Pure clear cell lesions of the thyroid gland are rare and prone to pose severe diagnostic challenges. We report the case of a 61-year-old man with a clear cell adenoma of the thyroid. The patient presented with a hypoechoic thyroid nodule. Fine needle aspiration cytology rendered the primary diagnosis of a follicular neoplasia, and right thyroid lobectomy was performed. By intra-operative frozen section, the diagnosis given was clear cell lesion of unknown malignant potential. Based on the light microscopic findings and the immunohistochemical profile, the lesion was diagnosed as clear cell follicular adenoma of the thyroid. A follicular thyroid lesion presenting with clear cell changes in fine needle aspiration cytology or intra-operative frozen section consultation constitutes a diagnostic challenge to every surgical pathologist. As immunohistochemistry of cytologic specimens is hampered by several methodological problems, any thyroid lesion with clear cell features warrants further histologic assessment to render the correct diagnosis.

Thyroid stimulating hormone (TSH) causes differentiation and epidermal growth factor (EGF) causes dedifferentiation of thyroid cells in vitro. In undifferentiated thyroid cancer cell lines, TSH stimulates tumor cell migration and invasion, a dedifferentiated function, presumably due to an escape of tumor cells from the control of differentiating growth factors. In a highly differentiated thyroid carcinoma cell line of Hürthle cell origin (XTC), we tested the hypothesis that TSH would stimulate thyroglobulin secretion (a differentiated function) more than EGF, and EGF would stimulate invasion (a de-differentiated function) more than TSH. Proliferation, adhesion, cell migration and invasion were measured by the MTT assay, human thyroglobulin by RIA and protease activity by substrate-gel zymography. TSH induced differentiated morphologic changes in XTC cells and stimulated secretion of human thyroglobulin in a dose dependent manner, whereas EGF did not. The effects of TSH on growth, adhesion, migration and invasion were dose dependent and biphasic, with an increase at low and a decrease at high concentrations of TSH. These effects were always more pronounced than those observed with EGE Gelatinolytic activity, consistent with metalloproteinase activity was revealed by zymography, but the pattern of secretion was not altered by neither TSH nor EGF. These results suggest, that TSH has pleiotropic effects on differentiated thyroid cancer cells in vitro that involve differentiated morphology and function but also affect features commonly associated with the malignant in vitro phenotype.

OBJECTIVE:The aim of this study was to compare the rate of hypocalcaemia after bilateral thyroid resection for Graves' Disease in patients with and without parathyroid gland autotransplantation (PTAT).PATIENTS AND METHODS:A total of 153 patients following thyroid surgery for Graves' disease were studied. Patients were divided into three groups. Subgroup I comprised of 129 patients without PTAT, Subgroup II comprised of 19 patients with PTAT of one parathyroid gland and Subgroup III comprised of 6 patients with PTAT of two parathyroid glands. Association between PTAT and postoperative hypocalcaemia (PH) after thyroidectomy was investigated.RESULTS:There were 27/128 (21.1%) patients with transient and 10/128 (7.8%) patients with permanent hypocalcemia within Subgroup I. Among Subgroup II 4/19 (21.1%) patients developed transient and no patient developed permanent hypocalcaemia, while in Subgroup III 2/6 (33.3 %) patients developed transient hypocalcaemia which became persistent. Thus, the frequency of permanent postthyroidectomy hypocalcaemia (PH) was significantly higher in the Subgroup III with PTAT of two parathyroid glands when compared to the Subgroup I without PTAT (p=0.032) and Subgroup II with PTAT of only one parathyroid glands (p=0.012).CONCLUSION:PTAT of one parathyroid gland is an effective procedure to reduce the incidence of permanent hypoparathyroidism after bilateral surgery for Graves disease. Transient hypoparathyroidism was not influenced by PTAT.

