Thyroglobulin Expression Research Articles

Pre-Therapeutic Measurements of Iodine Avidity in Papillary and Poorly Differentiated Thyroid Cancer Reveal Associations with Thyroglobulin Expression, Histological Variants and Ki-67 Index.

Simple SummaryIn the treatment of thyroid cancer, tumour uptake of iodine is important because it enables the use of radioactive iodine to kill cancer tissue. How radioactive iodine treatments are performed is usually determined by the size of the tumour. This study attempted to find new ways of predicting iodine uptake in advance of treatment. Several factors were found to predict uptake better than tumour size, all relatively simple to investigate in routine healthcare. This information can potentially be used to treat thyroid cancer patients more in accordance with how their tumours behave.Papillary thyroid cancer (PTC) and poorly differentiated thyroid cancer (PDTC) are treated with radioiodine to reduce recurrence and to treat the spread of disease. Adequate iodine accumulation in cancer tissue, iodine avidity, is important for treatment effect. This study investigated which clinical and histological tumour characteristics correlate with avidity. To quantify avidity in cancer tissue, tracer amounts of iodine-131 were given to 45 patients with cytologically confirmed thyroid cancer. At pathology grossing, representative samples of tumour and lymph nodes were taken and subjected to radioactivity quantification ex vivo to determine avidity. Afterwards, samples underwent extended pathology work-up and analysis. We found that tumoural Tg expression and Ki-67 index were correlated with avidity, whereas tumour size and pT stage were not. The histological variant of thyroid cancer was also correlated with iodine avidity. Variants associated with worse clinical prognoses displayed lower avidity than variants with better prognoses. This work provides new information on which tumours have low iodine avidity. Lower avidity in aggressive histological PTC variants may explain their overall poorer prognoses. Our findings also suggest that radioiodine dosage could be adapted to Tg expression, Ki-67 index or histological variant instead of pT stage, potentially improving the efficacy of radioiodine therapy.

Immunohistochemically detectable thyroglobulin expression in extrathyroidal cancer is 100% specific for thyroidal tumor origin

Thyroglobulin is a secreted 660 kDa glycoprotein produced by thyroid follicular cells used in diagnostic pathology to secure or exclude a thyroidal origin of metastases of unknown primary tumors. This study was performed to estimate specificity of thyroglobulin immunohistochemistry. 9974 tumor samples from 109 different tumor types and subtypes as well as 608 samples of 76 different normal tissue types were analyzed by immunohistochemistry in a tissue microarray format. Thyroglobulin was strongly expressed in all normal thyroid samples but not in any other normal tissues. Thyroglobulin immunostaining was detected in 99.1% of 106 thyroid adenomas, 98.1% of 364 papillary, 95.2% of 147 follicular, and 7.5% of 40 anaplastic thyroid cancers. Twelve of 15 thyroid samples that were thyroglobulin negative on TMAs showed at least a weak focal thyroglobulin positivity in corresponding large sections, suggesting higher sensitivity of large section analysis. Thyroglobulin positivity in one diffuse large B-cell lymphoma of the thyroid, one chondrosarcoma metastasis to the thyroid, and 42.4% of 92 medullary thyroid cancers was considered to be caused by diffusion of thyroidal colloid from destroyed or even intact adjacent follicles. Thyroglobulin positivity was, however, not seen in 6403 extrathyroidal tumors from 104 different tumor types and subtypes. Our data demonstrate a complete specificity of positive thyroglobulin immunostaining for thyroid origin in tumor tissues obtained from extrathyroidal locations. However, for all tumors located within the thyroid, false positivity can occur as a result of tissue contamination by thyroglobulin rich thyroid colloid from adjacent normal tissue.

Long Term Rescue of the TSH Receptor Knock-Out Mouse - Thyroid Stem Cell Transplantation Restores Thyroid Function.

