Evaluation of Betulinic Acid Derivatives as PET Tracers for Hypoxia-Induced Carbonic Anhydrase IX (CA IX) Expression.

Non-invasive visualisation of the expression of hypoxia-related proteins, such as carbonic anhydrase IX (CA IX), by positron emission tomography (PET) could provide important information on the oxygenation status of tumours. Since betulinic acid derivatives bind specifically to CA IX the aim of the study was the development betulinic acid-based 68Ga-labelled PET tracers and to evaluate the hypoxia detecting properties in vitro and in vivo. The binding of betulinic acid (B-DOTA) and betulinyl-3-sulfamate (BS-DOTA) was assessed in two rat tumour cell lines (AT1 prostate and Walker-256 mammary carcinomas). AT1 cells express CA IX in a hypoxia-dependent manner whereas Walker-256 cells, expressing almost no CA IX in wildtype, were transfected with the rat Car9 gene. In vivo measurements were carried out in a small animal PET/CT in AT1 tumours in rats breathing room air, 8% or 100% O2. In AT1 cells hypoxia-induced overexpression of CA IX led to a stronger binding of BS-DOTA but not of B-DOTA. The BS-DOTA binding correlated linearly with the CA IX protein expression and could be blocked by an excess of unlabelled tracer. In the transfected Walker-256 cells no specific binding of either of the tracers was seen. In vivo the intratumoral accumulation of BS-DOTA was increased in animals kept under inspiratory hypoxia and reduced by hyperoxia. Therefore, betulinyl-3-sulfamate could be used as a PET tracer of CA IX expression in tumours and to provide information about the oxygenation status. However, accumulation data indicated that binding not only depends on hypoxia-induce CA IX expression but also on the tumour-line-specific basal expression and on the initial oxygenation status of the tumour.

Laser capture microdissection is a valuable tool in the preoperative molecular screening of follicular lesions of the thyroid: an institutional experience.

OBJECTIVES: The application of molecular tests to thyroid fine needle aspiration (FNA) has been shown to be a valuable tool to better refine the pre-operative malignant risk of patients with indeterminate cytology results. In this study, we investigated the feasibility of using the laser capture microdissection (LCM) technique to obtain DNA and RNA for molecular tests in routine thyroid FNA smears. METHODS: Nine coupled FNA and histological retrospective cases and 31 prospective FNA cases with a follicular neoplasm/suspicious for a follicular neoplasm (FN/SFN) diagnosis were included in this study. Both cytological and histological specimens were investigated by direct sequencing and reverse transcription-polymerase chain reaction (RT-PCR) for BRAF and RAS mutations and for PAX8/PPARG and RET/PTC rearrangements, respectively. RESULTS: LCM yielded good DNA and RNA quality in all cases (100%) in both series, irrespective of the staining used (Giemsa, Papanicolaou, immunostain for thyroglobulin) and the cytology technique (conventional or liquid-based preparations). Total mutations found in the FNA and in the corresponding histological specimen in both series were: one PAX8/PPARG rearrangement in a follicular carcinoma (FC), four NRAS mutations [in two FCs, one papillary carcinoma and one follicular adenoma (FA)] and one HRAS mutation in one FA. The sensitivity was 67% and the specificity was 91%. CONCLUSIONS: LCM is a valuable tool to obtain good quality DNA and RNA for molecular tests in cytological material from thyroid FNA, and can be a useful option in the management of patients with an FN/SFN FNA diagnosis.

Controversial constitutive TSHR activity: patients, physiology, and in vitro characterization.

G protein-coupled receptors constitute a large family of transmembrane receptors, which activate cellular responses by signal transmission and regulation of second messenger metabolism after ligand binding. For several of these receptors it is known that they also signal ligand-independently. The G protein-coupled thyroid stimulating hormone receptor (TSHR) is characterized by a high level of constitutive activity in the wild type state. However, little is known yet concerning the physiological relevance of the constitutive wild type TSHR activity. Certainly, knowledge of the physiological relevance of constitutive wild type receptor activity is necessary to better understand thyroid physiology and it is a prerequisite for the development of better therapies for nonautoimmune hyperthyroidism and thyroid cancer. Based on a literature search regarding all published TSHR mutations, this review covers several mutations which are clearly associated with a hyperthyroidism-phenotype, but interestingly show a lack of constitutive activity determined by in vitro characterization. Possible reasons for the observed discrepancies between clinical phenotypes and in vitro characterization results for constitutive TSHR activity are reviewed. All current in vitro characterization methods for constitutive TSHR mutations are "preliminary attempts" and may well be revised by more comprehensive and even better approaches. However, a standardized approach for the determination of constitutive activity can help to identify TSHR mutations for which the investigation of additional signaling mechanisms would be most interesting to find explanations for the current clinical phenotype/in vitro discrepancies and thereby also define suitable methods to explore the physiological relevance of constitutive wild type TSHR activity.

miRNAs with the potential to distinguish follicular thyroid carcinomas from benign follicular thyroid tumors: results of a meta-analysis.

