Assessment of real-world usage of lanreotide AUTOGEL 120 in Polish acromegalic patients - results from the prospective 12-month phase of Lanro-Study.

AIM OF THE STUDY: To assess resource utilization and costs of treatment with lanreotide AUTOGEL 120 mg (ATG120) administered as part of routine acromegaly care in Poland. MATERIAL AND METHODS: A multicentre, non-interventional, observational study on resource utilization in Polish acromegalic patients treated with ATG120 at 4 weeks or extended (> 4 weeks) dosing interval. The study recruited adult acromegalic patients treated medically for ≥ 1 year including at least 3 injections of ATG120. Data on dosing interval, aspects of administration, and resource utilization were collected prospectively during 12 months. Costs were calculated in PLN from the public health-care payer perspective for the year 2013. RESULTS: 139 patients were included in the analysis. Changes in dosing regimen were reported in 14 (9.4%) patients. Combined treatment was used in 11 (8%) patients. Seventy patients (50%) received ATG120 at an extended dosing interval; the mean number of days between injections was 35.56 (SD 8.4). ATG120 was predominantly administered in an out-patient setting (77%), by health-care professionals (94%). Mean time needed for preparation and administration was 4.33 and 1.58 min, respectively, mean product wastage - 0.13 mg. Patients were predominantly treated in an out-patient setting with 7.06 physician visits/patient/year. The most common control examinations were magnetic resonance imaging of brain and brain stem (1.36/patient/year), ultrasound of the neck (1.35/patient/year), GH (1.69/patient/year), glycaemia (1.12/patient/year), IGF-1 (0.84/patient/year), pituitary-thyroid axis hormone levels assessment (TSH-0.58/patient/year, T4-0.78/patient/year). There were 0.43 hospitalizations/patient/year. For direct medical costs estimated at PLN 50 692/patient/year the main item was the costs of ATG120 (PLN 4103.87/patient/month; 97%). The mean medical cost, excluding pharmacotherapy, was PLN 1445/patient/year (out-patient care - 49%, hospitalization - 23%, diagnostics/laboratory tests - 28%). CONCLUSIONS: These results represent the current use of ATG120 in the population of Polish acromegalic patients in a realistic clinical setting. Findings that 50% of patients could be treated with dose intervals of longer than 28 days support the potential of ATG120 to reduce the treatment burden.

Antibody protection reveals extended epitopes on the human TSH receptor.

Stimulating, and some blocking, antibodies to the TSH receptor (TSHR) have conformation-dependent epitopes reported to involve primarily the leucine rich repeat region of the ectodomain (LRD). However, successful crystallization of TSHR residues 22-260 has omitted important extracellular non-LRD residues including the hinge region which connects the TSHR ectodomain to the transmembrane domain and which is involved in ligand induced signal transduction. The aim of the present study, therefore, was to determine if TSHR antibodies (TSHR-Abs) have non-LRD binding sites outside the LRD. To obtain this information we employed the method of epitope protection in which we first protected TSHR residues 1-412 with intact TSHR antibodies and then enzymatically digested the unprotected residues. Those peptides remaining were subsequently delineated by mass spectrometry. Fourteen out of 23 of the reported stimulating monoclonal TSHR-Ab crystal contact residues were protected by this technique which may reflect the higher binding energies of certain residues detected in this approach. Comparing the protected epitopes of two stimulating TSHR-Abs we found both similarities and differences but both antibodies also contacted the hinge region and the amino terminus of the TSHR following the signal peptide and encompassing cysteine box 1 which has previously been shown to be important for TSH binding and activation. A monoclonal blocking TSHR antibody revealed a similar pattern of binding regions but the residues that it contacted on the LRD were again distinct. These data demonstrated that conformationally dependent TSHR-Abs had epitopes not confined to the LRDs but also incorporated epitopes not revealed in the available crystal structure. Furthermore, the data also indicated that in addition to overlapping contact regions within the LRD, there are unique epitope patterns for each of the antibodies which may contribute to their functional heterogeneity.

Subunit interactions influence TSHR multimerization.

