United States and European Multicenter Prospective Study for the Analytical Performance and Clinical Validation of a Novel Sensitive Fully Automated Immunoassay for Calcitonin.

BACKGROUND: The objective of this study is the validation and proof of clinical relevance of a novel electrochemiluminescence immunoassay (ECLIA) for the determination of serum calcitonin (CT) in patients with medullary thyroid carcinoma (MTC) and in different diseases of the thyroid and of calcium homeostasis. METHODS: This was a multicenter prospective study on basal serum CT concentrations performed in 9 US and European referral institutions. In addition, stimulated CT concentrations were measured in 50 healthy volunteers after intravenous calcium administration (2.5 mg/kg bodyweight). RESULTS: In total, 1929 patients and healthy controls were included. Limits of blank, detection, and quantification for the ECLIA were 0.3, 0.5, and 1 ng/L, respectively. Highest intra- and interassay coefficients of variation were 7.4% (CT concentration, 0.8 ng/L) and 7.0% (1.1 ng/L), respectively. Medians (interval) of serum CT concentrations in 783 healthy controls were 0.8 ng/L (<0.5-12.7) and 3 ng/L (<0.5-18) for females and males, respectively (97.5th percentile, 6.8 and 11.6 ng/L, respectively). Diagnostic sensitivity and specificity were 100%/97.1% and 96.2%/96.4%, for female/males, respectively. Patients (male/female) with primary hyperparathyroidism, renal failure, and neuroendocrine tumors showed CT concentrations >97.5th percentile in 33%/4.7%, 18.5%/10%, and 8.3%/12%, females/males, respectively. Peak serum CT concentrations were reached 2 min after calcium administration (161.7 and 111.8 ng/L in males and females, respectively; P < 0.001). CONCLUSIONS: Excellent analytical performance, low interindividual variability, and low impact of confounders for increased CT concentrations in non-MTC patients indicate that the investigated assay has appropriate clinical utility. Calcium-stimulated CT results suggest good test applicability owing to low interindividual variability.

Increased chromogranin a and carcinoid syndrome-like symptoms in a patient treated with duloxetine.

OBJECTIVE: We report the case of a 50-year-old female patient who presented with symptoms suggestive of a serotonin-secreting neuroendocrine neoplasm. In addition, her serum chromogranin A (CA) level was elevated by more than 8-fold. METHODS: We present a case report with review of the relevant literature. RESULTS: No abnormalities could be detected in a complete conventional and functional morphological diagnostic work-up including a gallium-68-DOTA-d-Phe1-Tyr3-octreotide (Ga-68-DOTATOC) positron emission tomography-computed tomography (PET-CT) scan. These negative results prompted us to consider possible drug-related effects as the cause for these findings. The patient had started to take duloxetine, a second-generation antidepressant (SGA) and selective serotonin-norepinephrine reuptake inhibitor (SNRI), at a dose of 60 mg/day 2 months prior to her first visit at our department for pain relief. After withdrawal of duloxetine, her symptoms promptly ceased, and her CA levels fell to normal values within 7 weeks. CONCLUSION: We conclude that selective serotonin-norepinephrine reuptake inhibitors (SNRIs) can cause symptoms suggestive of serotonin-secreting neuroendocrine neoplasms, as well as elevated CA levels leading to unnecessary and expensive diagnostic workups. To our knowledge, the association between SNRI treatment and increased CA levels has not been described in the literature and needs to be further evaluated in well-controlled prospective studies.

Structural and functional MRI study of the brain, cognition and mood in long-term adequately treated Hashimoto's thyroiditis.

