Sudden onset of sarcoidosis after successful surgical treatment of Cushing's syndrome.

Not required for Clinical Vignette.

The Effect of Recombinant Human TSH on Sclerostin and Other Selected Bone Markers in Patients after Total Thyroidectomy for Differentiated Thyroid Cancer.

The direct effect of TSH on bone metabolism in vivo is difficult to capture as the changes of its concentrations are followed by respective alterations of thyroid hormone levels. We evaluated the effect of recombinant human TSH (rhTSH) on sclerostin and other bone markers in 29 patients after total thyroidectomy for differentiated thyroid cancer (DTC), without any signs of disease recurrence, who received L-thyroxine, most at non-suppressive doses. For two consecutive days, the patients were administered a standard dose of 0.9 mg rhTSH, i.m. Concentrations of sclerostin, osteocalcin, ß-CrossLaps, PTH, and some other parameters, were measured before and five days after the first rhTSH administration. The greater the increase in TSH concentration (∆TSH), the greater the decrease in: ∆sclerostin (r = -0.672; p < 0.001), ∆ß-CrossLaps (r = -0.580; p < 0.001) and ∆osteocalcin (r = -0.405; p = 0.029) levels, were recorded. The degree of TSH increase depended on the baseline PTH (r = 0.651; p < 0.001), age, and creatinine concentrations. rhTSH strongly inhibited bone turnover, thus, TSH-independently of thyroid hormones-exerted a direct protective effect on bone metabolism. Baseline PTH affected the magnitude of TSH increase and the degree of lowering in sclerostin and ß-CrossLaps that suggest factors affecting PTH may play a role in the effect of TSH on the bone.

Impact of factory-calibrated Freestyle Libre System with new glucose algorithm measurement accuracy and clinical performance in children with type 1 diabetes during summer camp.

BACKGROUND: Factory-calibrated intermittently-scanned Continuous Glucose Monitoring (isCGM) device FreeStyle Libre (FSL) has recently received improvements in its glucose tracking algorithm and calibration procedures, which are claimed to have improved its accuracy. OBJECTIVE: To compare the accuracy of two generations of 14-days FSL devices (A in 2016, B in 2019) to self-monitored blood glucose measurements (SMBG) in children with type 1 diabetes in real-life conditions during a summer camp. MATERIALS AND METHODS: Two largely independent groups of youth with type 1 diabetes took part in summer camps. In 2016 they used FSL-A, in 2019 FSL-B. On scheduled days, participants performed supervised 8-point glucose profiles with FSL and SMBG. The accuracy vs SMBG was assessed with mean absolute relative difference (MARD) and clinical surveillance error grid (SEG). RESULTS: We collected 1655 FSL-SMBG measurement pairs from 78 FSL-A patients (age 13 ± 2.3 years old; HbA1c: 7.6 ± 0.8%) and 1796 from 58 in FSL-B group (age 13.8 ± 2.3 years old, HbA1c: 7.5 ± 1.1%)-in total 3451 measurements. FSL-B displayed lower MARD than FSL-A (11.3 ± 3.1% vs 13.7 ± 4.6%, P = .0003), lower SD of errors (20.2 ± 6.7 mg/dL vs 24.1 ± 9.6 mg/dL, P = .0090) but similar bias (-7.6 ± 11.8 mg/dL vs -6.5 ± 8 mg/dL, P = .5240). Both FSL-A and FSL-B showed significantly higher MARD when glycaemia was decreasing >2 mg/dL/min (FSL-A:22.3 ± 18.5%; FSL-B:17.9 ± 15.8%, P < .0001) compared with stable conditions (FSL-A: 11.4 ± 10.4%, FSL-B:10.1 ± 9.1%) and when the system could not define the glycaemic trend (FSL-A:16.5 ± 16.3%; FSL-B:15.2 ± 14.9%, P < .0001). Both generations demonstrated high percentage of A-class and B-class results in SEG (FSL-A: 96.4%, FSL-B: 97.6%) with a significant shift from B (decrease by 3.7%) to A category (increase by 3.9%) between generations (FSL-A: 16/80.4%; FSL-B:12.3/85.3%, P = .0012). CONCLUSION: FSL-B demonstrated higher accuracy when compared to FSL-A However, when glycemia is decreasing or its trend is uncertain, the verification with a glucose meter is still advisable.