This study evaluated the role of EGF and the effects of EGF-targeting drugs (Cetuximab, AEE 788) on growth, apoptosis, and autocrine VEGF-secretion of thyroid cancer (TC) cells. Autocrine activation of the epidermal growth factor receptor (EGF-R) is commonly regarded to contribute to the malignant phenotype of TC cells and may therefore represent a rational therapeutic target. Out of a number of TC cell lines two anaplastic (Hth74, C643), one follicular (FTC133), and one papillary thyroid cancer cell line (TPC1) were analyzed in depth for VEGF-R-and EGF-R-expression, basal and EGF-stimulated (1–100 ng/ml) VEGF protein secretion and proliferation. Subsequently the antiprolifereative and antiangiogenic effect of cetuximab (Erbitux®), a monoclonal antibody that blocks the EGF-R and AEE 788, a novel dual-kinase inhibitor of EGF-R and VEGF-R were assessed, and the downstream EGF-R signal transduction was analyzed by means of detecting phosphorylated pEGF-R, pVEGF-R, pAkt, and p-MAPK. EGF stimulated VEGF-mRNA expression and protein secretion in all TC cell lines. The EGF-R antagonist Cetuximab consistently decreased VEGF secretion in all TC cell lines (min. 15%, n.s. in C643 cells and max. 90% in Hth74 cells, P < 0.05), but did not affect tumor cell proliferation in␣vitro. In contrast, the EGF-R- and VEGF-R-kinase inhibitor AEE 788 not only reduced VEGF secretion (min. 55%, P < 0.05 in C643 and max. 75%, P < 0.05, in FTC133), but also exhibited a dose-dependent inhibition of tumor cell proliferation (min. 75%, P < 0.05 in C643 and max. 95%, P < 0.05 in Hth74) and was a potent inductor of apoptosis in two of four TC cell lines. These effects were always accompanied by reduced levels of pEGF-R, pVEGF-R, pAkt, and pMAPK. Although inhibition of the EGF-receptor by Cetuximab potently disrupts autocrine secretion of VEGF, only the concurrent inhibition of the VEGF- and EGF receptor, e.g., by AEE 788 induces reduced proliferation and apoptosis in␣vitro. This suggests a particular rationale for the use of tyrosine kinase inhibitors with dual modes of action such as AEE 788 in thyroid cancer.

Inactivating mutations of wild-type p53 (WTp53) tumor suppressor gene are common in anaplastic thyroid cancer (ATC) and are associated with poor prognosis. Mutated p53 (MTp53) has been implicated with angiogenesis. Therefore, the potential of MTp53 knockout by oligodeo xyribonucleotide phosphorothioates (ODNs) to affect VEGF production of undifferentiated thryoid cancer cells with a recessive MTp53 mutation was evaluated. Transient transfection with 20 bp ODNs complementary to portions of exon 10 of p53 and a negative contr of ODN (HIV-RT) were carried out in FTC-133 cells. In vitro secretion of VEGF protein was quantified by EIA and correlated to cell numbers, which was evaluated by in vitro MTT assay. Transfection of undiffarentifiated thyroid cancer cells with ODN reduced VEGF secretion of FTC-133 cells following transfection by 34% as compared to the negative control (cell transfected with ODN-HIV; p=0.03). These results suggest that transient MTp53 knockout with ODNs complementary to p53 nucleotide sequences impair secretion of VEGF in the undifferentiated thyroid cancer cell line FTC-133.

