Germline ESR2 mutation predisposes to medullary thyroid carcinoma and causes up-regulation of RET expression.

Familial medullary thyroid cancer (MTC) and its precursor, C cell hyperplasia (CCH), is associated with germline RET mutations causing multiple endocrine neoplasia type 2. However, some rare families with apparent MTC/CCH predisposition do not have a detectable RET mutation. To identify novel MTC/CCH predisposition genes we undertook exome resequencing studies in a family with apparent predisposition to MTC/CCH and no identifiable RET mutation. We identified a novel ESR2 frameshift mutation, c.948delT, which segregated with histological diagnosis following thyroid surgery in family members and demonstrated loss of ESR2-encoded ERß expression in the MTC tumour. ERα and ERß form heterodimers binding DNA at specific oestrogen-responsive elements (EREs) to regulate gene transcription. ERß represses ERα-mediated activation of the ERE and the RET promoter contains three EREs. In vitro, we showed that ESR2 c.948delT results in unopposed ERα mediated increased cellular proliferation, activation of the ERE and increased RET expression. In vivo, immunostaining of CCH and MTC using an anti-RET antibody demonstrated increased RET expression. Together these findings identify germline ESR2 mutation as a novel cause of familial MTC/CCH and provide important insights into a novel mechanism causing increased RET expression in tumourigenesis.

The presentation, natural history and outcome of T1 a/b thyroid cancer with regard to new grading systems.

INTRODUCTION: The seventh edition tumor, node metastasis (TNM) classifies tumors <10 mm as T1a and tumors between 10 mm and 20 mm as T1b. Previous editions of the TNM staging system did not differentiate these lesions. We compare new classification in terms of presentation and outcome. METHODS: Early thyroid cancers that were previously staged under the TNM sixth edition were re-staged using the TNM seventh edition to identify whether the new staging system differentiated lesions in terms of presentation and outcome. Differences in presentation, rates of recurrent disease and surgical complications were recorded and compared. RESULTS: Of 1,415 thyroid operations, 369 were for malignant disease and there were 220 papillary carcinomas. There were 35 T1a/b lesions accounting for 11% of malignancies with no increasing incidence. Reclassification showed 64% T1a and 36% T1b lesions. There were no differences in presentation. Outcomes in terms of persistent or recurrent disease were the same after median follow up was 6.8 years. CONCLUSION: Isolated T1a/b thyroid cancer made up only a small proportion of malignancies, the majority of which were discovered incidentally. We have not seen the increase in early lesions reported elsewhere. In our series, the seventh TNM edition did not differentiate early lesions and we ultimately feel that parameters other than size of primary lesion are likely to be of greater importance in determining prognosis.

Expression of receptors for VEGFs on normal human thyroid follicular cells and their role in follicle formation.

Human thyroid follicular cells in culture expressed the mRNAs for the receptors for vascular endothelial growth factors (VEGFRs). The relative expression was neuropilin1 = neuropilin2 = VEGFR2 > VEGFR1 > VEGFR3. Western blotting for VEGFR2 showed labeling of proteins ~200-230 kDa. Clonal follicular thyroid cell lines (FRTL5 and FTC133) also expressed mRNAs for the VEGFR1 and 2 obviating concerns of endothelial cell contamination. In the primary cultures, TSH, which is essential for expression of differentiated function, reduced VEGFR2 mRNA levels by 60%. Immunostaining for VEGFRs and neuropilin2 (NRP2), showed expression on the plasma membrane but with the exception of neuropilin1 (NRP1), all VEGFRs were also found in the cytoplasm and nucleus. Antibody specific for phosphotyrosine 1214 in VEGFR2 showed that the receptor was phosphorylated in the primary cultures and the cell lines. When VEGFR signaling was blocked with a specific inhibitor, follicle formation in the primary cultures was enhanced suggesting that VEGFR activation was detrimental to follicle formation. Immunostaining of sections of normal thyroids and various pathologies showed staining for VEGFR2 and pVEGFR2. We conclude that normal thyroid follicular cell express VEGFRs. For VEGFR2 its subcellular localization suggests functions additional to that of a cell surface receptor and a role in follicular integrity.