The detection of somatic mutations in indeterminate or follicular proliferation fine-needle aspiration cytologies (FNACs) is able to clarify only a subgroup of those FNACs. Therefore, further markers to differentiate this problematic FNAC category by the identification of mutation negative thyroid cancers and benign nodules are urgently needed. Our objective was to evaluate previously published miRNA markers and discover novel ones from all publicly available miRNA expression profiling data sets. By literature review and data repository search we gathered 3 data sets describing human miRNA expression profiles of follicular thyroid cancer (FTC) and follicular adenoma (FA) samples. Literature review summarized 27 previously published miRNAs, which were validated in the 3 available data sets. By means of uniform statistical analysis 6 further miRNAs were identified and tested in an independent, previously published microarray data set. Meta-analysis confirmed 7 out of 27 previously published, and 4 out of 6 de novo identified miRNAs. The low confirmation rate of previously published miRNA markers was induced by low numbers of samples in the analyzed studies and high false discovery rates that were higher than 0.2. Finally, miR-637, miR-181c-3p, miR-206, and miR-7-5p were discovered as de novo potential FTC markers and validated in at least one independent, previously published data set. Two out of these new identified miRNAs (miR-7-5p and miR-206) were validated by qPCR in an independent sample set of 32 FTC and 46 FA samples. Especially miR-7-5p was able to differentiate benign and malignant thyroid tumors in several datasets.

Impact of different methodologies on the detection of point mutations in routine air-dried fine needle aspiration (FNA) smears.

Currently the best method to select suspicious thyroid nodules for surgery is fine needle aspiration (FNA) cytology. However, FNA cytology has some inherent limitations, which can partly be overcome by molecular analysis. Therefore, molecular testing for somatic mutations has emerged as the most promising approach for molecular FNA diagnostics. The objective of this methodological study was to evaluate the feasibility of detecting BRAF, NRAS, HRAS, and KRAS mutations from routine air-dried thyroid FNA smears, and to find an optimal method for detecting these mutations in FNA samples. DNA was extracted from 110 routine air-dried FNA smears and the corresponding surgically obtained formalin-fixed paraffin-embedded tissues. The presence of BRAF, NRAS, HRAS, and KRAS mutations was assessed by real-time PCRs and high resolution melting analysis, and/or pyrosequencing in comparison to real-time PCRs using hybridization probes and fluorescence melting curve analysis. The high-resolution melting-PCRs revealed a significantly lower number of PCR failures and questionable results, and detected more mutations than the PCRs using hybridization probes. The number of PCR failures ranging from 14-16% by high-resolution melting-PCRs could be further reduced to 5-14% by adding pyrosequencing assays. Moreover, pyrosequencing increased the specificity of the assays, up to 98-100%, while the sensitivity ranged between 32-63%. In summary, the mutation detection, especially in air-dried FNA samples, improves when using PCR assays in combination with high resolution melting analysis. Additional improvement can be obtained by subsequent pyrosequencing in comparison to previously described real-time PCRs using hybridization probes and fluorescence melting curve analysis.

Thyroid cancer in patients with hyperthyroidism.

Thyroid cancer can be associated with thyrotoxicosis caused by Graves' disease, toxic multinodular goiter, or autonomously functioning thyroid adenoma. The objective of this study was to summarize current evidence regarding the association of thyroid cancer and hyperthyroidism, particularly with respect to the type of hyperthyroidism found in some patients, and whether this affects the outcome of the patient. A PubMed search was performed up to August 2011. Articles were identified using combinations of the following keywords/phrases: thyroid cancer, papillary thyroid cancer, follicular thyroid cancer, medullary thyroid cancer, anaplastic thyroid cancer, hyperthyroidism, Graves' disease, auto-nomous adenoma, toxic thyroid nodule, and toxic multinodular goiter. Original research papers, case reports, and review articles were included. We concluded that the incidence, as well as the prognosis of thyroid cancer associated with hyperthyroidism is a matter of debate. It seems that Graves' disease is associated with larger, multifocal, and potentially more aggressive thyroid cancer than single hot nodules or multinodular toxic goiter. Patients with Graves' and thyroid nodules are at higher risk to develop thyroid cancer compared to patients with diffuse goiter. Every suspicious nodule associated with hyperthyroidism should be evaluated carefully.