The synergistic activation of transcription factors can lead to thyroid progenitor cell speciation. We have previously shown in vitro that mouse or human stem cells, expressing the transcription factors NKx2-1 and Pax8, can differentiate into thyroid neo-follicular structures (TFS). We now show that syngeneic mouse TFS when implanted into hypothyroid TSH receptor knockout (TSHR-KO) mice can ameliorate the hypothyroid state for an extended period. ES cells derived from heterozygous TSHR-KO blastocysts were stably transfected with Nkx2-1-GFP and Pax8-mcherry constructs and purified into 91.8% double positive cells by flow cytometry. After 5 days of activin A treatment these double positive cells were then induced to differentiate into neo-follicles in Matrigel for 21 days in the presence of 500μU/mL of TSH. Differentiated TFS expressing thyroglobulin mRNA were implanted under the kidney capsule of 4-6 weeks old TSHR-KO mice (n=5) as well as hind limb muscle (n=2) and anterior chamber of one eye (n=2). Five of the mice tested after 4 weeks were all rendered euthyroid and all mice remained euthyroid at 20 weeks post implantation. The serum T4 fully recovered (pre-bleed 0.62 ± 0.03 to 8.40 ± 0.57 µg/dL) and the previously elevated TSH became normal or suppressed (pre-bleed 391 ± 7.6 to 4.34 ± 1.25 ng/dL) at the end of the 20 week observation period. The final histology obtained from the implanted kidney tissues showed only rudimentary thyroid follicular structures but which stained positive for thyroglobulin expression. The presence of only rudimentary structures at the site of implant on these extended animals suggested possible migration of cells from the site of implant or an inability of TFCs to maintain proper follicular morphology in these external sites for extended periods. However, there were no signs of tumor formation and no immune infiltration. These preliminary studies show that TSHR-KO mice are a useful model for orthotropic implantation of functional thyroid cells without the need for thyroidectomy, radioiodine ablation or anti thyroid drug control of thyroid function. This approach is also proof of principle that thyroid cells derived from mouse ES cells are capable of surviving as functional neo-follicles in vivo for an extended period of 20 weeks.

THE PRINCIPLE OF COMBINED PREOPERATIVE DIAGNOSIS OF THYROID TUMORS

The aim of the research is to increase the effectiveness of preoperative diagnosis of patients with thyroid tumors and to assess the use of cancer-embryonic antigen and immunocytochemical research. Materials and methods: Patients were interviewed about their complaints and lifestyle; performed ultrasound with fine-needle aspiration, determination of the level of cancer-embryonic antigen (CEA), cytological and immunocytochemical researches. Results: The Benign process in the thyroid gland is low serum REA (less than 0.95 ng / ml), poor expression of thyroglobulin (77.8%), negative reaction with TTF-1 (100%) and cytokeratin-19 (55.6%). Differential-prognostic markers of thyroid neoplasms with risk of malignancy include increased serum REA (0.95 ng / ml and above), the presence of a moderate reaction with antibodies to thyroglobulin (80.0%), a positive reaction - to TTF-1 (100.0%) and E-cadherin (90.0%), with moderate or strong expression of cytokeratin-19 (90.0%). Statistically significant markers of malignant thyroid disease were determined: the presence of harmful factors at work (45.5%), smoking (27.3%), elevated serum REA (0.95 ng / ml and above), the presence of strong cytoplasmic expression of thyroglobulin (63.6%), moderate or strong expression of TTF-1 (90.9%) and cytokeratin-19 (81.8%). Conclusions: The most appropriate and practically significant for preoperative diagnosis of thyroid tumors is a set of several diagnostic methods, which are carried out in one hospital - ultrasound with fine-needle aspiration, cytomorphological, and immunocytochemical and REA levels in a primary screening.

Endocrine-disrupting activity of mancozeb.

The objective of the present study was to review the existing data on the mechanisms involved in the endocrine disrupting activity of mancozeb (MCZ) in its main targets, including thyroid and gonads, as well as other endocrine tissues that may be potentially affected by MCZ. MCZ exposure was shown to interfere with thyroid functioning through impairment of thyroid hormone synthesis due to inhibition of sodium-iodine symporter (NIS) and thyroid peroxidase (TPO) activity, as well as thyroglobulin expression. Direct thyrotoxic effect may also contribute to thyroid pathology upon MCZ exposure. Gonadal effects of MCZ involve inhibition of sex steroid synthesis due to inhibition of P450scc (CYP11A1), as well as 3β-HSD and 17β-HSD. In parallel with altered hormone synthesis, MCZ was shown to down-regulate androgen and estrogen receptor signaling. Taken together, these gonad-specific effects result in development of both male and female reproductive dysfunction. In parallel with clearly estimated targets for MCZ endocrine disturbing activity, namely thyroid and gonads, other endocrine tissues may be also involved. Specifically, the fungicide was shown to affect cortisol synthesis that may be mediated by modulation of CYP11B1 activity. Moreover, MCZ exposure was shown to interfere with PPARγ signaling, being a key regulator of adipogenesis. The existing data also propose that endocrine-disrupting effects of MCZ exposure may be mediated by modulation of hypothalamus-pituitary-target axis. It is proposed that MCZ neurotoxicity may at least partially affect central mechanisms of endocrine system functioning. However, further studies are required to unravel the mechanisms of MCZ endocrine disrupting activity and overall toxicity.