The current study investigated neuropsychological and underlying structural and functional brain alterations in long-term adequately treated patients with Hashimoto's thyroiditis in order to examine much discussed residual complaints in patients in relation to possible long-term neural alterations with a specific interest in the underlying autoimmune process. Eighteen patients with treated hypothyroidism due to Hashimoto's thyroiditis (mean age 32, range 18-54 years; two males; mean treatment duration 4.4 years) and 18 healthy matched control subjects underwent 3-Tesla magnetic resonance imaging (MRI). Voxel-based morphometry was used to investigate grey matter density, resting-state functional MRI to analyse the brain connectivity of areas known to be altered in hypothyroidism and event-related functional MRI to examine brain activity during associative memory encoding. Neuropsychological assessment included memory, working memory, psychomotor speed and attention. We previously reported subclinically reduced mood in this study population and investigated its neural correlates here. Thyroid stimulating hormone, free triiodthyronine, free thyroxine and thyroid peroxidase antibodies were measured in serum. We did not find cognitive deficits or alterations in grey matter density, functional connectivity or associative memory-related brain activity in comparison to the control group and cognition was unrelated to thyroid serum measures in the patient group. Thyroid peroxidase antibodies in the patient group correlated with increased grey matter density in right amygdala and enhanced connectivity between subcallosal and parahippocampal areas. Treatment duration was associated with brain structure in frontal and occipital cortex and connectivity between left amygdala and frontal cortex. Mood correlated with brain areas associated with distinct functional networks, but not with those most prominently affected in depression. In conclusion, no cognitive or neural alterations were detected in this young and otherwise healthy cohort of patients in comparison to a healthy control group and current mood status could not be related to depression-related networks. However, autoimmune activity and treatment duration showed a relationship with depression and hypothyroidism-related brain structure and function. They are thus promising factors to further investigate residual complaints despite biochemically adequate treatment in patients with Hashimoto's thyroiditis. Given the small sample size, all findings require replication.

Patient-reported outcomes in adequately treated hypothyroidism - insights from the German versions of ThyDQoL, ThySRQ and ThyTSQ.

BACKGROUND: Disease-specific patient-reported outcome measures (PROMs) have been developed as important research tools in the study of various diseases. For hypothyroidism there exist three validated disease-specific questionnaires in English: the Thyroid-Dependent Quality of Life Questionnaire (ThyDQoL), the Underactive Thyroid Symptom Rating Questionnaire (ThySRQ) and the Thyroid Treatment Satisfaction Questionnaire (ThyTSQ). We report psychometric properties of new German versions of the questionnaires including construct validity from two independent samples. METHODS: 230 envelopes with ThyDQoL, ThySRQ and ThyTSQ were given out to patients receiving levothyroxine for diagnosed hypothyroidism. Reliability and factor analyses were performed, correlations and hypothesised subgroup differences calculated to assess psychometric properties. Independently, 18 patients with treated hypothyroidism for autoimmune thyroiditis (Hashimoto's disease) and 18 healthy control subjects were enrolled in a clinical study. Participants filled in the above questionnaires alongside well-known generic PROMs, e.g. the Beck Depression Inventory, the 12-item Well-Being Questionnaire and the Short-Form-36. Two blood samples were taken. Groups were compared and correlations between disease-specific and generic instruments analysed. Relationships between PROMs and biochemically determined thyroid hormone status were investigated. RESULTS: 102 patients returned completed questionnaires (response rate 44%). The newly translated questionnaires had satisfactory psychometric properties. Cronbach's alpha was 0.92 for ThyDQoL, 0.81 for ThySRQ and 0.86 for ThyTSQ. For each of the questionnaires, a single factor structure explained the data best. Adequately treated patients with thyroid stimulating hormone levels in the upper normal range reported more symptoms in the ThySRQ. Those with autoimmune hypothyroidism reported being more bothered by depressive symptoms. Within the clinical sample, correlation with well-known generic instruments revealed good construct validity. In the clinical sample patients reported more symptoms in the ThySRQ, being more bothered by tiredness, higher depression and reduced well-being despite biochemically adequate treatment. Correlations between PROMs and biochemical thyroid hormone status revealed moderate though consistent associations. CONCLUSIONS: Psychometric properties including construct validity of German versions of the ThyDQoL, ThySRQ and ThyTSQ are satisfactory. Feasibility and sensitivity in a clinical sample could be shown. We encourage the use of disease-specific PROMs in future studies as important additions to generic instruments in clinical research on hypothyroidism.

Distinct pattern of oxidative DNA damage and DNA repair in follicular thyroid tumours.