A novel mechanism of sodium iodide symporter repression in differentiated thyroid cancer.

Differentiated thyroid cancers and their metastases frequently exhibit reduced iodide uptake, impacting on the efficacy of radioiodine ablation therapy. PTTG binding factor (PBF) is a proto-oncogene implicated in the pathogenesis of thyroid cancer. We recently reported that PBF inhibits iodide uptake, and have now elucidated a mechanism by which PBF directly modulates sodium iodide symporter (NIS) activity in vitro. In subcellular localisation studies, PBF overexpression resulted in the redistribution of NIS from the plasma membrane into intracellular vesicles, where it colocalised with the tetraspanin CD63. Cell-surface biotinylation assays confirmed a reduction in plasma membrane NIS expression following PBF transfection compared with vector-only treatment. Coimmunoprecipitation and GST-pull-down experiments demonstrated a direct interaction between NIS and PBF, the functional consequence of which was assessed using iodide-uptake studies in rat thyroid FRTL-5 cells. PBF repressed iodide uptake, whereas three deletion mutants, which did not localise within intracellular vesicles, lost the ability to inhibit NIS activity. In summary, we present an entirely novel mechanism by which the proto-oncogene PBF binds NIS and alters its subcellular localisation, thereby regulating its ability to uptake iodide. Given that PBF is overexpressed in thyroid cancer, these findings have profound implications for thyroid cancer ablation using radioiodine.

Adenoid Cystic Carcinoma of Trachea.

Adenoid cystic carcinoma (ACC) is the second most common primary malignant tumor of the trachea, after squamous cell carcinoma. Patients present with upper airway obstructive symptoms and signs including dyspnoea, cough, haemoptysis, and stridor which are often insidious, delaying diagnosis and optimal management. Described here is an unusual case of primary ACC cervical trachea with concomitant micropapillary thyroid carcinoma (microPTC) in a middle-aged lady presenting with cough and breathing difficulty since 3 months.

Pituitary tumour transforming gene (PTTG) induces genetic instability in thyroid cells.

Cancer reflects the progressive accumulation of genetic alterations and subsequent genetic instability of cells. Cytogenetic studies have demonstrated the importance of aneuploidy in differentiated thyroid cancer development. The pituitary tumour transforming gene (PTTG), also known as securin, is a mitotic checkpoint protein which inhibits sister chromatid separation during mitosis. PTTG is highly expressed in many cancers and overexpression of PTTG induces aneuploidy in vitro. Using fluorescent intersimple sequence repeat PCR (FISSR-PCR), we investigated the relationship between PTTG expression and the degree of genetic instability in normal and tumorous thyroid samples. The genomic instability index (GI index) was 6.7-72.7% higher in cancers than normal thyroid tissues. Follicular thyroid tumours exhibited greater genetic instability than papillary tumours (27.6% (n=9) versus 14.5% (n=10), P=0.03). We also demonstrated a strong relationship between PTTG expression and the degree of genetic instability in thyroid cancers (R2=0.80, P=0.007). To further investigate PTTG's role in genetic instability, we transfected FTC133 thyroid follicular cells and observed increased genetic instability in cells overexpressing PTTG compared with vector-only-transfected controls (n=3, GI Index VO=29.7+/-5.2 versus PTTG=63.7+/-6.4, P=0.013). Further, we observed a dose response in genetic instability and PTTG expression (GI Index low dose (0.5 microg DNA/ six-well plate) PTTG=15.3%+/-1.7 versus high dose (3 microg DNA) PTTG=50.8%+/-3.3, P=0.006). Overall, we describe the first use of FISSR-PCR in human cancers, and demonstrate that PTTG expression correlates with genetic instability in vivo, and induces genetic instability in vitro. We conclude that PTTG may be an important gene in the mutator phenotype development in thyroid cancer.