Costus Root Extract Preserves Thyroid Hormones Levels, Thyroglobulin Expression and Thyroid Tissues in Rats Receiving Valproate Sodium

BACKGROUND: Valproate sodium is an anticonvulsant drug. Saussurea lappa (costus) is a medicinal plant rich with antioxidants. This research aimed to assess the protective effect of costus root extract against valproate sodium-induced thyrotoxicity.METHODS: Eighty adult male albino rats were equally divided into four groups; group I: untreated control, group II: rats were given 200 mg/kg BW valproate sodium orally and daily for 8 weeks, group III: rats were given 300 mg/kg BW costus root extract orally and daily for 8 weeks, and group IV: rats were given combination of valproate sodium and costus root extract. After 8 weeks, blood samples were collected to evaluate T3, T4 and thyroid-stimulating hormone (TSH) levels. Thyroid gland samples were handled for light and electron microscopic investigation. The heights of follicular cells, area % of collagen fibers and color intensity of thyroglobulin immunoreaction were statistically analyzed. RESULTS: After being given valproate sodium as an induction, hormonal assay showed significant decrease in serum T3 and T4 and significant increase of TSH. Follicular and cellular alterations were shown by light and electron microscopes. Morphometric study revealed increased follicular cell height and area % of collagen fibers and decreased color intensity of thyroglobulin. In contrast, costus root extract appeared to have a protective role against valproate sodium-induced thyroid injury. Most of the changes induced by valproate sodium were not observed after supplementation with the plant root extract.CONCLUSION: Valproate sodium has serious effects on the function and structure of thyroid gland, and this study shows that costus root extract could have a protective effect against these effects.KEYWORDS: valproate sodium, rat, thyrotoxicity, Saussurea lappa

Papillary Thyroid Cancer Tumor Spheres Cultured by Passaging Without Sorting Exhibit Cancer Stemness.

Cancer stem-like cell (CSC) markers and the role of CSCs derived from papillary thyroid carcinoma (PTC) in pathogenesis are unclear. This study aimed to investigate CSC properties using tumor spheres from passaged PTC cells but without sorting CSCs. To identify the properties of CSCs derived from PTC, the expression of SRY-box transcription factor 2(SOX2), octamer-binding transcription factor 4 (OCT4), Nanog homeobox (NANOG), thyroglobulin (TG), thyroid-stimulating hormone receptor (TSHR), E-cadherin, YES-associated protein 1 (YAP1), and signal transducer and activator of transcription 3 (STAT3) was investigated in tumor spheres serially passaged without sorting CSCs. The cultured tumor spheres had cancer stemness; high expression of OCT4, SOX2, NANOG, and YAP1; low expression of E-cadherin; and varied expression of TG, TSHR, and STAT3. PTC tumor spheres transfected with small interfering RNA targeting YAP1 had fewer CSC properties than the non-transfected tumor spheres did. Tumor spheres derived from PTC cells by passaging without sorting CSCs have more stem-like cell properties, and less differentiation potential. Thus, this simple and cost-effective method can be used for the enrichment of PTC stemness for employment in cell-based models, reducing the need for use of animal models.

Acceleration of BRAFV600E-induced thyroid carcinogenesis by TGFβ signal deficiency in mice.