Late manifestation of subclinical hyperthyroidism after goitrogenesis in an index patient with a N670S TSH receptor germline mutation masquerading as TSH receptor antibody negative Graves' disease.

In 27 families with familial non-autoimmune hyperthyroidism (FNAH) reported up to date, the onset of hyperthyroidism varies from 18 months to 60 years. Also the manifestation of goitres is variable in these families. A 74-year-old woman first presented at the age of 69 years with tachyarrhythmia and hypertension. After initial treatment of her hypertension and oral anticoagulation for her intermittent atrial fibrillation, a thyroid workup revealed a suppressed TSH and normal fT3 and fT4. TPO, TSH receptor (TSHR), and thyroglobulin antibodies were negative. Thyroid ultrasound revealed a thyroid volume of 102 ml with several nodules with diameters of up to 2.6 cm right and up to 1.8 cm left. Scintigraphy showed a homogeneous Technetium-99 m ((99 m)Tc) uptake of 1.27%. She was subsequently treated with 1 GBq radioiodine ((131)I). At the age of 74, her thyroid function was normal and her thyroid volume decreased to 90 ml. Because of the diffuse (99 m)Tc uptake and the negative TPO, TSHR, and thyroglobulin antibodies, genetic analysis of her TSHR gene was performed, in spite of her negative family history for hyperthyroidism. Sequencing revealed a N670S TSHR germline mutation. Previous in vitro characterisation of this TSHR mutation suggests a weak constitutive activity, yet the experimental data are ambiguous. This case illustrates the necessity to analyse patients with hyperthyroidism accompanied by diffuse (99 m)Tc uptake and negative TPO, TSHR, and thyroglobulin antibodies for TSHR germline mutations. Moreover, it demonstrates that TSHR germline mutations may first lead to longstanding nodular goitrogenesis before the late manifestation of subclinical hyperthyroidism.

Improvement in thyroid ultrasound report quality with radiologists' adherence to 2015 ATA or 2017 TIRADS: a population study.

Objectives: There has been slow adoption of thyroid ultrasound guidelines with adherence rates as low as 30% and no population-based studies investigating adherence to guideline-based malignancy risk assessment. We therefore evaluated the impact of adherence to the 2015 ATA guidelines or 2017 ACR-TIRADS guidelines on the quality of thyroid ultrasound reports in our healthcare region. Methods: We reviewed 899 thyroid ultrasound reports of patients who received fine-needle aspiration biopsy and were diagnosed with Bethesda III or IV nodules or thyroid cancer. Ultrasounds were reported by radiology group 1, group 2, or other groups, and were divided into pre-2018 (before guideline adherence) or 2018 onwards. Reports were given a utility score (0-6) based on how many relevant nodule characteristics were included. Results: Group 1 had a pre-2018 utility score of 3.62 and 39.4% classification reporting rate, improving to 5.77 and 97.0% among 2018-onwards reports. Group 2 had a pre-2018 score of 2.8 and reporting rate of 11.5%, improving to 5.58 and 93.3%. Other radiology groups had a pre-2018 score of 2.49 and reporting rate of 32.2%, improving to 3.28 and 61.8%. Groups 1 and 2 had significantly higher utility scores and reporting rates in their 2018-onward reports when compared to other groups' 2018-onward reports, pre-2018 group 1 reports, and pre-2018 group 2 reports. Conclusions: Dedicated adherence to published thyroid ultrasound reporting guidelines can lead to improvements in report quality. This will reduce diagnostic ambiguity and improve clinician's decision-making, leading to overall reductions in unnecessary FNA biopsy and diagnostic surgery.

Evidence for a more pronounced effect of genetic predisposition than environmental factors on goitrogenesis by a case control study in an area with low normal iodine supply.