Tumor angiogenesis is believed to result from an imbalance of pro- and anti-angiogenic factors, some of which are candidates for targeted therapy. Such therapy has raised hopes for patients with undifferentiated thyroid carcinomas, who are facing a grave prognosis with a survival of only months. In this study, in vivo growth of xenografted human thyroid carcinomas unexpectedly responded quite differently to neutralizing anti-vascular endothelial growth factor (VEGF) antibody. In particular, lasting inhibition as well as accelerated growth occurred after treatment. Consequently, a panel of anti-angiogenic factors was addressed in a representative sample of thyroid carcinoma lines. VEGF, fibroblast growth factor (FGF-2), and endostatin were demonstrated by Western blotting and EIA, whereas PDGF-A, PDGF-B, and IL-6 were negative. Quantification of VEGF, FGF-2, and endostatin revealed a wide range of concentrations from 500 to 4,200 pg/ml VEGF, 5 to 60 pg/ml FGF-2, and 50 to 300 pg/ml endostatin, not related to a particular histologic thyroid carcinoma background. Angiostatin (kringles 1-3) was detected in all, but one of the cell lines. Finally, aaATIII was confirmed in FTC133 cells. These data highlight the complex regulation of angiogenesis in thyroid carcinoma cell lines and suggest that the array of angiogenic factors differs markedly between individual cell lines. For the first time, angiostatin, endostatin, and possibly also aaATIII are identified as novel candidate regulators of angiogenesis in thyroid carcinoma cells.

In thyroid cancer (TC), endothelial growth factor (EGF) has been associated with dedifferentiation, tumor cell proliferation, and angiogenesis. Vascular endothelial growth factor (VEGF) has been documented to be the main stimulator of angiogenesis in the thyroid gland. Patients with undifferentiated thyroid cancer are in desperate need of new therapeutic strategies because common protocols of therapy usually fail. The aim of this study, therefore, was to evaluate two tyrosine-kinase inhibitors (TKI, ZD 1839 gefitinib and ZD 6474 vandetanib), directed against the EGF/VEGF receptor for possible antitumor therapy in thyroid cancer.

BACKGROUND:The potential of MTp53 knockout by oligodesoxyribonucleotide phosphothioates (ODNs) to affect proliferation, apoptosis and chemosensitivity in undifferentiated thyroid cancer (UTC) cells with a recessive MTp53 mutation was evaluated.MATERIALS AND METHODS:Transient transfections with ODNs complementary to p53 and control ODN (HIV-RT) were carried out in FTC 133 cells. In vitro proliferation was evaluated by cell counting of 10 random fields and by the MTT assay. A single pulse of 100 microg/ml Cytarabine was added to each well and the cells were incubated for an additional day. Chemosensitivity was calculated as the ratio of apoptotic and necrotic cells versus viable cells by flow cytometry (FACS).RESULTS:Transfection of UTC cells with ODN decreased the cell number by up to 70% (p < 0.002). The proliferation rate also decreased up to 35% (p < 0.03), without inducing apoptosis. ODNs rendered FTC cells sensitive to treatment with Cytarabine, inducing apoptosis in 35% of cells, as compared to 17% of cells transfected with the reverse transcriptase gene of HIV (ODN-HIV) and less than 10% of non-transfected cells (p < 0.05).CONCLUSION:Transient MTp53 knockout with ODNs complementary to p53 nucleotide sequences inhibited proliferation and increased chemosensitivity in the UTC cell line FTC133.

Thyrotropin (TSH) is a thyroid-specific growth factor inducing differentiated function and growth of thyrocytes in vitro. In thyroid cancer, loss of TSH-receptor (TSHR) expression is a sign of de-differentiation and is believed to contribute to the malignant phenotype. The present studies aimed to determine the in vitro and in vivo effects of functioning tSHR in the follicular thyroid cancer cell line HTC, a subclone of FTC133 cells, lacking endogenous expression of TSHR, and HTCtshr+cells transfected with human TSHR-cDNA. HTCtshr+cells grew faster in vitro (doubling time 1.15 vs 1.56 d, p<0.05) and TSH caused a dose-dependent growth response. Adhesion to and invasion through reconstituted basement membrane were reduced in HTCtshr+cells, but when stimulated with TSH increased to levels comparable to naïve HTC cells. In vivo, tumor latency was 11 d for naïve HTC as compared to 21 d for HTCtshr+xenografts. Smaller tumor volumes were registered for HTCtshr+cells (250±217 vs 869±427 mm3, p<0.05). Angiogenesis, as determined by vascular surface density density (VSD) of experimental tumors, was enhanced in naïve HTC tumors (VSD 0.87±0.1 μm−1 vs 0.55±0.2μm−1 in HTCtshr+, p<0.05). VEGF secretion was more pronounced in naïve HTC cells stimulated with EGF, than in HTCtshr+cells stimulated with either TSH or EGF. In conclusion, regained expression of functional TSHR in the follicular thyroid cancer cell line HTC alters in vitro features commonly associated with the malignant phenotype. Smaller tumors and reduced angiogenesis of xenotransplanted HTC cells with functioning TSHR suggest a less aggressive in vivo phenotype. The present data highlight the pivotal role of TSHR to affect transformed thyrocytes in vitro and in vivo. They also suggest a role for EGF as a modulator of angiogenesis in thyrocytes devoid of TSHR.