Transforming growth factor-β (TGFβ) has pleiotropic actions, including both anti- and pro-tumorigenic abilities. We have previously shown no tumor development in the thyroid-specific TGFβ receptor type II knockout (Tgfβr2 KO) mice, indicating the insufficiency of defective TGFβ signal itself for thyroid cancer initiation. In the current study, we evaluated whether defective TGFβ signal accelerates BRAFV600E-mediated thyroid carcinogenesis in our mouse model, in which intrathyroidal injection of adenovirus expressing Cre under thyroglobulin (TG) promoter (Ad-TgP-Cre) into thyroid lobes of conditional BrafV600E knock-in mice (BrafCA) induces thyroid cancers 12 months later. BrafCA/wt;Tgfbr2floxE2/floxE2 mice were generated by crossing Tgfbr2floxE2/floxE2 and BrafCA mice, and Ad-TgP-Cre was injected into the left lobes of 4-6-week-old mice. Mice were sacrificed at 6 and 12 months, and the thyroid tissues were subjected to H&E and immune-histochemistry and -fluorecence. Thyroid tumors were observed in 8 of 10 mice at 6 months and 4 of 7 mice at 12 months. These tumors were judged to be malignant by H&E staining, because of the presence of papillary growth of atypical follicular cells, intranuclear cytoplasmic inclusions and so on. Immunohistochemical analyses using thyroid cancer tissues obtained at 6 months demonstrated variable levels of TG but steady levels of Paired Box-8 expression and higher Ki67 positivity. The degree of epithelial-to-mesenchymal transition could not be evaluated because normal thyroid tissues and thyroid cancers developed in BrafCA and BrafCA/wt;Tgfbr2floxE2/floxE2 mice were all E-cadherin+/vimentin-, that is, epithelial type. In a mouse model, defective TGFβ signaling pathway accelerates BRAFV600E-induced thyroid cancer development, which is occasionally accompanied by reduced TG expression implying dedifferentiation. The former finding is consistent with anti-tumorigenic ability of TGFβ in early tumorigenic process, but the latter is contradictory to generally accepted concept for TGFβ-induction of dedifferentiation.

Immunohistochemical Profile and 47-Gene Next-Generation Sequencing (NGS) Solid Tumor Panel Analysis of a Series of 13 Strumal Carcinoids

Strumal carcinoid is an extraordinary rare tumor of the ovary consisting of thyroid tissue intermixed with neuroendocrine tumor component. The cellular origin of strumal carcinoids has been an area of debate. There is also little data on detailed immunohistochemical and molecular characteristics of these neoplasms. For this reason, this series investigated the characteristics of a series of 13 strumal carcinoids using immunohistochemical markers and a 47-gene next-generation sequencing (NGS) solid tumor panel analysis. Both cellular components showed thyroglobulin expression in all tumors. TTF-1 expression was noted in both cellular components of 11 cases. Chromogranin A was positive in both components of most tumors (n = 12, 92.3% in the neuroendocrine component and n = 10, 76.9% in the thyroid follicular component). Synaptophysin stained the neuroendocrine component of all cases, and it was also identified in the follicular thyroid component of a single case. All tumors were negative for CDX2 and calcitonin. ISLET1 was positive in the neuroendocrine component of 8 cases (6.5%). With the exception of one case, all tumors were positive for SSTR2a. The tumors were associated with a low Ki67 labeling index. All cases were microsatellite stable and no pathogenic mutations were identified using a 47-gene NGS solid tumor analysis. This series underscored that strumal carcinoids are distinct neuroendocrine tumors. The synchronous expression for thyroid follicular epithelial and neuroendocrine differentiation biomarkers may suggest a precursor cell origin displaying mixed-amphicrine differentiation. While strumal carcinoids can be diagnosed by their typical morphology and immunohistochemical profile, frequent SSTR expression may serve as a potential theranostic biomarker in the management of affected patients. In addition, the absence of common driver mutations in the NGS solid tumor panel may suggest that these neoplasms seem to be genetically unrelated to follicular epithelial–derived thyroid tumors and potentially different than other commonly identified well-differentiated neuroendocrine neoplasms. Therefore, further studies focusing on molecular characteristics of this entity are still needed.

New Thieno-[2,3-d]pyrimidine-Based Functional Antagonist for the Receptor of Thyroid Stimulating Hormone.