Family and twin studies suggest a genetic predisposition for euthyroid goiters. However, iodine deficiency and smoking are important exogenous factors for goiter development. We investigated goiter predisposition by a matched case control study in a region with recently documented low normal iodine supply. A sum of 376 patients were included in the study. We matched 188 patients with euthyroid/subclinically hyperthyroid goiter (TSH 4.20-0.05 mU/l) with 188 euthyroid controls without thyroid enlargement for age and gender. Thyroid ultrasound was performed in all patients, whereby 50.5% of patients with goiters showed a positive family history for goiter. In contrast, only 25% of control patients had a positive family history (p<0.001; OR=3.1). Patients with goiters had a significantly higher proportion of parents (p<0.001; OR=3.6) or siblings (p=0.004; OR=2.5) with goiters. Children of parents with goiters showed a 2.7-fold increased risk for goiter development (goiter prevalence 73.3%). Patients with a positive goiter family history had a 4.1-fold increased goiter risk (p<0.001). The contribution of smoking, obesity, and pregnancies to goiter development was less important than the genetic predisposition (OR=1.7; p=0.06; OR=1.67; p=0.13; OR=0.8; p=0.56). In a region with low normal iodine supply, the significantly higher rate of positive family histories in patients with goiters as compared to the matched controls as well as the increased goiter prevalence in children of parents with goiters indicate the importance of genetic factors in goiter development.

Increased gallium-68 DOTATOC uptake in normal thyroid glands.

Increased Ga-68 DOTATOC uptake for normal and goitrous and/or nodular thyroids has previously been reported for a small number of patients. The aim of this study was to reevaluate these preliminary findings with larger patient numbers and to determine possible variables, which might influence the quantification of Ga-68 DOTATOC uptake in normal and pathologic thyroid tissues. Ga-68 DOTATOC PET scans of 165 patients classified for various thyroid pathologies were analyzed by the so-called region of interest technique for Ga-68 DOTATOC uptake and the patient's history, thyroid ultrasound, TSH, and anti-TPO antibodies. Thyroid glands without any pathology showed a clearly detectable uptake of Ga-68 DOTATOC with a large variability and significantly higher target to background ratios for men as compared to women. In 8 cases of normal thyroids with an increased uptake, follow-up examinations after 6-14 months did not show any thyroid pathology. An increased DOTATOC uptake (target to background ratio >3.4) was found in hot nodules, disseminated thyroid autonomy, and in most cases (5 of 8) of active Hashimoto's disease. In Ga-68 DOTATOC PET, normal thyroid glands show a clearly detectable radiotracer uptake with a large variability and significantly higher target to background ratios in male patients. All patients with thyroid autonomy and most patients with active Hashimoto's disease have an increased thyroid DOTATOC uptake.

Prolonged inappropriate TSH suppression during hypothyroidism after thyroid ablation in a patient with nonautoimmune familial hyperthyroidism.

Prolonged TSH suppression was reported in a patient with nonautoimmune hyperthyroidism. These observations were made during L-thyroxine treatment and it was not possible to investigate a possible increase in serum TSH concentrations to levels observed in untreated hypothyroidism. We describe nonautoimmune familial hyperthyroidism identified in an Israeli woman, which is remarkable for the prolonged inappropriate TSH suppression after thyroid ablation. After 2 radioiodine treatments for several years, her TSH was always lower than 0.03 mU/l with 1.6 µg/kg/day (100 µg) thyroxine. 14 years after the radioiodine treatments, she discontinued thyroxine for 3.5 months and developed myxoedema with fT4 <6.0 and fT3 1.3 pmol/l and TSH of only 4.4 mU/l, which rose to only 8.6 after TRH. Genomic analysis showed a germline substitution M626I in the TSHR gene. Both exons of the thyroid-releasing hormone receptor revealed no mutations in this gene. Functional in vitro characterization of M626I showed a cell surface expression of 70% compared with the wt (100%), a significant increase of basal activity (5-fold over wt basal), which was confirmed by linear regression analysis (LRA) (slope: M626I=7, wt=1). No TRH-receptor mutation was detected. Therefore, this is the first patient with nonautoimmune hyperthyroidism with unequivocal evidence for inappropriately prolonged TSH suppression documented by a clearly insufficient TSH increase during clinical hypothyroidism. The in vitro characterization of the TSH-receptor mutation did not show any explanations for the prolonged TSH suppression. Therefore, other possible candidate genes remain to be investigated for potential explanations for this prolonged TSH suppression.

High prevalence of TSHR/Gsα mutation-negative clonal hot thyroid nodules (HNs) in a Turkish cohort.

