph node metastases (p = 0.026). VEGF + 405 Galleles andVEGF +936 CC genotype were more common in PTC of advanced pathological staging (p = 0.018 and 0.017 respectively). Also, increased expression of VEGF-A mRNA was noted in PTC compared to control (p = 0.009). Within the group of patients with conventional PTC, those with lymph nodal metastases had a higher level of VEGF-A mRNA expression than other patients (p = 0.0003). CONCLUSION These findings suggest that VEGF polymorphisms and mRNA expression may predict the aggressiveness behaviour of thyroid cancer.

Association of FOXE1 Polyalanine Repeat Region with Papillary Thyroid Cancer

CONTEXT: Polyalanine tract variations in transcription factors have been identified for a wide spectrum of developmental disorders. The thyroid transcription factor forkhead factor E1 (FOXE1) contains a polymorphic polyalanine tract with 12-22 alanines. Single-nucleotide polymorphisms (SNP) close to this locus are associated with papillary thyroid cancer (PTC), and a strong linkage disequilibrium block extends across this region. OBJECTIVE: The objective of the study was to assess whether the FOXE1 polyalanine repeat region was associated with PTC and to assess the effect of polyalanine repeat region variants on protein expression, DNA binding, and transcriptional function on FOXE1-responsive promoters. DESIGN: This was a case-control study. SETTING: The study was conducted at a tertiary referral hospital. PATIENTS AND METHODS: The FOXE1 polyalanine repeat region and tag SNP were genotyped in 70 PTC, with a replication in a further 92 PTC, and compared with genotypes in 5767 healthy controls (including 5667 samples from the Wellcome Trust Case Control Consortium). In vitro studies were performed to examine the protein expression, DNA binding, and transcriptional function for FOXE1 variants of different polyalanine tract lengths. RESULTS: All the genotyped SNP were in tight linkage disequilibrium, including the FOXE1 polyalanine repeat region. We confirmed the strong association of rs1867277 with PTC (overall P = 1 × 10(-7), odds ratio 1.84, confidence interval 1.31-2.57). rs1867277 was in tight linkage disequilibrium with the FOXE1 polyalanine repeat region (r(2) = 0.95). FOXE1(16Ala) was associated with PTC with an odds ratio of 2.23 (confidence interval 1.42-3.50; P = 0.0005). Functional studies in vitro showed that FOXE1(16Ala) was transcriptionally impaired compared with FOXE1(14Ala), which was not due to differences in protein expression or DNA binding. CONCLUSIONS: We have confirmed the previous association of FOXE1 with PTC. Our data suggest that the coding polyalanine expansion in FOXE1 may be responsible for the observed association between FOXE1 and PTC.

Use of fine-needle aspirate calcitonin to detect medullary thyroid carcinoma: A systematic review.

BACKGROUND: The measurement of calcitonin in washout fluids of thyroid nodule aspirate (FNA-calcitonin) has been reported as accurate to detect medullary thyroid carcinoma (MTC). The results from these studies have been promising and the most updated version of ATA guidelines quoted for the first time that "FNA findings that are inconclusive or suggestive of MTC should have calcitonin measured in the FNA washout fluid." Here we aimed to systematically review published data on this topic to provide more robust estimates. RESEARCH DESIGN AND METHODS: A comprehensive computer literature search of the medical databases was conducted by searching for the terms "calcitonin" AND "washout." The search was updated until April 2015. RESULTS: Twelve relevant studies, published between 2007 and 2014, were found. Overall, 413 thyroid nodules or neck lymph nodes underwent FNA-calcitonin, 95 were MTC lesions and 93 (97.9%) of these were correctly detected by this measurement regardless of their cytologic report. CONCLUSIONS: The present study shows that the above ATA recommendation is well supported. Almost all MTC lesions are correctly detected by FNA-calcitonin and this technique should be used to avoid false negative or inconclusive results from cytology. The routine determination of serum calcitonin in patients undergoing FNA should improve the selection of patients at risk for MTC, guiding the use of FNA-calcitonin in the same FNA sample and providing useful information to the cytopathologist for the morphological assessment and the application of tailored ancillary tests.

Correlation between BRAF mutation and the clinicopathological parameters in papillary thyroid carcinoma with particular reference to follicular variant

Mutation of the BRAF gene is common in thyroid cancer. Follicular variant of papillary thyroid carcinoma is a variant of papillary thyroid carcinoma that has created continuous diagnostic controversies among pathologists. The aims of this study are to (1) investigate whether follicular variant of papillary thyroid carcinoma has a different pattern of BRAF mutation than conventional papillary thyroid carcinoma in a large cohort of patients with typical features of follicular variant of papillary thyroid carcinoma and (2) to study the relationship of clinicopathological features of papillary thyroid carcinomas with BRAF mutation. Tissue blocks from 76 patients with diagnostic features of papillary thyroid carcinomas (40 with conventional type and 36 with follicular variant) were included in the study. From these, DNA was extracted and BRAF V600E mutations were detected by polymerase chain reaction followed by restriction enzyme digestion and sequencing of exon 15. Analysis of the data indicated that BRAF V600E mutation is significantly more common in conventional papillary thyroid carcinoma (58% versus 31%, P = .022). Furthermore, the mutation was often noted in female patients (P = .017), in high-stage cancers (P = .034), and in tumors with mild lymphocytic thyroiditis (P = .006). We concluded that follicular variant of papillary thyroid carcinoma differs from conventional papillary thyroid carcinoma in the rate of BRAF mutation. The results of this study add further information indicating that mutations in BRAF play a role in thyroid cancer development and progression.