Evaluation of the effectiveness of iodine prophylaxis in Poland based on over 20 years of observations of iodine supply in school-aged children in the central region of the country.

INTRODUCTION: Due to the mild-to-moderate iodine deficiency in Poland, in 1997 iodine prophylaxis based on obligatory salt iodization was introduced. We attempted to evaluate the effectiveness of such prophylaxis, based on over 20 years of observations of iodine supply in school-aged children in Opoczno district (Central Poland). MATERIAL AND METHODS: A group of 603 children (316 girls and 287 boys), aged 6-14, was examined at 4 time points: in the years 1994, 1999, 2010 and 2016. The children were tested for urine iodine concentration (UIC) and in each child the thyroid volume was measured ultrasonographically. RESULTS: The median UIC in 1994 (45.5 µg/l) indicated moderate iodine deficiency, while after introducing prophylaxis it corresponded to adequate values (1999 - 101.1 µg/l, 2010 - 100.6 µg/l, 2016 - 288.3 µg/l); however, the last value was higher than the previous two. The thyroid size, assessed by ultrasonography and presented as volume/body surface area (V/BSA), in 1994 was 6.55 × 10-6 m; this value was higher than at other time points (2.73 × 10-6 m in 1999, 2.73 × 10-6 m in 2010, and 2.70 × 10-6 m in 2016). CONCLUSIONS: Iodine prophylaxis has proved effective in eliminating iodine deficiency. In recent years, the diversification of iodine sources, despite the reduction of salt consumption, has led to an increase in median UIC to values close to the upper limit of UIC, accepted as normal. Further increase in iodine supply may be unfavourable for health; therefore constant monitoring of iodine prophylaxis is required.

Angiotensinogen gene T174M polymorphism is related to Hashimoto's thyroiditis.

OBJECTIVES: The Hashimoto thyroiditis is found to be Th1-related autoimmunity. Recently, it has been proved that the renin-angiotensin-aldosterone system (RAAS) may be involved in promoting Th1-mediated autoimmune diseases. However, the role of RAAS in HT pathogenesis remains still unknown. The aim of this study was to determine whether the polymorphisms of ACE, AGTR1 and AGT genes are associated with HT. MATERIAL AND METHODS: Polymerase Chain Reaction (PCR) was performed to determine ACE I/D, AGTR1 A1166C and AGT T174M polymorphisms and next chi-square test was used to compare allele frequencies of genes between HT patients (n=53) and the control group (n=31). RESULTS: TM genotype of AGT gene has been more often presented in HT patients (p <0.05). No others statistically significant differences were found in the distribution of I/D ACE and A1166C, AGTR1 genes polymorphisms between studied groups. CONCLUSION: Our study has examined for the first time the association of genes related to RAAS with autoimmune thyroid disease and results suggest that AGT TM genotype individuals might be at higher risk of HT. Although in the present study we have not found any association between increased activation of RAAS and the risk of HT, still this issue seems to be interesting and worthy further research, considering patients with thyroid cancers.

Differences of the Structure of Immune Regulatory Cell Populations between Cellular Material from Sonographically Detected Focal Thyroid Lesions and Peripheral Blood in Humans.

Focal thyroid lesions are common ultrasound findings with the estimated prevalence up to 67% of the population. They form characteristically enveloped regions with individual encapsulated microenvironment that may involve the specific distribution of immune system compounds-especially antigen presenting cells (APC). We analyzed and compared the most potent APC-plasmacytoid and conventional dendritic cells (DCs) subpopulations and three monocyte subpopulations as well as other immune cells-in peripheral blood and local blood of thyroid gland obtained parallelly in patients with focal thyroid lesions using flow cytometry. The analysis revealed significant differences in the distribution of main subsets of assessed cells between peripheral blood and biopsy material. The results support the existence of local, organ-specific immune reaction control networks within thyroid nodules.

Relationship between urine lipid peroxidation, anthropometric parameters and parameters associated with goitre formation in school-age children.