Whereas the majority of hot thyroid nodules are caused by somatic TSH-receptor mutations, the percentage of TSH-receptor mutation negative clonal hot nodules (HN) and thus the percentage of hot nodules likely caused by other somatic mutations are still debated. This is especially the case for toxic multinodular goiter (TMNG). 35 HNs [12 solitary hot nodules (SHN), 23 TMNG] were screened for somatic TSHR mutations in the exons 9 and 10 and for Gsα mutations in the exons 7 and 8 using DGGE. Determination of X-chromosome inactivation was used for clonality analysis. Overall TSHR mutations were detected in 14 out of 35 (40%) HNs. A nonrandom X-chromosome inactivation pattern was detected in 18 out of 25 (72%) HNs suggesting a clonal origin. Of 15 TSHR or Gsα mutation negative cases 13 (86.6%) showed nonrandom X-chromosome inactivation, indicating clonal origin. The frequency of activating TSHR and/or Gsα mutations was higher in SHNs (9 of 12) than in TMNGs (6 of 23). There was no significant difference for the incidence of clonality for HNs between TMNGs or SHNs (p: 0.6396). Activating TSHR and/or Gsα mutations were more frequent in SHNs than in TMNG. However, the frequency of clonality is similar for SHN and TMNG and there is no significant difference for the presence or absence of TSHR and/or Gsα mutations of clonal or polyclonal HNs. The high percentage of clonal mutation-negative HNs in SHN and TMNG suggests alternative molecular aberrations leading to the development of TSHR mutation negative nodules.

Comparison of Color Flow Doppler Sonography (CFDS) and immunohistologic detection of microvessels for the assessment of the malignancy of thyroid nodules.

The assessment of tumor vascularization by color flow Doppler sonography (CFDS) has been suggested for the distinction between benign and malignant thyroid nodules. Our objective was to investigate if the CFDS results reflect the percentage of histologically determined microvessels in adenomas (As), adenomatous nodules (ANs), and papillary carcinomas (PCs). Tissue sections from 10 adenomas, 8 ANs and 13 PC and surrounding tissue of 10 PCs and 2 benign nodules were immunostained for CD34. A computerized image analysis was used to determine the microvessel density in four hot spots and ten systematically selected fields. Preoperatively CFDS was performed and classified according to Frates et al. We found a consistent percentage increase of CD34 stained microvessels in PCs (83 and 96%) as compared to adenomas and ANs (38 and 49%) determined by the hot spot analysis and systematic field analysis. A ROC analysis on the basis of the histologically determined number of microvessels demonstrated 70% microvessels as an optimal cut point for the diagnosis of PC with the highest sensitivity of 92% and highest specificity of 89%. The analysis of the CFDS-classification IV for the distinction between PCs and adenomas and ANs showed a sensitivity of 62% with a specificity of 100%. The lower sensitivity of the CFDS classification as compared with the immunohistologic determination of the microvessel density indicates that the CFDS classification detects the pathognomonic intranodular microvessels only incompletely. The higher CFDS specificity is most likely due to the detection of other vascular aspects of malignancy in addition to intranodular microvessels.

Lack of consistent association of thyrotropin receptor mutations in vitro activity with the clinical course of patients with sporadic non-autoimmune hyperthyroidism.

BACKGROUND: Up to date, 14 patients with sporadic non-autoimmune hyperthyroidism (SNAH) caused by sporadic germline mutations in the TSH receptor (TSHR) gene have been reported. Despite considerable differences in the activity of hyperthyroidism, all SNAH case reports concluded that the demonstrated constitutive activity explains the phenotype. AIM: Recently, linear regression analysis (LRA) of constitutive activity as a function of TSHR expression determined by 125I-bTSH binding or fluorescence activated cell sorting analysis was described as a more reliable way of characterizing the in vitro activity (IVA) of a constitutively activating TSHR mutation. Therefore, we analyzed a possible genotype-phenotype correlation in a systematic review of the case reports and investigated the TSHR mutation's LRA in selected cases. MATERIAL AND METHODS: We determined the LRA for all sporadic germline mutations which had not previously been reported. Moreover, we systematically evaluated all case reports of SNAH for evidence of an association of the clinical course (CC) with the IVA of the mutated TSHR. RESULTS: The LRA determined were: M453T (5.2+/-0.8), L512Q (4.5+/-0.7), I568T (25.6+/-6.3), F631L (45.9+/-9.4), T632I (14.5+/-2.7), D633Y (16.4+/-6.4). None of the 10 examined clinical signs showed a significant association with the LRA. Moreover, the comparison of the CC of patients harboring the same mutation (S281N, M453T, I568T, S505N) also showed no relation of the clinical activity with a high LRA. CONCLUSION: Considering the different diagnostic circumstances, therapeutic strategies and the limitations of a systematic analysis of case reports due to the restricted number of case reports and limited follow-up we found no consistent relation of the TSHR mutation's IVA determined by LRA with the CC of patients with SNAH. This may also be due to the action of genetic, epigenetic, and environmental modifiers.