Increased oxidative stress has been linked to thyroid carcinogenesis. In this paper, we investigate whether oxidative DNA damage and DNA repair differ in follicular adenoma (FA) and follicular thyroid carcinoma (FTC). 7,8-Dihydro-8-oxoguanine (8-OxoG) formation was analysed by immunohistochemistry in 46 FAs, 52 FTCs and 18 normal thyroid tissues (NTs). mRNA expression of DNA repair genes OGG1, Mut Y homologue (MUTYH) and endonuclease III (NTHL1) was analysed by real-time PCR in 19 FAs, 25 FTCs and 19 NTs. Induction and repair of oxidative DNA damage were studied in rat FRTL-5 cells after u.v. irradiation. Moreover, activation of DNA damage checkpoints (ataxia telangiectasia mutated (ATM) and H2A histone family, member X (H2AFX (H2AFX))) and proliferation index (MIB-1) were quantified in 28 non-oxyphilic and 24 oxyphilic FTCs. Increased nuclear and cytosolic 8-OxoG formation was detected in FTC compared with follicular adenoma, whereby cytosolic 8-OxoG formation was found to reflect RNA oxidation. Significant downregulation of DNA repair enzymes was detected in FTC compared with FA. In vitro experiments mirrored the findings in FTC with oxidative stress-induced DNA checkpoint activation and downregulation of OGG1, MUTYH and NTHL1 in FRTL-5 cells, an effect that, however, was reversible after 24  h. Further analysis of FTC variants showed decreased oxidative DNA damage, sustained checkpoint activation and decreased proliferation in oxyphilic vs non-oxyphilic FTC. Our data suggest a pathophysiological scenario of accumulating unrepaired DNA/RNA damage in FTC vs counterbalanced DNA/RNA damage and repair in FA. Furthermore, this study provides the first evidence for differences in oxidative stress defence in FTC variants with possible implications for therapeutic response and prognostic outcome.

[Thyroid diseases and pregnancy]. / Schilddrüsenerkrankungen und Schwangerschaft.

Thyroid diseases in pregnancy must be recognized as a specific challenge for the clinician. Any pregnancy is causing alterations in thyroid hormone metabolism which have to be differentiated from pathologic states of thyroid function. Any thyroid disease of the mother with disturbances in the functional state of the gland could induce an adverse influence on the course of pregnancy. Furthermore, it can be associated with adverse consequences on fetal development. Especially hypothyroidism has to be avoided during pregnancy due to a danger of affected neurocognitive development of the offspring. Yet also maternal hyperthyroidism can lead to impairments in the course of pregnancy and to fetal thyroid dysfunction. Further clinical attention should be given to thyroid autoimmunity. There is a clear relationship between autoimmune thyroid disease and decreased fertility and an increase in the rate of spontaneous miscarriages. Furthermore, it displays an increased risk for the manifestation of postpartum thyroiditis. The management of nodular thyroid disease and malignancy does not differ from that of nonpregnant women/patients. Thyroid scintiscan and radioiodine therapy must be avoided during pregnancy and lactation. This review deals with the broad variety of thyroid disorders and function disturbances during and after pregnancy. All described diagnostic and therapeutic procedures are based upon the recent Clinical Practice Guideline of the Endocrine Society published in August 2007.

FOXO3a: a novel player in thyroid carcinogenesis?

The forkhead box transcription factor FOXO3a has recently been identified as central mediator of the cellular response to oxidative stress inducing cell cycle arrest or apoptosis. The aim of our study was to investigate the regulation of FOXO3a in the thyroid and to determine whether alterations in FOXO3a activity occur in thyroid carcinogenesis. In vitro, we demonstrate that FOXO3a activity is negatively regulated by the PI3K/Akt cascade promoting increased phosphorylation and cytoplasmatic accumulation of FOXO3a with decreased transcription of the target genes p27kip (CDKN1B) and Bim (BCL2L11), but increased expression of GADD45A. By contrast, we show that H(2)O(2) exposure activates FOXO3a in thyrocytes with JNK (MAPK8)-mediated nuclear accumulation of FOXO3a and increased expression of the cell cycle arrest genes p27kip and GADD45A. In vivo, we observed a marked cytoplasmatic accumulation of FOXO3a in differentiated thyroid cancers versus an exclusive nuclear accumulation in follicular adenoma and normal thyroid tissue. Moreover, this cytosolic accumulation of FOXO3a correlated with an increased phospho-Akt expression in thyroid malignancies and was accompanied by decreased expression of the FOXO targets p27kip and Bim and an increase in GADD45A mRNA expression in the thyroid cancers. Our data suggest FOXO3a as a novel player of cellular stress response in the thyroid, mediating the thyrocyte's fate either to survive or to undergo apoptosis. Furthermore, PI3K-dependent FOXO3a inactivation may be a novel pathomechanism for the escape from apoptosis in thyroid cancer cells, in particular in follicular thyroid carcinoma.

Forkhead box-O transcription factor: critical conductors of cancer's fate.