Thyroid stimulating hormone (TSH) receptor antagonists are required for the treatment of TSH-dependent tumors and Graves disease. We developed the compound 5-amino-N-(tert-butyl)-4-(4-iodophenyl)-2-(methylthio)thieno[2,3-d]pyrimidine-6-carboxamide (TP48) and showed that it reduces the TSH-stimulated adenylate cyclase activity in rat thyroid membranes. Pretreatment of rats with compound TP48 (ip, 40 mg/kg) reduced the increase in the levels of total and free thyroxin in blood and the increase in the expression of thyroglobulin and D2 deiodinase genes in the thyroid gland, which are responsible for the synthesis of thyroid hormones, which were caused by intranasal administration of thyroliberin to animals (300μg/kg). These data indicate that compound TP48 is a functional antagonist of the TSH receptor and can be used to correct the thyroid status in hyperthyroidism.

Thyrotropin, but Not Thyroid-Stimulating Antibodies, Induces Biphasic Regulation of Gene Expression in Human Thyrocytes.

Background: Thyrotropin (TSH) and thyroid-stimulating antibodies (TSAbs) activate TSH receptor (TSHR) signaling by binding to its extracellular domain. TSHR signaling has been studied extensively in animal thyrocytes and in engineered cell lines, and differences in signaling have been observed in different cell systems. We, therefore, decided to characterize and compare TSHR signaling mediated by TSH and monoclonal TSAbs in human thyrocytes in primary culture. Methods: We used quantitative reverse transcription-polymerase chain reaction to measure mRNA levels of thyroid-specific genes thyroglobulin (TG), thyroperoxidase (TPO), iodothyronine deiodinase type 2 (DIO2), sodium-iodide symporter (NIS), and TSHR after stimulation by TSH or two monoclonal TSAbs, KSAb1 and M22. We also compared secreted TG protein after TSHR activation by TSH and TSAbs using an enzyme-linked immunosorbent assay. TSHR cell surface expression was determined using fluorescence activated cell sorting (FACS). Results: We found that TSH at low doses increases and at high doses (>1 mU/mL) decreases levels of gene expression for TSHR, TG, TPO, NIS, and DIO2. The biphasic effect of TSH on signaling was not caused by downregulation of cell surface TSHRs. This bell-shaped biphasic dose-response curve has been termed an inverted U-shaped dose-response curve (IUDRC). An IUDRC was also found for TSH-induced regulation of TG secretion. In contrast, KSAb1- and M22-induced regulation of TSHR, TG, TPO, NIS, and DIO2 gene expression, and secreted TG followed a monotonic dose-response curve that plateaus at high doses of activating antibody. Conclusions: Our data demonstrate that the physiological activation of TSHRs by TSH in primary cultures of human thyrocytes is characterized by a regulatory mechanism that may inhibit thyrocyte overstimulation. In contrast, TSAbs do not exhibit biphasic regulation. Although KSAb1 and M22 may not be representative of all TSAbs found in patients with Graves' disease, we suggest that persistent robust stimulation of TSHRs by TSAbs, unrelieved by a decrease at high TSAb levels, fosters chronic stimulation of thyrocytes in Graves' hyperthyroidism.

Ультразвукові та цитологічні характеристики радіойодчутливості метастазів папілярного раку щитоподібної залози