The TSH receptor (TSHR) is the key molecule influencing thyroid growth and development and is an antigenic target in autoimmune thyroid disease. The TSHR exists in monomeric and multimeric forms, and it has been shown previously that multimeric complexes of the TSHR preferentially localize in lipid rafts. However, unlike other glycoprotein hormone receptors, the TSHR exists in several forms on the cell membrane due to intramolecular cleavage of its ectodomain, which causes the production of α- and ß-subunits of various lengths. After cleavage and reduction of disulfide bonds, α-subunits consisting of the receptor ectodomain may be lost from the cell surface by receptor shedding, leading to accumulation of excess ß-subunits within the membrane. Because cell surface expression of these various forms of the TSHR is critical to receptor signaling and autoimmune responses, we set out to model the influence of ß-subunits on full-length TSHRs. To study this interaction, we generated three truncated ectodomain ß-subunits linked to green fluorescent protein (named ß-316, -366, and -409) as examples of native cleaved forms of the TSHR. These constructs were transfected into human embryonic kidney 293 cells in the presence and absence of the full-length receptor. Whereas the ß-316 and ß-366 forms showed cell surface expression, the expression of ß-409 was primarily intracellular. Cotransfection of the ß-subunits with a full-length hemagglutinin-tagged wild-type (WT) receptor (HT-WT-TSHR) in both transient and stable systems caused a significant decrease in surface expression of the full-length WT receptors. This decrease was not seen with control plasmid consisting of a plasma membrane-targeted protein tagged to red fluorescent protein. To ascertain if this response was due to homointeraction of the truncated ß-constructs with the WT-TSHRs, we immunoprecipitated membranes prepared from the cotransfected cells using antihemagglutinin and then probed with anti-green fluorescent protein. These studies confirmed dimerization of the ß-subunits with the WT full-length receptor, and this interaction was further observed in vivo by fluorescence resonance energy transfer. We then studied the functional consequences of this interaction on TSHR signaling by examining Gαs-mediated signals. The well-expressed truncated constructs, when coexpressed with full-length TSHR, did not alter constitutive cAMP levels, but there was a significant decrease in TSH-induced cAMP generation. Furthermore, we observed that truncated ß-316 and ß-366 had faster internalization rate, which may lead to a significant decrease in the expression of the full-length receptor on the cell surface, thus contributing to the decreased signaling response. However, the decrease in surface receptors may also be due to inhibition of newly formed receptors reaching the surface as result of receptor-receptor interaction. It is well known that under normal physiological conditions both cleaved and uncleaved TSHR forms coexist on the cell surface of normal thyrocytes. Our studies allow us to conclude, therefore, that multimerization of cleaved/ truncated forms of the ß-subunits with the full-length TSHR has a profound influence on TSHR internalization and signaling. Hence, the degree of intramolecular cleavage must also modulate TSHR signaling.

A tyrosine residue on the TSH receptor stabilizes multimer formation.

BACKGROUND: The thyrotropin stimulating hormone receptor (TSHR) is a G protein coupled receptor (GPCR) with a large ectodomain. The ligand, TSH, acting via this receptor regulates thyroid growth and thyroid hormone production and secretion. The TSH receptor (TSHR) undergoes complex post-translational modifications including intramolecular cleavage and receptor multimerization. Since monomeric and multimeric receptors coexist in cells, understanding the functional role of just the TSHR multimers is difficult. Therefore, to help understand the physiological significance of receptor multimerization, it will be necessary to abrogate multimer formation, which requires identifying the ectodomain and endodomain interaction sites on the TSHR. Here, we have examined the contribution of the ectodomain to constitutive multimerization of the TSHR and determined the possible residue(s) that may be involved in this interaction. METHODOLOGY/PRINCIPAL FINDINGS: We studied ectodomain multimer formation by expressing the extracellular domain of the TSHR linked to a glycophosphotidyl (GPI) anchor in both stable and transient expression systems. Using co-immunoprecipitation and FRET of tagged receptors, we established that the TSH receptor ectodomain was capable of multimerization even when totally devoid of the transmembrane domain. Further, we studied the effect of two residues that likely made critical contact points in this interaction. We showed that a conserved tyrosine residue (Y116) on the convex surface of the LRR3 was a critical residue in ectodomain multimer formation since mutation of this residue to serine totally abrogated ectodomain multimers. This abrogation was not seen with the mutation of cysteine 176 on the inner side of the LRR5, demonstrating that inter-receptor disulfide bonding was not involved in ectodomain multimer formation. Additionally, the Y116 mutation in the intact wild type receptor enhanced receptor degradation. CONCLUSIONS/SIGNIFICANCE: These data establish the TSH receptor ectodomain as one site of multimerization, independent of the transmembrane region, and that this interaction was primarily via a conserved tyrosine residue in LRR3.

TSH receptor autoantibodies.