Die vorliegenden Untersuchungen bestätigen die Erfahrung aus anderen Tumorentitäten, dass das EGF ein potenter Stimulus von Tumorwachstum von VEGF-assoziierter Tumorangiogenese ist. Auch beim Schilddrüsenkarzinom findet sich eine Assoziation zu Tumorzellproliferation und VEGF-Sekretion in vitro. Allerdings zeigt sich, dass hier deutliche zelllinienspezifische Unterschiede in der Regulation durch EGF bestehen. So kann man zusammenfassen, dass es sich hierbei offensichtlich eher um ein mehr zelllinientypisches als histotypisches Phänomen handelt. Durch Anwendung eines dualen Tyrosinkinaseinhibitors (AEE 788®) scheint es möglich, sowohl die EGF-assoziierten Wirkungen inklusive der Wirkung auf die VEGF-Sekretion, als auch die VEGF-rezeptorvermittelten Wirkungen zu inhibieren. So konnte neben der Wachstumshemmung in vitro und der Reduktion der VEGF-Sekretionsleistung auch die Hemmung der rezeptorassoziierten Signaltransduktionskaskade durch AEE 788® gezeigt werden. Eine Bestätigung dieser Effekte in vivo vorausgesetzt, scheint der Einsatz des dualen Tyrosinkinaseinhibitors AEE 788® ein vielversprechender neuer Therapieansatz in der Behandlung wenig differenzierter und anaplastischer Schilddrüsentumoren zu sein.

Hemangioendotheliomas of the thyroid are neoplasms that are rarely encountered in the Alpine region in Europe. The endemic goiter in this iodine-deficient region can be considered as a predisposing factor. We report the second case of an epithelioid hemangioendothelioma (EHE) of the thyroid gland and the first clinical case with malignant behavior. A 73-year-old woman with a long history of multinodular goiter presented with a rapidly growing thyroid mass, free of distant metastases. A total thyroidectomy was performed. Histologically the lesion had characteristic epithelioid and spindle cell proliferation in a background of chondromyxoid stroma. Cytoplasmic vacuolization in the epithelioid and spindle cells were present. Tumor cells were positive for factor VIII-related antigen, MNF116, PAN-ceratin, CD 34, and CD 31 and negative for cytokeratin allowing the diagnosis of EHE. Nine months after the primary resection the tumor locally recurred. A palliative resection was performed in a second operation followed by interferon-alpha therapy. The patient rapidly developed a second local relapse and died 13 months after the diagnosis of an EHE had been made. An improvement of the very poor prognosis of this rare tumor may depend on early tumor diagnosis and surgery combined with a multimodal treatment.

Aim: The EGF/EGF-receptor (EGFR) system in thyroid neoplasia has long since been associated with de-differentiation, enhanced proliferation and angiogenesis. Angiogenesis is essential for tumor growth and VEGF is the main stimulator of angiogenesis in the thyroid. This study aimed to evaluate the EGF/EGFR system to regulate VEGF in thyroid cancer cell lines and to evaluate the potential of inhibitors to affect such regulation.