Follicular variant of papillary thyroid carcinoma : a diagnostic challenge for clinicians and pathologists

The follicular variant of papillary thyroid carcinoma (FVPTC) presents a type of papillary thyroid cancer that has created continuous diagnosis and treatment controversies among clinicians and pathologists. In this review, we describe the nomenclature, the clinical features, diagnostic problems and the molecular biology of FVPTC. It is important for clinicians to understand this entity as the diagnosis and management of this group of patient may be different from other patients with conventional PTC. The literature suggests that FVPTC behaves in a way similar, clinically, to conventional papillary thyroid carcinoma. However, there are some genotypic differences which may characterise this neoplasm. These parameters may account for the phenotypic variation described by some scientists in this type of cancer. Further understanding can only be achieved by defining strict pathological criteria, in-depth study of the molecular biology and long term follow-up of the optional patients with FVPTC.

IGF-1 or insulin, and the TSH cyclic AMP cascade separately control dog and human thyroid cell growth and DNA synthesis, and complement each other in inducing mitogenesis.

The regular doubling of cell mass, and therefore of cell protein content, is required for repetitive cell divisions. Preliminary observations have shown that in dog thyrocytes insulin induces protein accumulation but not DNA synthesis, while TSH does not increase protein accumulation but triggers DNA synthesis in the presence of insulin. We show here that EGF and phorbol myristate ester complement insulin action in the same way. HGF is the only factor activating both protein accumulation and DNA synthesis. The effects of insulin on protein accumulation and in permitting the TSH effect are reproduced by IGF-1 and are mediated, at least in part by the IGF-1 receptor. The concentration effect curves are similar for both effects. Similar results are obtained in human thyrocytes. They reflect true cell growth, as shown by increases in RNA content and cell size. Carbachol and fetal calf serum also stimulate protein synthesis and accumulation without triggering DNA synthesis, but they are not permissive for the mitogenic effects of TSH or of the general adenylate cyclase activator, forskolin. Moreover the mitogenic effect of TSH greatly decreased in cells deprived of insulin for 2 days although these cells remain hypertrophic. Hypertrophy may therefore be necessary for cell division, but it is not sufficient to permit it. Three different mechanisms can therefore be distinguished in the mitogenic action of TSH: (1) the increase of cell mass (hypertrophy) induced by insulin or IGF-1; (2) the permissive effect of insulin or IGF-1 on the mitogenic effect of TSH which may involve both the increase of cell mass and the induction of specific proteins such as cyclin D3 and (3) the mitogenic effect of the TSH cyclic AMP cascade proper.

Thyroid function and prevalent and incident metabolic syndrome in older adults: the Health, Ageing and Body Composition Study.

OBJECTIVE: Both subclinical hypothyroidism and the metabolic syndrome have been associated with increased risk of coronary heart disease events. It is unknown whether the prevalence and incidence of metabolic syndrome is higher as TSH levels increase, or in individuals with subclinical hypothyroidism. We sought to determine the association between thyroid function and the prevalence and incidence of the metabolic syndrome in a cohort of older adults. DESIGN: Data were analysed from the Health, Ageing and Body Composition Study, a prospective cohort of 3075 community-dwelling US adults. PARTICIPANTS: Two thousand one hundred and nineteen participants with measured TSH and data on metabolic syndrome components were included in the analysis. MEASUREMENTS: TSH was measured by immunoassay. Metabolic syndrome was defined per revised ATP III criteria. RESULTS: At baseline, 684 participants met criteria for metabolic syndrome. At 6-year follow-up, incident metabolic syndrome developed in 239 individuals. In fully adjusted models, each unit increase in TSH was associated with a 3% increase in the odds of prevalent metabolic syndrome (OR, 1.03; 95% CI, 1.01-1.06; P = 0.02), and the association was stronger for TSH within the normal range (OR, 1.16; 95% CI, 1.03-1.30; P = 0.02). Subclinical hypothyroidism with a TSH > 10 mIU/l was significantly associated with increased odds of prevalent metabolic syndrome (OR, 2.3; 95% CI, 1.0-5.0; P = 0.04); the odds of incident MetS was similar (OR 2.2), but the confidence interval was wide (0.6-7.5). CONCLUSIONS: Higher TSH levels and subclinical hypothyroidism with a TSH > 10 mIU/l are associated with increased odds of prevalent but not incident metabolic syndrome.