INTRODUCTION: Oxidative stress has been implicated in the normal ageing process and the pathogenesis of several diseases, including goitre. The aim of the study was to evaluate the relationship between urine lipid peroxidation (LPO) and anthropometric parameters as well as the parameters associated with goitre formation in children. MATERIAL AND METHODS: The subjects included 172 healthy children (93 girls and 79 boys) aged 8-15, divided into 4 age groups - group I (8-9 years), group II (10-11 years), group III (12-13 years) and group IV (14-15 years) - and into 2 groups based on the BSA: the BSA-1 group (≤ 0.55 m2) and the BSA-2 group (> 0.55 m2). RESULTS: The value of LPO was the highest in group I but the difference between the groups was not statistically significant (p = 0.074). In the BSA-1 group, the LPO was higher than in the BSA-2 group (12.75 ±6.90 nmol/ml and 10.79 ±4.86 nmol/ml, respectively; p = 0.023). We found a weak, negative linear correlation between LPO and age (r = -0.216; p < 0.005), body mass (r = -0.153; p < 0.05), height (r = -0.152; p < 0.05) and BSA (r = -0.151; p < 0.05). CONCLUSIONS: Anthropometric parameters of school-age children independently of age are negatively associated with oxidative damage to membrane lipids, whereas factors promoting goitrogenesis do not contribute to this process.

In children with autoimmune thyroiditis CTLA4 and FCRL3 genes--but not PTPN22--are overexpressed when compared to adults.

BACKGROUND: Numerous genetic studies revealed several susceptibility genes of autoimmune thyroid diseases (AITD), including CTLA4, PTPN22 and FCRL3. These immune-modulating genes are involved in genetic background of AITD among children and adult patients. However, possible age-related differences in overexpression of these genes remain unclear. PURPOSE: The goal of this single centre cohort study was evaluation of expression levels of three (3) genes CTLA4, PTPN22 and FCRL3 in adult patients and children with autoimmune thyroiditis. METHODS: A total of 47 patients--24 adults (mean age--47.7 years) and 23 children (mean age--12.4 years) with autoimmune thyroiditis were assessed for the level of expression of CTLA4, PTPN22 and FCRL3 genes, utilizing ABI PRISM' 7500 Sequence Detection System (Applied Biosystem, Foster City, CA, USA). RESULTS: The overexpression of PTPN22 (mean RQ = 2.988) and FCRL3 (mean RQ = 2.544) genes were confirmed in adult patients with autoimmune thyroiditis, at the same time the expression level of CTLA4 gene was significantly decreased (mean RQ = 0.899) (p < 0.05). Similar discrepancies were not observed in children with autoimmune thyroiditis in whom overexpression of all three genes--CTLA4, PTPN22 and FCRL3--was observed. Differences in CTLA4 and FCRL3 genes expression levels in patients with autoimmune thyroiditis were found depending on the age, with increased expression levels of CTLA4 (mean RQ = 3.45 1) and FCRL3 (mean RQ = 7.410) in children when compared to adults (p < 0.05) (Mann-Whitney's U-test). There were moderate negative linear correlations between two genes in question (CTLA4 and FCRL3) expression level and patients' age [correlation coefficient (r) = -0.529 (p < 0.0002) and -0.423 (p < 0.0032), respectively; Spearman's rank correlation test]. CONCLUSION: Our results are consistent with the hypothesis that there are few age-dependent genetic differences as regards autoimmune thyroiditis in adults and children. Accordingly, CTLA4 and FCRL3 genes overexpression may play an important role in children suffering from autoimmune thyroiditis.

Mechanisms of Normalisation of Bone Metabolism during Recovery from Hyperthyroidism: Potential Role for Sclerostin and Parathyroid Hormone.