Adhesion of tumor cells to the extracellular matrix (ECM) is a crucial step for the development of metastatic disease and is mediated by specific integrin receptor molecules (IRM). The pattern of metastatic spread differs substantially among the various histotypes of thyroid cancer (TC). However, IRM have only occasionally been characterized in TC until now. IRM expression was investigated in 10 differentiated (FTC133, 236, 238, HTC, HTC TSHr, XTC, PTC4.0/4.2, TPC1, Kat5) and two anaplastic TC cell lines (ATC, C643, Hth74), primary cultures of normal thyroid tissue (Thy1,3), and thyroid cancer specimens (TCS). Expression of 16 IRM (beta1-4, beta7, alpha1-6, alphaV, alphaIIb, alphaL, alphaM, alphaX) and of four IRM heterodimers (alpha2beta1, alpha5beta1, alphaVbeta3, alphaVbeta5), was analyzed by fluorescent-activated cell sorter (FACS) and immunohistochemical staining. Thyroid tumor cell adhesion to ECM proteins and their IRM expression in response to thyrotropin (TSH) was assessed. Follicular TC cell lines presented high levels of integrins alpha2, alpha3, alpha5, beta1, beta3 and low levels of alpha1, whereas papillary lines expressed a heterogenous pattern of IRM, dominated by alpha5 and beta1. ATC mainly displayed integrins alpha2, alpha3, alpha5, alpha6, beta1 and low levels of alpha1, alpha4 and alphaV. Integrin heterodimers correlated with monomer expression. Evaluation of TCS largely confirmed these results with few exceptions, namely alpha4, alpha6, and beta3. The ability of TC cell lines to adhere to purified ECM proteins correlated with IRM expression. TSH induced TC cell adhesion in a dose-dependent fashion, despite an unchanged array of IRM expression or level of a particular IRM. Thyroid carcinoma cell lines of different histogenetic background display profoundly different patterns of IRM expression that appear to correlate with tumor aggressiveness. In vitro adhesion to ECM proteins and IRM expression concur. Finally, TSH-stimulated adhesion of thyroid tumor cell lines to ECM may not be associated with altered IRM expression.

Thyroid tumor growth requires angiogenesis, and vascular endothelial growth factor (VEGF) has been shown to be the most important endothelial mitogen. TSH is the major thyrotropic hormone, but its impact to modulate VEGF production has not yet been studied. Several other growth factors have also been shown to affect thyroid cancer cell growth and function in vitro. Therefore, the aim of the current study was to 1) establish the effect of TSH on VEGF as well as 2) evaluate the TSH signal transduction of this effect, and 3) screen other growth factors for the ability to modulate VEGF in thyroid cancer cell lines. HTC, a follicular cancer cell line lacking endogenous TSH receptor (TSHr), its receptor positive variant (HTC TSHr), and a cell line of Huerthle cell origin (XTC) were used. After stimulation with growth factors in vitro [TSH; epidermal growth factor (EGF), IGF, placenta growth factor, TGF-alpha, TGF-beta1, fibroblast growth factor, platelet-derived growth factor, and hepatocyte growth factor] cells were analyzed for VEGF gene expression by Northern blotting and for VEGF protein by enzyme immunoassay. TSHr signal transduction was evaluated by analyzing the effect of stimulators (cholera toxin, 8-bromo-cAMP, forskolin, and 12-O-tetradecanoyl-phorbol-13-acetate) and inhibitors (2',5'-dideoxyadenosine and staurosporine) on VEGF protein levels under basal and TSH-stimulated conditions. TSH increased VEGF mRNA and protein in a dose-dependent manner in HTC TSHr and XTC cells by up to 40%. The effects of TSH were mediated by protein kinase C (PKC), rather than protein kinase A (PKA), stimulation, because inhibition of PKC by staurosporine resulted in a decrease in VEGF production of up to 65%, whereas inhibition of the PKA signal transduction pathway (2',5'-dideoxyadenosine) resulted in only a minor decrease. TSH was not the most powerful stimulator of VEGF production. TGF-beta1 and EGF were 1.5- to 2-fold more potent. Placenta growth factor and TGF-alpha did not induce VEGF production in TSHr-positive HTC cells, whereas they did induce VEGF production in TSHr-negative HTC cells. In thyroid cancer cell lines, TSH induces VEGF production involving the PKC, rather than the PKA, pathway. However, EGF and TGF-beta increase the capacity of thyroid cancer cells to provide VEGF more effectively than TSH. In the absence of a functioning TSHr, additional growth factors, such as TGF-alpha, increase capacity for VEGF stimulation.

