M Sequeira, B Jasani, D Fuhrer, M Wheeler and M Ludgate
OBJECTIVE: Thyroid function and growth are controlled by TSH. Hyperthyroidism can be due to Graves' Disease (GD), in which thyroid-stimulating antibodies mimic TSH, or gain-of-function mutations in the TSH receptor (TSHR). These activating mutations have poor surface expression when assessed in non-thyroidal cells in vitro but nothing is known of their in vivo behaviour. Several TSHR antibodies have been produced but none has been applied to thyroid paraffin sections. This study aimed to develop a technique suitable for use on paraffin sections and apply it to investigate TSHR expression in thyroids harbouring activating TSHR germline mutations compared with normal and GD thyroids. DESIGN AND METHODS: Immunocytochemistry coupled with antigen retrieval, using a spectrum of antibodies to the TSHR, was applied to paraffin sections of GD thyroid tissue. Subsequently, TSHR immunoreactivity was examined in three normal thyroids, three patients with GD and three patients with familial hyperthyroidism, due to different gain-of-function TSHR germline mutations, using the optimised protocol. RESULTS: Two antibodies, A10 and T3-495, to the extracellular domain (ECD) and membrane spanning region (MSR) of the TSHR respectively, produced specific basolateral staining of thyroid follicular cells. In normal and GD thyroids, basolateral staining with T3-495 was generally more intense than with A10, suggesting a possible surfeit of MSR over ECD. Graves' Disease thyroids have more abundant TSHR than normal glands. In contrast, thyroids harbouring gain-of-function mutations have the lowest expression in vivo, mirroring in vitro findings. CONCLUSIONS: The development of an immunocytochemical method applicable to paraffin sections has demonstrated that different molecular mechanisms causing hyperthyroidism result in the lowest (mutation) and highest (autoimmunity) levels of receptor at the thyrocyte surface.
D Fuhrer, M Eszlinger, S Karger, K Krause, C Engelhardt, D Hasenclever, H Dralle and R Paschke
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.
V F H Brauer, H Below, A Kramer, D Führer and R Paschke
Objective: Thiocyanate (SCN−) has concentration dependent antithyroid properties and a role in the etiology of goiter has been suggested in several studies. In 1991 an epidemiological survey conducted in the region of Halle/Leipzig (Saxony), an area with significant air pollution, suggested an inverse relationship between urinary iodine (I−)/SCN− excretion and goiter prevalence. 10 years later, we reinvestigated the same industrial area to clarify if the situation has changed after the elimination of most industrial waste products and moreover, if SCN− excretion levels alone or in combination with air pollution or smoking as a SCN− source are critical for thyroid function.
Design and methods: We investigated a cohort of 708 probands for I−, SCN− and creatinine excretion in spot urine samples and determined the prevalence of goiter and thyroid nodules by high resolution ultrasonography.
Results: Probands with goiter (n = 79, 11%) had significantly higher urinary SCN− excretions than probands without (3.9 ± 2.8 vs 3.1 ± 3.4 mg SCN−/g creatinine) and significantly lower urinary I−/SCN− ratios than patients without thyroid disorders (41 ± 38 vs 61 ± 71 μg I−/mg SCN−/l). Mean urinary I− excretions were not different between probands with or without goiter. Smokers showed significantly elevated urinary SCN−/creatinine ratios in comparison to non-smokers (4.3 ± 4.3 vs 2.4 ± 2.1 mg SCN−/g creatinine). ANOVA revealed a prediction of thyroid volume through age (P < 0.001), gender (P < 0.001), body weight (P < 0.05) and smoking (P < 0.05).
Conclusions: In our investigation, age, gender and smoking (raising SCN− levels by CN− inhalation) were predictive for thyroid volume and the urinary I−/SCN− ratios were able to detect probands with an increased risk of developing goiter in contrast to urinary I− excretion levels alone. These data suggest, that in an era and area of decreased cyanide pollution, SCN− may remain a cofactor in the multifactorial aetiology of goiter.