Sclerostin, a protein expressed by osteocytes, is a negative regulator of bone formation. The aim of the study was to investigate the relationship between parathyroid hormone (PTH) and markers of bone metabolism and changes of sclerostin concentrations before and after treatment of hyperthyroidism. Patients and Methods. The study involved 33 patients (26 women), age (mean ± SD) 48 ± 15 years, with hyperthyroidism. Serum sclerostin, PTH, calcium, and bone markers [osteocalcin (OC) and collagen type I cross-linked C-telopeptide I (CTX)] were measured at diagnosis of hyperthyroidism and after treatment with thiamazole. Results. After treatment of hyperthyroidism a significant decrease in free T3 (FT3) and free T4 (FT4) concentrations was accompanied by marked decrease of serum sclerostin (from 43.7 ± 29.3 to 28.1 ± 18.4 pmol/L; p < 0.001), OC (from 35.6 ± 22.0 to 27.0 ± 14.3 ng/mL; p < 0.001), and CTX (from 0.49 ± 0.35 to 0.35 ± 0.23 ng/dL; p < 0.005), accompanied by an increase of PTH (from 29.3 ± 14.9 to 39.8 ± 19.8; p < 0.001). During hyperthyroidism there was a positive correlation between sclerostin and CTX (r s = 0.41, p < 0.05) and between OC and thyroid hormones (with FT3 r s = 0.42, with FT4 r s = 0.45, p < 0.05). Conclusions. Successful treatment of hyperthyroidism results in a significant decrease in serum sclerostin and bone markers concentrations, accompanied by an increase of PTH.

Assessment of adequacy of vitamin D supplementation during pregnancy.

INTRODUCTION: Deficiency of vitamin D in pregnancy leads to higher incidences of preeclampsia, gestational diabetes, preterm birth, bacterial vaginosis, and also affects the health of the infants. According to Polish recommendations published in 2009, vitamin D supplementation in pregnant women should be provided from the 2nd trimester of pregnancy in daily dose of 800-1000 IU. The aim of the presented study is: 1) to estimate how many pregnant women comply with those recommendations and 2) to determine the 25(OH)D levels in pregnant women. PATIENTS AND METHODS: The study included 88 pregnant women, aged 20-40 years, between 12-35 week of gestation. Vitamin D concentrations [25(OH)D] were measured by a direct electrochemiluminescence immunoassay (Elecsys, Roche). RESULTS: 31 of 88 pregnant women (35.2%) did not use any supplementation. Mean level of 25(OH)D was 28.8 ± 14.8 ng/mL (range from 4.0 - 77.5 ng/mL). Vitamin D deficiency, defined as 25(OH)D concentration below 20 ng/mL, was found in 31.8% of the women (28/88). Insufficiency of vitamin D [25(OH)D concentration between 20-30 ng/mL] was present in 26.1% of the women (23/88). Optimal level of 25(OH)D (over 30 ng/mL) was present in 37/88 (42.0% women). Hence, in 46.2% of women taking vitamin D supplementation, the levels of 25(OH)D were still below 30 ng/mL. CONCLUSIONS: Supplementation of vitamin D in the investigated group was inadequate. More than 35% of pregnant women did not take any supplements, while half of the subjects who had declared taking vitamin D, failed to achieve optimal serum 25(OH)D concentration.

Assessment of cyclooxygenase-1 and 2 gene expression levels in chronic autoimmune thyroiditis, papillary thyroid carcinoma and nontoxic nodular goitre.

INTRODUCTION: The cyclooxygenases are a group of enzymes catalyzing the formation of prostaglandins from arachidonic acid. Cyclooxygenase-1 (COX-1) is a constitutive form, thought to be a "housekeeping gene", with constant levels of expression in most tissues. COX-1 expression in the thyroid gland, except for medullary thyroid carcinoma, has not been a subject of much interest. Cyclooxygenase-2 (COX-2) can be expressed in response to various stimuli, such as mitogens, hormones, cytokines, growth factors. The product of COX-2 activity has been implicated in carcinogenesis. Recent studies have shown that up-regulation of COX-2 is associated with numerous neoplasms. Hereby, we present a study analysing COX-1 and COX-2 expression in papillary thyroid carcinoma (PTC), Hashimoto thyroiditis (HT) and nontoxic nodular goitre (NNG) in fine needle aspiration biopsy (FNAB) washouts and in postoperative tissue. MATERIAL AND METHODS: Cytological specimens from 120 patients (105 females and 15 males) have been studied, including patients with HT, PTC and NNG. Moreover, we have examined postoperative tissue specimens from 51 patients with PTC and NNG. The methods of molecular analysis have included extraction of total RNA from FNAB cytological material and postoperative tissues, spectrophotometric assessment of the RNA purity, cDNA synthesis in reverse transcription reaction and an analysis of genes expression data by real-time PCR. RESULTS: The performed analysis has revealed statistically significant higher expression level of the COX-2 gene in PTC group, in comparison with HT and NNG groups (in both cytological and postoperative material). In PTC patients, COX-2 gene expression levels in the material obtained by FNAB were similar to those in the postoperative thyroid tissue. No correlations between COX-2 gene expression level and TNM staging in PTC samples have been observed. There were no correlations between COX-2 expression and anti-TPO antibodies level, or patient's sex or age in the studied groups. Also, there were no correlations of COX-1 gene expression level among PTC, HT and NNG groups. CONCLUSIONS: Our results suggest that COX-2 gene does not participate in the mechanisms involved in molecular association of HT with PTC. However, in case of PTC itself, it may play some role in neoplastic transformation.

