John C. Watkinson

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 PTTGs 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 μg DNA/ six-well plate) PTTG=15.3%±1.7 versus high dose (3 μg 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.

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.

Papillary carcinoma arising in a thyroglossal duct cyst : a case report and literature review

Papillary carcinoma arising in a thyroglossal duct cyst is a rare finding. Less than 100 cases have been reported in the English literature. In most cases the diagnosis is only established after excision of a clinically benign thyroglossal duct cyst. The aetiology of such tumours is unclear but de novo origin and spread from a primary thyroid gland tumour has been suggested. This has important implications for therapeutic approaches. A further case of thyroglossal duct carcinoma is presented and the management is discussed on the basis of the current rationale for treatment of thyroid cancer.

Investigating the thyroid nodule

#### Summary points Thyroid nodules are common: 4-7% of the adults have a palpable nodule, and up to 50-70% will have nodules on high definition ultrasonography, which may cause considerable concern to patients. In this article, we present an evidence based guide to investigating and managing thyroid nodules and we discuss the myths about nodules. Where relevant, we also highlight the differences between the two most widely used guidelines on this topic: the recently issued second edition of the British Thyroid Association’s guidelines on thyroid cancer1 and the American Thyroid Association’s guidelines.2 These guidelines were formulated by two large committees of experts, who reviewed all the available evidence, which mainly consists of prospective and retrospective cohorts; where there was no evidence, expert consensus opinion was used. Traditional teaching states that 20-40% of thyroid nodules are malignant.3 However, this pertains to a highly selected group of patients, with solitary cold nodules on scintigraphy.4 The risk of malignancy for a thyroid nodule identified on ultrasonography is much lower: 4-7%.5 6 Little evidence is available on referral times. Relying on expert opinion, the British Thyroid Association’s guidelines say that most thyroid nodules are benign and therefore do not require urgent referral to secondary care.1 This advice …

PTTG and PBF repress the human sodium iodide symporter.

The ability of the thyroid to accumulate iodide provides the basis for radioiodine ablation of differentiated thyroid cancers and their metastases. Most thyroid tumours exhibit reduced iodide uptake, although the mechanisms accounting for this remain poorly understood. Pituitary tumour transforming gene (PTTG) is a proto-oncogene implicated in the pathogenesis of thyroid tumours. We now show that PTTG and its binding factor PBF repress expression of sodium iodide symporter (NIS) messenger RNA (mRNA), and inhibit iodide uptake. This process is mediated at least in part through fibroblast growth factor-2. In detailed studies of the NIS promoter in rat FRTL-5 cells, PTTG and PBF demonstrated specific inhibition of promoter activity via the human upstream enhancer element (hNUE). Within this ∼1 kb element, a complex PAX8-upstream stimulating factor 1 (USF1) response element proved critical both to basal promoter activity and to PTTG and PBF repression of NIS. In particular, repression by PTTG was contingent upon the USF1, but not the PAX8, site. Finally, in human primary thyroid cells, PTTG and PBF similarly repressed the NIS promoter via hNUE. Taken together, our data suggest that the reported overexpression of PTTG and PBF in differentiated thyroid cancer has profound implications for activity of the NIS gene, and hence significantly impacts upon the efficacy of radioiodine treatment.

Squamous cell carcinoma of the oral cavity rarely harbours oncogenic human papillomavirus

Although it is now well established that a significant proportion of oropharyngeal squamous cell carcinomas (SCC) harbour oncogenic human papillomavirus (HPV) sequences, the frequency with which these sequences are detected in oral SCC (excluding oropharyngeal subsites) is highly variable. In an attempt to establish the true prevalence of HPV-16 and HPV-18 subtypes in oral SCC, we screened 142 consecutive cases from a UK cohort using both conventional PCR with consensus primers and type-specific quantitative PCR (Q-PCR), while at the same time employing a rigorous protocol to avoid sample contamination. Q-PCR revealed HPV sequences in five cases; two contained HPV-16 alone, two HPV-18 alone, and one sample carried both genotypes. However, only two of these cases (both HPV-16-positive) had moderate viral loads (51 and 91 viral copies per 100 cells respectively) and were positive for HPV DNA by conventional PCR. Both cases contained HPV DNA in tumour cells as shown by Q-PCR analysis of micro-dissected tissue and by in situ hybridisation. The remaining three cases had only very low viral loads (between 3 and 7 viral copies per 100 cells), were negative by conventional PCR and lacked HPV DNA in tumour cells. Our data provide strong evidence that oncogenic HPV is uncommon in oral SCC and that routine HPV testing of these tumours cannot be advocated.

Postoperative hypocalcemia—The difference a definition makes

Review of the literature reveals considerable variability in the definitions and criteria used for reporting postoperative hypocalcemia. The lack of standardization prevents a meaningful comparison of results and performance locally with the national standard. It also prevents the pooling of data when performing meta‐analysis, and may affect the comparison of research results.

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.

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.