Detection and surgical treatment of cervical lymph nodes in differentiated thyroid cancer.

There is considerable controversy regarding the treatment of patients with cervical metastases from differentiated thyroid cancer. Most have papillary carcinoma and the main areas of contention relate to methods of assessment and staging, surgical management and mode of follow up. there is little evidence to support elective anatomical imaging with CT or MRI in those patients with suspected or proven malignancy at the primary site as indicated by fine needle aspiration cytology (FNAC) but who have no clinical evidence of nodal disease. The role of routine ultrasound (US) in the pre-operative assessment of suspected or known malignancy is developing but is largely unproven. When it is performed, high risk areas for metastatic neck disease (levels II-V) should be assessed. Suspicious nodes on US should be further evaluated by FNAC. Suspected or proven neck disease may be further assessed pre-operatively with CT or MRI and then treated surgically. Disease in the central compartment requires a total thyroidectomy and level VI central compartment neck dissection. Suspected or proven lateral compartment cancer should be treated by selective neck dissection (at least levels III, IV, and V) below the accessory nerve. There is no role for 'Berry picking' and clinically node negative high risk patients should have an elective central compartment level VI neck dissection. Sentinel node biopsy lays no role and neither does elective lateral compartment surgery in patients with no clinical or radiological evidence of disease. For follow up, US represents the most sensitive means of detecting neck recurrences and in the presence of an elevated serum thyroglobulin, imaging may also include whole body iodine-131 scanning and anatomical imaging with CT or MRI. The role of PET remains controversial but is likely to develop further as the technique becomes more widely available. In the future, the concentration of patients with this disease in large center can only improve the way we treat differentiated thyroid cancer.

Complete inhibition of goiter in mice requires combined gene therapy modification of angiopoietin, vascular endothelial growth factor, and fibroblast growth factor signaling.

In goiter, increased expression of growth factors and their receptors occurs. We have inhibited the action of some of these growth factors, alone and in combination, to determine which are important in goitrogenesis. Recombinant adenovirus vectors (RAds) expressing truncated, secreted forms of human Tie2 (RAd-sTie2) and vascular endothelial growth factor receptor 1 (RAd-sVEGFR1) or a truncated, dominant-negative fibroblast growth factor receptor 1 (RAdDN-FGFR1) were used. Goiters in mice were induced by feeding an iodide-deficient diet, containing methimazole and sodium perchlorate. RAds were administered to mice simultaneously with the goitrogenic regimen, which was continued for 14 d. RAd treatment did not significantly affect increases in TSH or reductions in thyroid hormone or thyroid hyperactivity seen in goitrogen-treated controls mice, suggesting no effect on pituitary or thyroid responses to hypothyroidism. In control goiters, a 4-fold increase in vascular volume accompanied a 2-fold increase in thyroid mass. Complete inhibition of these increases was found when animals were treated with the three RAds in combination. In thyroids from three RAd-treated animals, there was marked, significant inhibition of Tie2, FGFR1, VEGFR1, FGF-2, and VEGF expression, compared with control goiters. When used individually, RAdDN-FGFR1 partially prevented goiter and RAd-sVEGFR1 partially reduced vascular volume. Their effects were not additive. RAd-sTie2 did not reduce goiter mass or vascular volume when used alone but was essential for complete goiter inhibition. VEGF and VEGFR1 expression was reduced in these thyroids. Limitation of physiologic organ growth is complex, requiring inhibition of multiple, interdependent growth factor axes.

Adenovirus-mediated expression of dominant negative fibroblast growth factor (FGF) receptor 1 in thyroid cells blocks FGF effects and reduces goitrogenesis in mice.