Distribution of subpopulations of dendritic cells in peripheral blood of patients treated with exogenous thyrotropin.

BACKGROUND: Dendritic cells (DCs) play a major role as regulators of inflammatory events associated with thyroid pathology. The immunoregulatory function of DCs depends strongly on their subtype, as well as maturation and activation status. Numerous hormonal factors modulate the immune properties of DCs, however, little is known about effects exerted by the hypothalamus-pituitary-thyroid-axis. Recently, we have shown a direct regulatory influence of thyroid hormones (TH) on human DCs function. The aim of the present study was to analyze the effect of systemically administered thyrotropin (TSH) on human blood DCs ex vivo. METHODS: Blood samples for the cytometric analysis of peripheral blood plasmacytoid and myeloid DCs subtypes were collected from patients subjected to total thyroidectomy because of differentiated thyroid carcinoma at 2 time points: (i) directly before the commencement of TSH administration and (ii) 5 days after first TSH injection. The whole blood quantitative and phenotypic analysis of plasmacytoid and myeloid DCs subtypes was performed by flow cytometry. RESULTS: Administration of TSH did not influence the percentage of plasmacytoid DCs in peripheral blood of study participants. Also the percentage of the two main myeloid DCs subpopulations - CD1c/BDCA1+ DCs and CD141/BDCA3+ DCs did not change significantly. TSH administration had no effect on the surface expression of CD86 - one of the major costimulatory molecules - neither in the whole peripheral blood mononuclear cell (PBMC) fraction nor in particular DCs subtypes. CONCLUSIONS: In the present study, we demonstrated no influence of systemic TSH administration on human peripheral blood DCs subtypes. These results are in accordance with our previous work suggesting the direct effect of TH on human DCs ex vivo.

The role of phosphoinositide 3-kinase subunits in chronic thyroiditis.

BACKGROUND: The risk of neoplastic transformation in patients with chronic thyroiditis (Hashimoto's thyroiditis - HT) is slightly increased. Genetic background of this observation is still unclear. PI3K isoforms are linked with inflammatory and neoplastic processes, thus they appear to be interesting subjects of a research in this respect. The aim of our study was to assess the PIK3CA, PIK3CB, PIK3CD and PIK3CG genes expression levels in HT. METHODS: Following conventional cytological examination, 67 thyroid FNAB specimens, received from patients with HT, were quantitatively evaluated regarding PIK3CA, PIK3CB, PIK3CD and PIK3CG expression levels by real-time PCR in the ABI PRISM ®7500 Sequence Detection System. RESULTS: The performed analysis has revealed significantly higher expression levels (RQ) of PIK3CD, PIK3CG and PIK3CA genes in comparison with PIK3CB gene (p<0.05) and significantly higher gene expression level of PIK3CD in comparison with PIK3CA (p<0.05). CONCLUSION: The observed increased PIK3CD, PIK3CG genes expression in HT is probably related to lymphocyte infiltration commonly seen in this condition, however, the role of increased PIK3CA gene expression in the multi-step carcinogenesis process cannot be excluded.

Evaluation of efficacy of iodine prophylaxis in Poland based on the examination of schoolchildren living in Opoczno Town (Lodz Voivodship).