At times, the diagnosis of acute appendicitis may be difficult. However, for minimum morbidity to be obtained, early and accurate diagnosis is essential. This study aimed to validate a scoring system proposed by Eskelinen et al. as an aid in making the diagnosis of appendicitis.

Background. Blind, randomised trials are conceived as the gold standard in clinical research, but this ideal, in its strict sense, can rarely be achieved in surgical settings. One way to strengthen the study design is to check for observer bias in the assessment and evaluation of surgical outcome. Method. In a randomised, prospective trial comparing nasogastric versus gastrostomy tubes the primary endpoint was the subjective inconvenience induced by the tube system and was assessed in the context of a standardised face-to-face interview. These interviews were tape-recorded on a pocket memo. Two independent raters listened to these interviews and judged-on the basis of how the interviewer formulated the questions-which treatment arm they thought the patients were assigned to and how confident they were in their judgement. Results. The overall proportion of correct judgements was 50.5% for rater 1 and 53.2% for rater 2. In other words, both judgement performances were not greater than chance. Nevertheless, the raters' confidence in their judgements increased significantly (P<0.05) in the course of the rating procedure, whereas the actual proportion of correct judgements did not. There was no overlap between the two raters [kappa = 0.022, not significant (NS)] and between actual group assignment and both raters' judgements (kappa = 0.012, NS and kappa = 0.110, NS). Conclusion. The two independent raters were not able to detect systematic variations in the interviewing style that were contingent on treatment arm assignment. This gives further credence to the results of the randomised trial showing greater patient-reported discomfort and inconvenience with the nasogastric tube than with the gastrostomy tube. The present report describes a feasible method to monitor subtle biases that may occur in trial settings. This helps to strengthen the design of randomised clinical trials in surgery.

BACKGROUND AND AIM:At times, the diagnosis of acute appendicitis may be difficult. However, for minimum morbidity to be obtained, early and accurate diagnosis is essential. This study aimed to validate a scoring system proposed by Eskelinen et al. as an aid in making the diagnosis of appendicitis.PATIENTS AND METHODS:The prospectively documented data of a consecutive series of 2,359 patients admitted for suspicion of appendicitis were used for validation. Accuracy and positive predictive value were defined as the main overall performance parameters, as was the rate of unnecessary operations to assess changes of patient management. Overall performance was assessed by receiver-operator characteristics (ROC) analysis.RESULTS:Of 2,359 patients, 662 were proven to have acute appendicitis (prevalence of 28%). The overall sensitivity, specificity, positive and negative predictive value, and accuracy of the score were 0.79, 0.85, 0.68, 0.91 and 0.835 at a cut-off value of 55. Calibration of the score's cut-off value to 57 yielded more favourable results (0.72, 0.91, 0.76, 0.9 and 0.86), and the rate of unnecessary operations declined from 26.6% to 15.4% ( P<0.05, chi2). ROC analysis revealed an area index of 0.91.CONCLUSION:The Eskelinen score delivered acceptable clinical results only after calibration to a cut-off value of 57. The data from this study suggest the investigation of whether a calibrated score might be particularly instrumental in the pre-admission evaluation of the patient in whom appendicitis is suspected.