Levels of fibroblast growth factor 2 (FGF-2) and its receptor, FGFR1, are elevated in goiter, but whether this is a direct effect of TSH is unknown. We have determined the regulation of FGF-2 and FGFR1 synthesis by TSH in a rat thyroid cell line (FRTL5) and have used a replication-defective adenovirus (RAd) expressing dominant negative FGFR1 (RAdDN-FGFR1) to examine the role of FGFR signaling in vitro and in goiter induced in mice. TSH induced FGF-2 and increased the expression of FGFR1 in FRTL5 cells. Infection of TSH-stimulated FRTL5 cells with RAdDN-FGFR1 inhibited growth and prevented FGF-2-mediated inhibition of (125)I uptake. Similar effects were found in primary cultures of human thyroid follicular cells. For in vivo experiments, male BALB/c mice were injected systemically with RAdDN-FGFR1 or RAd encoding green fluorescent protein, and goiter was simultaneously induced. Mouse thyroid follicles were shown to be transduced with RAd encoding green fluorescent protein. Circulating TSH was elevated comparably in the two groups. In the RAdDN-FGFR1-injected animals, goiter induced over 14 d was significantly smaller, and the vascular volume increase seen in goiter was also diminished. We conclude that the FGF axis is important in thyroid growth and that RAdDN-FGFR1 effectively blocks FGF actions, offering a means to control goitrogenesis.

The PTTG1-binding factor (PBF/PTTG1IP) regulates p53 activity in thyroid cells.

The PTTG1-binding factor (PBF/PTTG1IP) has an emerging repertoire of roles, especially in thyroid biology, and functions as a protooncogene. High PBF expression is independently associated with poor prognosis and lower disease-specific survival in human thyroid cancer. However, the precise role of PBF in thyroid tumorigenesis is unclear. Here, we present extensive evidence demonstrating that PBF is a novel regulator of p53, a tumor suppressor protein with a key role in maintaining genetic stability, which is infrequently mutated in differentiated thyroid cancer. By coimmunoprecipitation and proximity-ligation assays, we show that PBF binds specifically to p53 in thyroid cells and significantly represses transactivation of responsive promoters. Further, we identify that PBF decreases p53 stability by enhancing ubiquitination, which appears dependent on the E3 ligase activity of Mdm2. Impaired p53 function was evident in a transgenic mouse model with thyroid-specific PBF overexpression (transgenic PBF mice), which had significantly increased genetic instability as indicated by fluorescent inter simple sequence repeat-PCR analysis. Consistent with this, approximately 40% of all DNA repair genes examined were repressed in transgenic PBF primary cultures, including genes with critical roles in maintaining genomic integrity such as Mgmt, Rad51, and Xrcc3. Our data also revealed that PBF induction resulted in up-regulation of the E2 enzyme Rad6 in murine thyrocytes and was associated with Rad6 expression in human thyroid tumors. Overall, this work provides novel insights into the role of the protooncogene PBF as a negative regulator of p53 function in thyroid tumorigenesis, in which PBF is generally overexpressed and p53 mutations are rare compared with other tumor types.

Regulation of human thyroid follicular cell function by inhibition of vascular endothelial growth factor receptor signalling.

The potential autocrine role of human thyroid vascular endothelial growth factors (VEGFs) was examined using the VEGF receptor (VEGFR) inhibitor, ZM306416HCl. ZM306416HCl reduced VEGFR2 phosphorylation and inhibited endogenous, steady-state levels of p42/44 MAPK phosphorylation. It potently inhibited the secretion of plasminogen activators (PA) and increased (125)I uptake. Cell survival was compromised but rescued with insulin and TSH. Although the EGF receptor remained responsive to challenge by EGF in p42/44 MAPK assays, stimulatory effects of EGF on PA production were prevented by ZM306416HCl and those of protein kinase C stimulator, TPA reduced. In assays of (125)I uptake, ZM306416HCl prevented the inhibitory effects of EGF but not those of TPA. We conclude that autocrine VEGF may modulate thyroid function and that VEGFR inhibition increases iodide uptake and decreases PA production through regulation of p42/44 MAPK phosphorylation. VEGFR inhibition may have effects on thyroid function which may contribute to "off target" effects in clinical trials.

Proto-oncogene PBF/PTTG1IP regulates thyroid cell growth and represses radioiodide treatment.