BACKGROUND: In 1997 a currently obligatory model of iodine prophylaxis, based on mandatory iodization of household salt with 30 mg KI/kg, was introduced. The aim of our study was to assess the iodine intake among school-age children living in Opoczno in 3 subsequent time points - in 1994, before establishment of currently operating model of iodine prophylaxis, in 1999 - 2 years after implementation of iodine prophylaxis and in 2010, - 14 years after its implementation. METHODS: We assessed goitre incidence and urine iodine concentration (UIC) in 104 children in 1994, 207 children in 1999 and 174 children in 2012. Age of examined children ranged from 6 to 15 years. The thyroid volumes evaluated by ultrasound were compared to reference values for thyroid volume proposed by Zimmermann at al. Moreover, we have introduced a new index - V/BSA ratio (comparison of thyroid volume to the body surface area), which to our belief allows for more accurate assessment of thyroid volume. RESULTS: The median of UICs was 45.5 µg/L (1994), 101.1 µg/L (1999) and 100.6 µg/L (2010). The distribution of obtained results has changed as well - iodine concentrations below 50 µg/L were present in 59.1% children in 1994, in 12.6% children - in 1999 and in 7.1% children - in 2010.Although a significant decrease in goitre incidence with regard to age - 92.6% (1994) vs 18.5% (1999) and 15.8% (2010), as well as with regard to BSA - 95.4% (1994) vs 15.2% (1999) and 11.6% (2010) was observed, it still points to the iodine deficiency, which is in contradiction with UICs as they are within normal limits. V/BSA ratio avoids such discrepancy. The values of ratio V/BSA were higher in 1994 (7.079 ± 2.775) than in 1999 (2.935 ± 1.112) (p<0.05) and in 2010 (2.846 ± 1.029) (p<0.05). CONCLUSIONS: Hitherto model of iodine prophylaxis has proved to be effective in eliminating the iodine deficiency. The iodine intake is now more even, homogenous, which translates into smaller scatter of UICs and less percentage of children, in whom UIC is less than 50 µg/L. However, the iodine intake only slightly exceeds the recommended values, so median of UICs oscillates around the lower limit of references values.

Successful treatment of thyrotoxicosis is accompanied by a decrease in serum sclerostin levels.

UNLABELLED: Sclerostin, a product of a SOST gene, is a protein expressed by osteocytes that inhibits osteoblastic bone formation. Several hormones, including PTH and glucocorticosteroids, have been suggested to be possible regulators of sclerostin production. The influence of thyroid hormones on sclerostin synthesis has not been investigated, so far. The aim of the study was to evaluate sclerostin concentrations in patients before and after treatment of thyrotoxicosis. PATIENTS AND METHODS: The study involved 15 patients (4 men), mean age 51.8±15.3 years, mean BMI value - 24.7±3.5, with thyrotoxicosis due to Graves' disease or toxic multinodular goitre. Serum sclerostin was measured by immunoassay at diagnosis of thyrotoxicosis and after 6-10 weeks of treatment with thiamazole. The data were analysed by means of simple descriptive statistics of location and dispersion and Mann-Whitney U test for pairs of results, before and after thiamazole therapy. Association between variables was evaluated with use of Spearman`s correlation coefficient. RESULTS: There was a significant decrease in free T3 (FT3) and free T4 (FT4) concentrations (from 8.74±4.79 pg/ml to 3.54±2.40 pg/ml, and from 4.48±2.21 ng/ml to 1.02±1.07 ng/ml, respectively, p<0.001). This was accompanied by a marked decrease of serum sclerostin levels from 55.46±20.90 pmol/l to 35.73±15.70 pmol/l, p<0.0015). Interestingly, enough, sclerostin levels did not correlate with serum FT3 or FT4 concentrations. CONCLUSIONS: Restoration of a euthyroid state in patients with thyrotoxicosis results in a significant decrease in serum sclerostin concentrations. The above mentioned phenomenon may reflect lowering of bone metabolism, but a possible direct influence of thyroid hormones on SOST gene needs to be investigated.

Is the PIK3CA gene expression level in FNAB washouts equivalent to that in postoperative tissue specimens of papillary thyroid carcinoma?