Cells have evolved elaborated mechanisms to coordinate the cellular answer of either survival or apoptosis. Recent concepts of human carcinogenesis have suggested disturbances in these cellular relays as a potential link to cellular dedifferentiation and uncontrolled proliferation. Forkhead box-O transcription factors (FOXOs) play an important role in tumour suppression by regulating the expression of genes involved in stress resistance, DNA damage repair, cell cycle arrest and apoptosis. The specific regulation of FOXO function is tightly controlled by posttranslational modifications such as phosphorylation, acetylation and ubiquitination. Loss of FOXO function has recently been identified in several human cancers. In this review, we will give an overview about recent progress in the understanding of function and regulation of FOXOs, as well as their role in carcinogenesis. Furthermore, we will discuss a potential clinical use of FOXOs by therapeutically restoring their tumour suppressive properties.

Evidence for a role of the amyloid precursor protein in thyroid carcinogenesis.

We have recently found an increased expression of amyloid precursor protein (APP) in cold thyroid nodules that are difficult to classify as a truly benign thyroid neoplasm or a lesion with the potential for further dedifferentiation. Since differences in APP activity have been found in other human cancers, we asked whether thyroid carcinogenesis might be associated with an altered APP expression and function. APP regulation was studied in vitro in differentiated (FRTL-5) and dedifferentiated follicular thyroid carcinomas (FTC-133) thyroid cells after specific inhibition or activation of the cAMP-PKA, the PI3K/AKT or the protein kinase c (PKC) cascades. In vivo analysis of APP expression and downstream signalling was performed in benign and malignant thyroid tissues. We found that upregulation of APP expression and sAPP secretion is induced by TSH in differentiated thyroid cells and by insulin in thyroid cancer cells. PKC is a strong activator of APP cleavage and in FTC-133 confers prolonged release of the APP ectodomain. FTC-133 but not FRTL-5 cells show a prominent cell surface expression of the APP ectodomain, which has been suggested to function as an autocrine growth factor. Thyroid cancers are characterized by APP upregulation, increased membrane targeting of the APP ectodomain and significantly increased mRNA levels of the APP scaffold proteins JIP1, ShcA and Fe65.

TFF3-based candidate gene discrimination of benign and malignant thyroid tumors in a region with borderline iodine deficiency.

BACKGROUND: With the advent of microarray technology, increasing numbers of marker genes are proposed to distinguish benign and malignant thyroid lesions. However, most markers await confirmation through independent studies. In this paper, we re-evaluate the diagnostic potential of 10 proposed candidate genes in benign and malignant thyroid pathologies in a region with borderline iodine deficiency. METHODS: Quantitative real-time PCR was performed for CCND2, PLAB, PCSK2, HGD1, TFF3, B4GALT, LGALS3, ETS1, ADM3, and TG in 150 thyroid specimens, including 52 benign thyroid nodules (28 follicular adenoma and 24 adenomatous nodules), 52 corresponding normal thyroid tissues, 20 follicular carcinomas, 20 papillary carcinomas, and six undifferentiated carcinomas. RESULTS: On a single-gene basis, significant differences in mRNA expression were found for TFF3, PLAB, and ADM3 in benign thyroid nodules and thyroid malignancy. Using two-marker gene sets, we identified 11 combinations, which allowed both a distinction of benign and malignant thyroid nodules and a discrimination of follicular adenoma and carcinoma. However, for cancer prediction, analysis of a minimum of six genes per sample was necessary and allowed correct prediction of a benign thyroid lesion and thyroid cancer with 94% accuracy in the most discriminative set (TFF3/PLAB/TG/ADM3/HGD1/LGALS3). CONCLUSION: We confirm the applicability of a number of recently proposed marker genes for the distinction of benign and malignant thyroid tumor and suggest that their diagnostic usefulness is independent of the iodide supply. We propose that the most discriminative marker set identified in our validation study together with marker combinations proposed by other investigators should now be evaluated in multicenter trials.

Characterisation of DEHAL1 expression in thyroid pathologies.