Pituitary tumor transforming gene (PTTG)-binding factor (PBF or PTTG1IP) is a little characterized proto-oncogene that has been implicated in the etiology of breast and thyroid tumors. In this study, we created a murine transgenic model to target PBF expression to the thyroid gland (PBF-Tg mice) and found that these mice exhibited normal thyroid function, but a striking enlargement of the thyroid gland associated with hyperplastic and macrofollicular lesions. Expression of the sodium iodide symporter (NIS), a gene essential to the radioiodine ablation of thyroid hyperplasia, neoplasia, and metastasis, was also potently inhibited in PBF-Tg mice. Critically, iodide uptake was repressed in primary thyroid cultures from PBF-Tg mice, which could be rescued by PBF depletion. PBF-Tg thyroids exhibited upregulation of Akt and the TSH receptor (TSHR), each known regulators of thyrocyte proliferation, along with upregulation of the downstream proliferative marker cyclin D1. We extended and confirmed findings from the mouse model by examining PBF expression in human multinodular goiters (MNG), a hyperproliferative thyroid disorder, where PBF and TSHR was strongly upregulated relative to normal thyroid tissue. Furthermore, we showed that depleting PBF in human primary thyrocytes was sufficient to increase radioiodine uptake. Together, our findings indicate that overexpression of PBF causes thyroid cell proliferation, macrofollicular lesions, and hyperplasia, as well as repression of the critical therapeutic route for radioiodide uptake.

Fifteen years' experience in thyroid surgery.

INTRODUCTION: Thyroid disease is common, thyroid cancer is uncommon. Most goitres are investigated using blood tests, fine needle aspiration cytology together with ultrasound. Surgery usually entails either lobectomy or total thyroidectomy, and for malignancy, patients may need a neck dissection. Recently, significant advances have been made regarding mechanisms involved in both thyroid growth and function (goitrogenesis) and carcinogenesis at a molecular level. PATIENTS AND METHODS: In the study cohort, 1113 patients had benign disease and 387 malignancy. For benign disease, 716 patients had lobectomy or isthmusectomy, 44 had near-total thyroidectomy and 318 a total thyroidectomy. For malignancy, patients received initial lobectomy (180) or total thyroidectomy (152). One hundred and eleven had completion surgery. Thirty patients had extensive surgery. Thyroid growth and function was investigated using 500 human thyroid cell primary cultures obtained at surgery, as well as in three animal models. The role of pituitary tumour transforming gene (PTTG), PTTG binding factor (PBF) and sodium iodide symporter (NIS) in thyroid cell function was then evaluated. RESULTS: Temporary and permanent recurrent laryngeal nerve palsy rates were 2.4% and 0.4%. Other complications included temporary (21%) and permanent (3%) hypoparathyroidism, wound infection (1.2%), haematoma (1.2%) and poor scar (0.8%). Six patients have died. Regarding thyroid growth and function, TSH represents (either directly or indirectly) the main factor mediating thyroid follicular cell growth. For carcinogenesis, over-expression of the proto-oncogenes PTTG and PBF induces tumours in nude mice, and PTTG can induce proliferation of human thyroid cells and, in addition, both repress expression and function of NIS.

Regulation of plasminogen activators in human thyroid follicular cells and their relationship to differentiated function.