INTRODUCTION: Papillary thyroid carcinoma (PTC) is the most common malignant tumor of the thyroid gland. The pathogenesis of PTC remains still mostly enigmatic, although PI3K/PTEN/AKT pathway has been proposed to play a role in development of PTC. Moreover, the significance of genetic analysis in the material from fine-needle aspiration biopsy (FNAB) in PTC patients has recently been demonstrated. Hereby, we present a study analyzing expression of PIK3CA in FNAB washouts of PTC and a comparison of the level of that expression with respective expression in postoperative PTC tissue. Furthermore, we have assessed correlation between tumor size, evaluated according to pTNM scale, and level of PIK3CA gene expression in postoperative PTC tissue. METHODS: Total RNA was extracted by use of an RNeasy Micro Kit (Qiagen, Hilden, Germany) in FNAB material, and RNeasy Midi Kit (Qiagen, Hilden, Germany) in tissue material. The purity of total RNA was assessed by NanoDrop® ND-100 spectrophotometr. One hundred nanograms of total RNA were used in the first strand cDNA synthesis with TaqMan® Reverse Transcripton Reagents (Applied Biosystems, Branchburg, New Jersey, USA). The gene expression level of PIK3CA was analyzed by real-time PCR in the ABI PRISM ®7500 Sequence Detection System in the 21 (17 women, 4 men) FNAB and 20 (16 women, 4 men) postsurgical specimens of PTC. pTNM staging of PTC was assessed based on UICC classification. RESULTS: Overexpression of PIK3CA was confirmed in FNAB washout specimens and in postoperative tissues of PTC, in comparison to macroscopically unchanged thyroid tissue. Furthermore, statistically significant differences in PIK3CA gene expression levels between both examined groups were not confirmed. Moreover, correlation between pTNM staging and level of PIK3CA gene expression in PTC samples was not found. CONCLUSION: The genetic analysis of overexpression of PIK3CA in FNAB washout specimens may be equivalent of postsurgical PTC tissue. A possibility of its future clinical application in FNAB specimens - adequate or undetermined for cytological analysis - awaits for evaluation. The level of expression of PIK3CA is independent of primary thyroid tumour size, evaluated according pTNM scale.

COX-2 expression in papillary thyroid carcinoma (PTC) in cytological material obtained by fine needle aspiration biopsy (FNAB).

BACKGROUND: COX-2 is an enzyme isoform that catalyses the formation of prostanoids from arachidonic acid. An increased COX-2 gene expression is believed to participate in carcinogenesis. Recent studies have shown that COX-2 up-regulation is associated with the development of numerous neoplasms, including skin, colorectal, breast, lung, stomach, pancreas and liver cancers. COX-2 products stimulate endothelial cell proliferation and their overexpression has been demonstrated to be involved in the mechanism of decreased resistance to apoptosis. Suppressed angiogenesis was found in experimental animal studies as a consequence of null mutation of COX-2 gene in mice. Despite the role of COX-2 expression remains a subject of numerous studies, its participation in carcinogenesis or the thyroid cancer progression remains unclear. METHODS: Twenty three (23) patients with cytological diagnosis of PTC were evaluated. After FNAB examination, the needle was washed out with a lysis buffer and the obtained material was used for COX-2 expression estimation. Total RNA was isolated (RNeasy Micro Kit), and RT reactions were performed. ß-actin was used as endogenous control. Relative COX-2 expression was assessed in real-time PCR reactions by an ABI PRISM 7500 Sequence Detection System, using the ΔΔCT method. RESULTS: COX-2 gene expression was higher in patients with PTC, when compared to specimens from patients with non-toxic nodular goitre (NTG). CONCLUSIONS: The preliminary results may indicate COX-2 role in thyroid cancer pathogenesis, however the observed variability in results among particular subjects requires additional clinical data and tumor progression analysis.

Assessment of RET/PTC1 and RET/PTC3 rearrangements in fine-needle aspiration biopsy specimens collected from patients with Hashimoto's thyroiditis.