American Association of Clinical Endocrinologists, Associazione Medici Endocrinologi, and European Thyroid Association medical guidelines for clinical practice for the diagnosis and management of thyroid nodules: Executive Summary of recommendations.

American Association of Clinical Endocrinologists, Associazione Medici Endocrinologi, and European Thyroid Association medical guidelines for clinical practice for the diagnosis and management of thyroid nodules are systematically developed statements to assist health care professionals in medical decision making for specific clinical conditions. Most of the content herein is based on literature reviews. In areas of uncertainty, professional judgment was applied. These guidelines are a working document that reflects the state of the field at the time of publication. Because rapid changes in this area are expected, periodic revisions are inevitable. We encourage medical professionals to use this information in conjunction with their best clinical judgment. Any decision by practitioners to apply these guidelines must be made in light of local resources and individual patient circumstances.

Characterization of NVX-207, a novel betulinic acid-derived anti-cancer compound.

BACKGROUND: Development of betulinic acid derivatives for clinical use has been hampered by adverse pharmacological and physico-chemical characteristics of this class of compounds. We here present a novel semi-synthetic betulinic acid-derived drug candidate well suited for further clinical development. MATERIALS AND METHODS: In vitro activity and mode of action of NVX-207 were determined using normal as well as cancer cell lines. Gene expression profiling was performed with Affymetrix U133 microarrays. NVX-207 binding partners were identified using a heterobifunctional chemical crosslinker system. Potential binding proteins were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis. Clinical studies were conducted in canine cancer patients suffering from spontaneously arising pre-treated tumours. RESULTS: NVX-207 showed anti-tumour activity (mean IC(50) = 3.5 microM) against various human and canine cell lines. NVX-207-induced apoptosis was associated with activation of the intrinsic apoptotic pathway via cleavage of caspases -9, -3, -7 and of poly (ADP-ribose) polymerase (PARP). Global gene expression profiling demonstrated regulation of genes associated with lipid metabolism, most notably an upregulation of genes coding for insulin-induced gene 1 (Insig-1), low-density lipoprotein receptor (LDL-R) and of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA). NVX-207 bound to apolipoprotein A-I, a major regulator of lipid metabolism and cholesterol transport. A phase I/II study in dogs suffering from naturally occurring cancer receiving local treatment of NVX-207 (10 mg mL(-1)) showed excellent clinical responses including a complete remission in so far 5/5 treated animals. CONCLUSIONS: NVX-207 is well tolerated and has significant anti-cancer activity in vitro and in vivo in dogs with treatment-resistant malignancies.

Preferences of transmembrane helices for cooperative amplification of G(alpha)s and G (alpha)q signaling of the thyrotropin receptor.

The majority of constitutively activating mutations (CAMs) of the thyroid-stimulating hormone receptor display a partially activated receptor. Thus, full receptor activation requires a multiplex activation process. To define impacts of different transmembrane helices (TMHs) on cooperative signal transduction, we combined single CAMs in particular TMHs to double mutations and measured second messenger accumulation of the G(alpha)s and the G(alpha)q pathway. We observed a synergistic increase for basal activity of the G(alpha)s pathway, for all characterized double mutants except for two combinations. Each double mutation, containing CAMs in TMH2, 6 and 7 showed the highest constitutive activities, suggesting that these helices contribute most to G(alpha)s-mediated signaling. No single CAM revealed constitutive activity for the G(alpha)q pathway. The double mutations with CAMs from TMH1, 2, 3 and 6 also exhibited increase for basal G(alpha)q signaling. Our results suggest that TMH2, 6, 7 show selective preferences towards G(alpha)s signaling, and TMH1, 2, 3, 6 for G(alpha)q signaling.

Molecular and structural effects of inverse agonistic mutations on signaling of the thyrotropin receptor--a basally active GPCR.