UNLABELLED: Iodotyrosine dehalogenase 1 (DEHAL1) is a transmembrane protein involved in the recycling of iodide in the human thyroid. The aim of the present study was (I) to investigate whether DEHAL1 expression is different in differentially functioning thyroid pathologies and (II) to evaluate DEHAL1 as a possible marker of thyroid cell differentiation. DESIGN AND METHODS: Real-time PCR for DEHAL1 and its isoform DEHAL1B was performed in a series of 105 thyroid specimens, including toxic thyroid nodules (TTN), Graves' disease (GD) thyroids, benign cold thyroid nodules (CTN), normal thyroid tissues and thyroid cancers (follicular thyroid carcinomas (FTC), papillary thyroid carcinomas (PTC), partially differentiated thyroid cancers (PDTC) and anaplastic thyroid carcinomas (ATC)). In addition, DEHAL1 protein expression was studied by immunohistochemistry in 163 benign and malignant thyroid pathologies and normal thyroids. RESULTS: (I) The highest DEHAL1 mRNA levels were found in GD thyroids, while downregulation of DEHAL1 and DEHAL1B mRNA occurred in PTC and ATC (P<0.001 and <0.05 respectively); (II) DEHAL1 protein was overexpressed in TTNs and GD thyroids with predominant apical staining in all samples; (III) a weaker and patchy staining pattern was found in CTNs and normal thyroids; (IV) in differentiated thyroid cancers (FTC and PTC), a diffuse cytoplasmic DEHAL1 expression was found; and (V) in PDTC and ATC, DEHAL1 expression was faint or absent. CONCLUSION: Upregulation of DEHAL1 protein expression and sublocalisation of DEHAL1 at the apical cell pole in TTNs and GD thyroids is consistent with increased thyroid hormone turnover during thyrotoxicosis. Diffuse cytoplasmatic localisation or downregulation of DEHAL1 expression in thyroid cancers suggests alteration or loss of DEHAL1 function during thyroid cell dedifferentiation.

Proteomic profiling of cold thyroid nodules.

Cold thyroid nodules (CTNs) represent a frequent endocrine disorder accounting for up to 85% of thyroid nodules in a population living in an iodine-deficient area. Benign CTNs need to be distinguished from thyroid cancer, which is relatively rare. The molecular etiology of benign CTNs is unresolved. To obtain novel insights into their pathogenesis, protein expression profiling was performed in a series of 27 solitary CTNs (10 follicular adenoma and 20 adenomatous nodules) and surrounding normal thyroid tissues using two-dimensional gel electrophoresis combined with mass spectrometry analysis, Western blotting, and immunohistochemistry. The proteome analysis revealed a specific fingerprint of CTNs with up-regulation of three functional systems: 1) thyroid cell proliferation, 2) turnover of thyroglobulin, and 3) H2O2 detoxification. Western blot analysis and immunohistochemistry confirmed the proteome data and showed that CTNs exhibit significant up-regulation of proteins involved in thyroid hormone synthesis yet are deficient in T4-containing thyroglobulin. This is consequential to intranodular iodide deficiency, mainly due to cytoplasmic sodium iodide symporter localization, and portrays the CTN as an activated proliferating lesion with an intranodular hypothyroid milieu. Furthermore, we provide preliminary evidence that up-regulation of H2O2 generation in CTNs could override the antioxidative system resulting in oxidative stress, which is suggested by the finding of raised 8-oxo-guanidine DNA adduct formation in CTNs.

Impaired enteral levothyroxine absorption in hypothyroidism refractory to oral therapy after thyroid ablation for papillary thyroid cancer: case report and kinetic studies.

We present a 49-year-old patient suffering from hypothyroidism (thyrotropin [TSH], 20-80 mU/L) refractory to oral levothyroxine (LT(4)) substitution after total thyroidectomy and radioiodine therapy for papillary thyroid cancer. Extensive, repetitive work-up excluded small bowel, liver, and pancreatic disease and drug interactions. Triiodothyronine/thyroxine (T(3)/T(4)) antibodies were negative and reverse T(3) levels were normal. Supervised absorption tests of several LT(4) preparations in increasing dosages (250-1500 microg/d) confirmed an insufficient rise in serum T(4) levels following oral drug administration. Thus, intravenous LT(4) application (5 times per week) was commenced to restore at least normal range TSH levels. Repetition of absorption tests 1 year later, after a documented period of more than 3 months of stable euthyroidism, showed a considerable improvement of intestinal LT(4) uptake. Subsequently, the patient was managed on large doses of oral LT(4) (1500-2100 microg/d) alone, but this was found to be insufficient so that continuous intravenous LT(4) was administered via a morphine pump device to ultimately achieve stable euthyroidism. Of note, at 4-year follow-up there was no evidence of recurrent or metastasized papillary thyroid cancer. The reason for the disturbed intestinal LT(4) absorption in this patient remains unresolved. However, we would like to suggest the possibility of a specific intestinal uptake deficit, which will only become apparent in the case of previous thyroid ablation.