Thyrotropin receptor autoantibodies (TSHR-Abs) of the stimulating variety are the hallmark of Graves' disease. The presence of immune defects leading to synthesis of TSHR-Abs causes hyperthyroidism and is associated with other extrathyroidal manifestations. Further characterization of these antibodies has now been made possible by the generation of monoclonal antibodies with this unique stimulating capacity as well as similar TSHR-Abs not associated with hyperthyroidism. Their present classification divides TSHR-Abs into stimulating, blocking (competing with TSH binding) and neutral (no signaling). Recent studies using monoclonal TSHR-Abs has revealed that stimulating and blocking antibodies bind to the receptor using mostly conformational epitopes, whilst neutral antibodies utilize exclusively linear peptides. Subtle differences in epitopes for stimulating and blocking antibodies account for the diversity of their biological actions. Recently non-classical signaling elicited by neutral antibodies has also been described, raising the need for a new classification of TSHR-Abs.

[Influence of the endogene TSH stimulation of thyroid volume increase in the patients after total thyroidectomy due to differentiated thyroid cancer]. / Wplyw stymulacji endogennego TSH na wzrost resztkowej objetosci tarczycy u chorych po calkowitej tyreoidektomii z powodu raka zróznicowanego tarczycy.

INTRODUCTION: The treatment-of-choice for differentiated thyroid carcinoma (DTC) is a total thyroidectomy with subsequent radioiodine therapy. In order to increase an iodine uptake in thyroid tissue remnants, the L-thyroxine withdrawal is required. It is recommended to achieve TSH levels higher than 25 mU/ml. As TSH is a known key factor in thyroid cell proliferation regulation, prolonged stimulation of the cells during L-thyroxine withdrawal can be a causative factor for a re-growth. Our aim was to assess the degree of thyroid re-growth in the patients after total thyroidectomy due to DTC and its possible clinical implications. MATERIAL AND METHODS: 23 patients operated due to papillary and follicular thyroid cancer were included into the study. Biochemical determinations and ultrasound thyroid imaging were performed (TSH, Tg) during suppressive L-thyroxine therapy as well as 4-5 weeks after the withdrawal. RESULTS: The mean volume of thyroid tissue remnants increased after withdrawal for substantial 30.1%. The difference was extremely significant. CONCLUSIONS: L-Thyroxine withdrawal in the patients after total thyroidectomy due to DTC can cause re-growth of the tissue remnants. The phenomenon may be of a clinical significance in the selected cases influencing therapeutic decisions.

Clinical significance of the insulin-like growth factor I gene promoter (P1) polymorphism in thyroid nodular disease.

OBJECTIVES: Due to the recent increase of incidence of thyroid nodules and the known risk of malignant transformation, there is an elevated risk of thyroid cancer in Poland. Several approaches, including molecular, have been proposed to support fine needle aspiration biopsy in the early detection of malignant lesions. Although the IGF-I system in thyroid cancer has been studied, little is known about the gene and its promoter structure changes. Our aim was to assess, whether the analysis of the IGF-I gene promoter region and 5'UTR exon 1 structure may be useful in assessing the risk of thyroid carcinoma. MATERIAL: Our study included 46 patients that underwent strumectomy due to a presence of thyroid nodules. METHODS: All patients underwent clinical examination and laboratory investigations to assess their thyroid structure and function. Tissues obtained during the surgery were used for DNA extraction, PCR, SSCP and direct sequencing. RESULTS: Among 46 patients, 14 had a nucleotide difference in one of the examined regions. In our study we revealed no significant difference between carcinomatous and non-carcinomatous groups of patients in terms of presence of nucleotide change, but Fisher's exact test gave a significant result in terms of the efficacy of detecting follicular adenoma. Moreover, the patients with nucleotide change had thyroid glands significantly smaller in volume. CONCLUSIONS: We conclude that the molecular analysis of the IGF-I gene promoter is thought to be of a functional significance, but probably could not be considered useful in the assessment of risk of thyroid cancer in thyroid nodules.

[Urinary cotinine in patients with hyperthyroidism]. / Kotynina w moczu u pacjentów z nadczynnoscia tarczycy.