Human thyroid cells in culture take up and organify (125)I when cultured in TSH (acting through cAMP) and insulin. They also secrete urokinase (uPA) and tissue-type (tPA) plasminogen activators (5-100 IU/10(6)cells/day). TSH and insulin both decreased secreted PA activity (PAA), uPA and tPA protein and their mRNAs. Autocrine fibroblast growth factor increased secreted PAA and inhibited thyroid cell (125)I uptake. Epidermal growth factor (EGF) and the protein kinase C (PKC) activator, TPA significantly increased PAA and inhibited thyroid differentiated function, (TPA > EGF). For TPA, effects were rapid, increased PAA secretion and decreased (125)I uptake being seen at 4 h whereas for EGF, a 24 h incubation was required. qRT-PCR showed significantly increased mRNA expression of uPA with lesser effects on tPA. Aprotinin, which inhibits PAA, increased (125)I uptake but did not abrogate the effects of TPA and EGF. The MEKK inhibitor, PD98059 partially reversed the effects of EGF and TPA on PAA, and largely reversed the effects of EGF but not TPA on differentiated function. PKC inhibitors bisindoylmaleimide 1, and the specific PKCbeta inhibitor, LY379196 completely reversed the effects of TPA on (125)I uptake and PAA whereas EGF effects were unaffected. TPA inhibited follicle formation and this effect was blocked by LY379196 but not PD98059. We conclude that in thyroid cells, MAPK activation inversely correlates with (125)I uptake and directly correlates with PA expression, in contrast to the effects of cAMP. TPA effects on iodide metabolism, dissolution of follicles and uPA synthesis are mediated predominantly through PKCbeta whereas EGF exerts its effects through MAPK but not PKCbeta.

Role and regulation of the fibroblast growth factor axis in human thyroid follicular cells.

Thyroidal levels of fibroblast growth factor-2 (FGF-2) and fibroblast growth factor receptor 1 (FGFR1) are elevated in human thyroid hyperplasia. To understand the significance of this, effects of FGFR1 activation on normal human thyrocyte growth and function in vitro and the regulation of FGF-2 and FGFR1 expression have been examined. FGF-2 stimulated cell growth, as measured by cell counting, and inhibited thyroid function as measured by 125I uptake. Sensitivity to FGF-2 disappeared after 7 days, although FGFR1 expression was maintained. Thyroid-stimulating hormone (TSH, 300 mU/l) increased FGFR1 mRNA expression within 4 h and protein expression by 8 h. Exogenous FGF-2 decreased FGFR1 protein. Endogenous FGF-2 levels were low (approximately 1-2 pg/microg protein), and TSH treatment decreased these by 50%. Protein kinase C (PKC) activation increased FGF-2 mRNA and FGF-2 secretion within 2 h. This effect was enhanced (4.4-fold) when cells were cultured in TSH. We conclude that TSH stimulates FGFR1 but not FGF-2 expression. PKC activation stimulates FGF-2 synthesis and secretion, and TSH synergizes with PKC activators. Increases in FGFR1 or FGF-2 or in both may contribute to goitrogenesis.

Isolation and characterization of human thyroid endothelial cells.

From collagenase digests of human thyroid, endothelial cells were separated from follicular cells by their greater adherence to gelatin-coated plates. Endothelial cells were further purified using fluorescence-activated cell sorting, selecting for cells expressing factor VIII-related antigen. Isolated cells were negative for thyroglobulin and calcitonin when examined by immunostaining. The receptor for the angiopoietins, Tie-2, was expressed by the cells, and expression was increased by agents that elevate cAMP. Nitric oxide synthase (NOS) 3, the endothelial form of NOS, was expressed by the cells and similarly regulated. Cells responded strongly to the mitogen fibroblast growth factor (FGF)-2 in growth assays but only weakly to vascular endothelial growth factor (VEGF). VEGF was, however, able to stimulate nitric oxide release from the cells consistent with their endothelial origin. The FGF receptor (FGFR1) was full length (120 kDa) and immunolocalized to the cytosol and nucleus. Thyrotropin (TSH) did not regulate FGFR1, but its expression was increased by VEGF. Thrombospondin, a product of follicular cells, was a growth inhibitor, but neither TSH nor 3,5,3'-triiodothyronine had direct mitogenic effects. Thyroid follicular cell conditioned medium contained plasminogen activator activity and stimulated the growth of the endothelial cells, but when treated with plasminogen to produce the endothelial-specific inhibitor, angiostatin, growth was inhibited. Human thyroid endothelial cell cultures will be invaluable in determining the cross talk between endothelial and follicular cells during goitrogenesis.