Several mutations that decrease the basal signaling activity of G-protein coupled receptors (GPCRs) with pathogenic implications are known. Here we study the molecular mechanisms responsible for this phenotype and investigate how basal and further activated receptor conformations are interrelated. In the basally active thyroid stimulating hormone receptor (TSHR) we combined spatially-distant mutations with opposing effects on basal activity in double-mutations and characterized mutant basal and TSH induced signaling. Mutations lowering basal activity always have a suppressive influence on TSH induced signaling and on constitutively activating mutations (CAMs). Our results suggest that the conformation of a basally 'silenced' GPCR might impair its intrinsic capacity for signaling compared to the wild-type. Striking differences in conformation and intramolecular interactions between TSHR models built using the crystal structures of inactive rhodopsin and partially active opsin help illuminate the molecular details underlying mutations decreasing basal activity.

Myocardial Infarction after Long-Term Treatment with a Tyrosine Kinase Inhibitor (TKI) with Anti-VEGF Receptor Activity.

TKIs including anti-VEGF receptor activity have been approved for the treatment of patients with radioiodine resistant thyroid carcinomas. For lenvatinib arterial thromboembolic events are listed as adverse events of special interest with lenvatinib. In the phase III study, arterial thromboembolic events were reported in 3% of lenvatinib-treated patients and 1% in the placebo group. Most of the patients had predisposing factors. Only one myocardial infarct was reported in the lenvatinib phase III study. We report a 73-year-old female patient with metastatic thyroid papillary carcinoma who was treated with total thyroidectomy. The operation was followed by four radioiodine therapies over a period of 6 years. At 6 years she developed lung metastasis without radioiodine uptake, one solitary liver metastasis and one solitary right renal metastasis. One year after the first diagnosis of radioiodine resistant lung metastasis the lung metastasis showed progression according to RECIST criteria. This treatment was resulting in prolonged partial response with disappearance of a hepatic and renal metastasis. A myocardial infarction occurred after 39 months of lenvatinib treatment resulting in implantation of 3 stents and a two chamber pacemaker. The treatment was discontinued. Except for well controlled hypertension there were neither predisposing diseases like diabetes nor symptoms of cardiac ischemia on exertion. However, the family history for cardiovascular diseases was positive for cardiac infarction reported for one brother. Another brother was treated for hypertension and the patient's mother suffered from a cerebral infarction at the age of 60. While only one myocardial infarct was reported in the lenvatinib phase III study with 392 patients this case suggests that long-term treatment with lenvatinib may be associated with an increased risk for myocardial infarct also in patients with no predisposing diseases except well controlled hypertension and positive family history for cardiovascular diseases.

[Speech therapy after thyroid gland operations in Germany: analysis of routine data from 50,676 AOK patients]. / Logopädie nach Schilddrüsenoperationen in Deutschland: eine Routinedatenanalyse von 50.676 AOK-Patienten.

BACKGROUND: This study investigated the frequency of postoperative speech therapy in the context of vocal cord palsy after thyroid surgery based on nationwide routine data. Additionally, volume-outcome relationships were examined. MATERIAL AND METHODS: Nationwide routine data from insured patients of the Local Health Insurance Fund (AOK) who underwent thyroid surgery for a benign thyroid disease between 2013 and 2015 were analyzed. Postoperative speech therapy was determined based on prescription data. Transient and permanent vocal cord palsy were determined using indicators. The effect of hospital volumes (volume quintiles) on prescription of postoperative speech therapy was determined by multivariate logistic regression. RESULTS: A total of 50,676 thyroid gland operations were identified. The overall frequency of postoperative speech therapy prescription was 6.5%. In AOK patients with transient or permanent vocal cord palsy, the frequencies of postoperative speech therapy prescription were 56.1% and 75.2%, respectively. The prescription volume of the normal case (≥21 units of speech therapy) was exceeded in 0.7% of the AOK patients. In the two lowest case volume categories the risk of postoperative speech therapy exceeding the prescription volume of the normal case was significantly higher compared to the highest case volume hospitals (odds ratios: 1.2 and 1.8, respectively). CONCLUSION: This study presents the reality of healthcare with respect to the frequency of speech therapy prescription after thyroid gland surgery in Germany. In addition, it was determined that the risk of postoperative speech therapy prescription exceeding the volume of the normal case after thyroid gland operations decreases with increasing case volumes of hospitals.