The aim of our study was an analysis of cotynine, the main nicotine metabolite in the urine among hyperthyroid patients. The study group included 39 females and 4 males. The mean age was 35.59+/-14.22 yrs. (range: 18-73 yrs.; median: 32 yrs) among hyperthyroid patients suffering from: Graves-Basedow disease (GB), Graves' Ophtalmopathy (GO) and toxic nodular goiter (TNG). To evaluate the nicotine smoking intensity and ETS Environmental Tobacco Smoke, the urine analysis of cotynine level were performed. According to the statistical analysis using the Mann-Whitney test, the statistically significant difference between the level of cotynine among smokers suffering from GO and Graves-Basedow disease was revealed (p = 0.03). Similar results were obtained among the GO and TNG (p=0.02) using t-Student test with Welsch correction. To compare, there was no stastistically significant difference between the GB and TNG series (p=0.4). In the group of smoking patients with GO we found out incresed level of urine cotinine than in smoking patients with GB and TNG. We didn't found differences between GB and TNG in depends on an urine analysis of cotynine level.

[The endocrinological management of thyroid associated orbitopathy in relation to severity and activity of the disease]. / Postepowanie endokrynologiczne w orbitopatii tarczycowej w zaleznosci od zaawansowania i aktywnosci choroby.

The complete diagnosis of thyroid associated orbitopathy (TAO) comprises endocrinological and ophthalmological assessment of patients. This approach facilitates classification of patients and qualification of patients for the proper treatment of eye changes. The aim can be obtained by assessment of severity and activity of the disease. One can distinguish at least 4 clinical situations (1. severe and active TAO, 2. severe and inactive TAO, 3. non-severe and active TAO, 4. non-severe and non-active TAO). Methods of reestablishing euthyreosis are proposed in this article, in relation to disease activity and severity of the disease. The two case-reports are described in order to express proposed theses.

Efficacy of procalcitonin measurement in patients after total thyroidectomy due to medullary thyroid carcinoma.

Procalcitonin (PCT) is a protein synthetized by the thyroid C cells, inside which it is cut into calcitonin (CT) and catacalcin. It remains undetectable in serum in normal conditions. Its level increases during inflammation and in small cell lung cancer. There have been studies suggesting that the PCT level increases in medullary thyroid carcinoma (MTC). So far there have been no reports that would assess the usefulness of PCT detection in MTC. Our aim was to evaluate the usefulness of serum PCT assays in patients with MTC. We investigated 24 patients at 17-78 years of age, all after total thyroidectomy due to MTC. All patients had serum CT concentrations measured by radioimmune assay. The upper limit of the CT level was 60 pg/ml. The serum PCT was evaluated with an immunochromatographic kit. The reaction was considered positive when the PCT level exceeded 0.5 ng/ml. In all cases the C-reactive protein (CRP) serum level was measured. The statistical analysis was performed with Statistica 5.1G. The CT levels in all patients varied from 0 to 1410, mean 603.8 pg/ml. In 8 patients the CT level was within normal range, in 6 patients it was marginally, and in 10 patients markedly elevated. The PCT test was considered positive in 16 patients. There was correlation among serum PCT and CT concentrations (Spearman test, p<0.0001). The PCT levels varied considerably among patients with normal, marginally and markedly elevated CT levels (Kruskal-Wallis test, p=0.0013). All patients had normal CRP values. Fisher's exact test revealed a correlation between serum PCT and CT increase (p=0.04). Further studies on a larger group of patients should be considered; thus, the PCT assay can be considered useful in cases of unclear CT concentration.

[Usefulness of serum calcitonin, CEA and AFP assays in the early detection of medullary thyroid carcinoma relapse]. / Wartosc oznaczen kalcytoniny, CEA i AFP w surowicy we wczesnym wykryciu wznowy raka rdzeniastego tarczycy.

The medullary thyroid carcinoma (MTC) occurs in the two major forms, the sporadic medullary carcinoma (SMTC) and hereditary medullary carcinoma, to which belong familial form (FMTC) and the element of Multiple Endocrine Neoplasia Syndrome MEN 2A and 2B. The method-of-choice of treatment is total thyroidectomy with following radiotherapy in selected cases. Among the follow-up methods, there are two used the most frequently: the pentagastrin and omeprazole stimulation test of calcitonin (CT), and CEA antigen assay. The aim of this study is to evaluate usefulness of plasma CT, CEA and AFP assay in the early detection of relapse or metastasis of MTC. 18 patients (14 females and 4 males) were investigated. The following procedures were performed in all the patients: plasma CT assays in the basal conditions and after stimulation tests, CEA and AFP. The results were analysed according to TNM staging and neck USG results. Our conclusion is that calcitonin stimulation tests, and CEA assays are useful methods to estimate the presence of relapse or metastases in the patients after surgical treatment due to MTC. Assays of plasma AFP concentration are not useful in a follow-up of patients operated on MTC.