Diagnostic and prognostic implications of the PAX8-PPARγ translocation in thyroid carcinomas-a TMA-based study of 226 cases

AIMS Follicular thyroid carcinoma (FTC) has been a diagnostic challenge for decades. The PAX8-PPARγ rearrangement has been detected in FTC and classic papillary thyroid carcinomas (PTCs). The aims of this study were to assess the presence of PAX8-PPARγ by using tissue microarrays in a large cohort of different thyroid neoplasms, and to assess its diagnostic and prognostic implications. METHODS AND RESULTS Fluorescence in-situ hybridization (FISH) analysis for PAX8-PPARγ was performed on 226 thyroid tumours, comprising FTCs (n = 59), PTCs (n = 126), poorly differentiated thyroid carcinomas (PDs; n = 34), follicular thyroid adenomas (FTAs; n = 5), and follicular tumours of unknown malignant potential (FTUMPs; n = 2). PAX8-PPARγ was detected in 12% of FTCs, 1% of PTCs, 7% of PDs, and in both cases of FTUMP. There was no correlation between the extent of capsular or vascular invasion and PAX8-PPARγ, or between lymph node or haematogenous metastasis and PAX8-PPARγ. Overall survival (OS), tumour-specific survival (TSS) and relapse-free-survival (RFS) were not influenced by PAX8-PPARγ. CONCLUSIONS In this study, we demonstrate for the first time the presence of PAX8-PPARγ in PDs and FTUMPs, whereas in FTCs and PTCs the prevalence of PAX8-PPARγ is lower than previously reported. PAX8-PPARγ did not correlate with invasiveness or affect prognosis in any tumour type.

Phase I clinical trials in 56 patients with thyroid cancer: the M. D. Anderson Cancer Center experience.

INTRODUCTION: Thyroid cancer is the most common endocrine malignancy. The outcomes of patients with relapsed thyroid cancer treated on early-phase clinical trials have not been systematically analyzed. PATIENTS AND METHODS: We reviewed the records of consecutive patients with metastatic thyroid cancer referred to the Phase I Clinical Trials Program from March 2006 to April 2008. Best response was assessed by Response Evaluation Criteria in Solid Tumors. RESULTS: Fifty-six patients were identified. The median age was 55 yr (range 35-79 yr). Of 49 patients evaluable for response, nine (18.4%) had a partial response, and 16 (32.7%) had stable disease for 6 months or longer. The median progression-free survival was 1.12 yr. With a median follow-up of 15.6 months, the 1-yr survival rate was 81%. In univariate analysis, factors predicting shorter survival were anaplastic histology (P = 0.0002) and albumin levels less than 3.5 g/dl (P = 0.05). Among 26 patients with tumor decreases, none died (median follow-up 1.3 yr), whereas 52% of patients with any tumor increase died by 1 yr (P = 0.0001). The median time to failure in our phase I clinical trials was 11.5 months vs. 4.1 months for the previous treatment (P = 0.04). CONCLUSION: Patients with advanced thyroid cancer treated on phase I clinical trials had high rates of partial response and prolonged stable disease. Time to failure was significantly longer on the first phase I trial compared with the prior conventional treatment. Patients with any tumor decrease had significantly longer survival than those with any tumor increase.

Paediatric thyroid surgery is safe - experiences at a tertiary surgical centre.

PRINCIPLES Thyroidectomy in children is rare and mostly performed because of thyroid neoplasms. The aim of this study based on prospective data acquisition was to evaluate whether thyroid surgery in children can be performed as safely as in adults when undertaken by a team of adult endocrine surgeons and paediatric surgeons. METHODS Between 2002 and 2012, 36 patients younger than 18 years underwent surgery for thyroid gland pathologies. All surgical procedures were performed by an experienced endocrine surgeon and a paediatric surgeon. Baseline demographic data, surgical procedure, duration of operation, length of hospital stay, and postoperative morbidity and mortality were analysed. RESULTS The median age of all patients was 13 years (range 2-17 years), with predominantly female gender (n = 30, 83%). The majority of operations were performed because of benign thyroid disease (n = 27, 75%) and only a minority because of malignancy or genetic abnormality with predisposition for malignant transformation (MEN) (n = 9, 25%). Total thyroidectomy was performed in the majority of the patients (n = 24, 67%). The median duration of the surgical procedure was 153 minutes (range 90-310 minutes). The median hospital stay was 5 days (3-1 days). One patient developed persistent hypoparathyroidism after neck dissection due to cancer. One persistent and two temporary recurrent nerve palsies occurred. CONCLUSION This study demonstrated that paediatric thyroidectomy is safe as performed by this team of endocrine and paediatric surgeons, with acceptable morbidity even when total thyroidectomy was performed in the case of benign disease.

What you need to know about thyroid cancer.

"P620"--P. [4] of cover.