BACKGROUND: RET/PTC rearrangements are the most frequent molecular changes in papillary thyroid carcinoma (PTC). So far, 15 main RET/PTC rearrangements have been described, among which RET/PTC1 and RET/PTC3 are the most common in PTC - especially in radiation-induced tumours. RET/PTC1 and RET/PTC3 are the result of intrachromosomal paracentric inversions in chromosome 10, where RET and the activating genes (H4 and ELE1, respectively) are located. Recently, RET/PTC rearrangements have been shown not only in PTC but also in benign thyroid lesions, including Hashimoto's thyroiditis (HT). The aim of study was an assessment of RET/PTC1 and RET/PTC3 rearrangements in patients with Hashimoto's thyroiditis. MATERIALS AND METHODS: Thyroid aspirates, eligible for the study, were obtained from 26 patients with Hashimoto's thyroiditis by fine-needle aspiration biopsy (FNAB). Each aspirate was smeared for conventional cytology, while its remaining part was immediately washed out of the needle. The cells, obtained from the needle, were used in further investigation. Total RNA from FNAB was extracted by use of an RNeasy Micro Kit, based on modified Chomczynski and Sacchi's method and reverse transcription (RT-PCR) was done. Quantitative evaluation of RET/PTC1 and RET/PTC3 rearrangements by real-time PCR was performed by an ABI PRISM® 7500 Sequence Detection System. In the study, PTC tissues with known RET/PTC1 and RET/PTC3 rearrangements served as a reference standard (calibrator), while ß-actin gene was used as endogenous control. RESULTS: Amplification reactions were done in triplicate for each examined sample. No RET/PTC1 and RET/PTC3 rearrangements were found in the examined samples. CONCLUSIONS: Our results indicate that RET/PTC1 and RET/PTC3 rearrangements in Hashimoto's thyroiditis, if any, are rather rare events and further investigations should be conducted in order to determine molecular changes, connecting Hashimoto's thyroiditis with PTC.

Metastases of breast cancer to the thyroid gland in two patients - a case report.

INTRODUCTION: Metastatic cancer is rarely found in the thyroid (only 2-3% of malignant tumours found in that gland); primary sources usually including breast, kidney, and lung tumours. CASES REPORTS: Two cases of advanced breast cancer with thyroid metastases in female patients are presented. The similarities between these two cases included: 1) postmenopausal age; 2) diagnosis based on result of FNAB (numerous groups of cells with epithelial phenotype strongly implying metastatic breast cancer); 3) thyroid function - overt hyperthyroidism in the first woman and subclinical hyperthyroidism in the second one; 4) the presence of nodular goitre in clinical examination, the occurrence of many nodular solid normoechogenic lesions with calcifications in both thyroid lobes in US; and 5) negative antithyroid antibodies. The main difference was the time of establishing diagnosis; in the first woman - before mammectomy, parallel to diagnostics of breast tumour, and in the second woman four years after mammectomy, during cancer dissemination (with right pleural effusion and lung metastasis). In the first case, mammectomy was followed two weeks later by thyroidectomy. The second patient was disqualified from thyroid surgery due to systemic metastatic disease. CONCLUSIONS: 1. Fine needle aspiration biopsy of the thyroid gland should obligatorily be performed in patients with breast cancer and nodular goitre, even without any clinical data of metastatic disease. 2. The clinical context of cytological findings is of critical value. 3. In patients with breast cancer accompanied by multinodular goitre, we recommend that more punctures be performed during FNAB than is routinely done. (

Relative quantification of PIK3CA gene expression level in fine-needle aspiration biopsy thyroid specimens collected from patients with papillary thyroid carcinoma and non-toxic goitre by real-time RT-PCR.

BACKGROUND: Recent studies have shown that the phosphatidylinositol 3-kinase (PI3K) signaling pathway is important regulator of many cellular events, including apoptosis, proliferation and motility. PI3K pathway alterations (PIK3CA gene mutations and/or amplification) have been observed in various human tumours. In the majority of diagnosed cases, mutations are localized in one of the three "hot spots" in the gene, responsible for coding catalytic subunit α of class I PI3K (PIK3CA). Mutations and amplification of PIK3CA gene are characteristic for thyroid cancer, as well. METHODS: The aim of our study was to examine a gene expression level of PIK3CA in fine-needle aspiration biopsy (FNAB) thyroid specimens in two types of thyroid lesions, papillary thyroid carcinoma (PTC) and non-toxic goitre (NTG). Following conventional cytological examination, 42 thyroid FNAB specimens, received from patients with PTC (n = 20) and NTG (n = 22), were quantitatively evaluated regarding PIK3CA expression level by real-time PCR in the ABI PRISM® 7500 Sequence Detection System. RESULTS: Significantly higher expression level (RQ) of PIK3CA in PTC group has been noted in comparison with NTG group (p < 0.05). CONCLUSION: These observations may suggest role of PIK3CA alterations in PTC carcinogenesis.