Мета — дослідження ультразвукових і цитологічних характеристик метастазів, виявлених у поопераційний період, залежно від їх йоднакопичувальної здатності. Матеріал і методи. Ультразвукові, цитологічні, імуноцитохімічні дослідження проводили на групі метастазів папілярного раку щитоподібної залози, виявлених у поопераційний період (після тиреоїдектомії та радіойодтерапії): 30 радіойодчутливих метастазів і 65 радіойодрезистентних. Оцінювали такі ультразвукові характеристики, як локалізація метастазів, чи є вони множинними або поодинокими, ехогенність, ехоструктуру та наявність гіперехогенних включень. Цитологічні та імуноцитохімічні дослідження проводили на пункційному матеріалі даних метастазів, забарвленому за Романовським. Використовували непрямий імунопероксидазний метод із моноклональними антитілами до тиреоїдної пероксидази та тиреоглобуліну. Статистичний аналіз проводили за непараметричним критерієм Манна – Уїтні та методом χ2 у програмі Statistica 11.0. Результати. Проведені дослідження ультразвукових і цитологічних характеристик поопераційних метастазів папілярних карцином щитоподібної залози не виявили значущої різниці між групами радіойодрезистентних і радіойодчутливих метастазів за такими ультразвуковими характеристиками, як ехогенність, ехоструктура та наявність гіперехогенних включень. Показано, що у випадках виявлення гіпоехогенних метастазіву латеральних лімфатичних колекторах імовірність радіойодрезистентності метастазів становить 93±2%. Ультразвукову ознаку кістозної дегенерації виявляли в 1,8 раза частіше в радіойодрезистентних метастазах порівняно з радіойодчутливими. Цитологічні дослідження демонструють фенотипову гетерогенность епітелію радіойодрезистентних метастазів порівняно з радіойодчутливими та вірогідну різницю в експресії тиреоглобуліну й тиреоїдної пероксидази. Висновки. За даними ультразвукового дослідження не виявлено достовірних змін, які дозволили б проводити диференційну діагностику між радіойодрезистентними та радіойодчутливими метастазами папілярного раку щитоподібної залози. Визначено цитологічні ознаки радіойодрезистентних метастазів папілярного тиреоїдного раку, які дозволяють їх передопераційне прогнозування. Продемонстровано значущу редукцію експресії тиреоїдної пероксидази та тиреоглобуліну в пунктатах радіойодрезистентних метастазів порівняно з радіойодчутливими, що є підставою передопераційного прогнозування радіойодрезистентності поопераційних метастазів папілярного тиреоїдного раку. Виявлені за даними ультразвукової діагностики метастатичні лимфатичні вузли шиї дозволяють проводити подальші імуноцитохімічні та цитологічні дослідження, що може підвищити ефективність діагностики метастазів високодиференційованого раку щитоподібної залози й полегшити вибір подальшого лікування.

Expression of thyroglobulin by regulatory T cells in thyroid tissue.

The follicles are the minimal functional unit of the thyroid; the morphology and the function of each follicle can vary significantly. However, the reasons for the apparent follicular heterogeneity are poorly understood. Some tissue-resident regulatory T cells (Tregs) have a special phenotype that expresses unique molecules related to local tissue and regulates the tissue functions. The aim of this study was to identify the phenotype of thyroid Tregs and the roles of thyroid Tregs in thyroid physiological regulation. Thyroid tissue and peripheral blood samples were obtained from patients with benign thyroid nodules. Microarray-based gene expression, flow cytometry, immunofluorescence microscopy, and functional analysis of thyroid Tregs were performed. Here, we demonstrated that human thyroid Tregs expressed high level of thyroglobulin (Tg), both gene and protein. The immunofluorescence microscopy of thyroid section showed that the FOXP3+Tg+ cells concentrated in some of the thyroid follicles, at the side of the thyroid follicle. The peripheral blood Tregs expressed minimal levels of Tg, and low levels of Tg could effectively induce peripheral blood Tregs to express Tg, which was independent of thyrotropin simulation. Furthermore, the Tg secreted freely from thyroid Tregs that negatively regulated some thyroid-related genes expression. Our results revealed that the thyroid Tregs was a distinct population of Tregs, which expressed high level of Tg. The thyroid Tregs regulate thyroid function by Tg that is paracrine from the cells.

Rare and common genetic variations in the Keap1/Nrf2 antioxidant response pathway impact thyroglobulin gene expression and circulating levels, respectively

Nuclear factor, erythroid 2-like 2 (Nrf2) is a transcription factor that has been gaining attention in the field of pharmacology and especially in the chemoprevention of diseases such as cancer, metabolic and neurodegenerative diseases, etc. This is because natural compounds such as sulforaphane, which is found in broccoli sprout extracts, can activate Nrf2. The repertoire of the roles of Nrf2 is ever increasing; besides its traditional antioxidant and cytoprotective effects, Nrf2 can have other functions as a transcription factor. We have recently shown that Nrf2 directly regulates the expression of thyroglobulin (Tg), which is the most abundant thyroidal protein and the precursor of thyroid hormones. Two functional binding sites for Nrf2 (antioxidant response elements, AREs) were identified in the regulatory region of the TG gene. Interestingly, we then observed that one of these AREs harbors a rare single-nucleotide polymorphism (SNP). Also recently, we performed the first genome-wide association study (GWAS) for common SNPs that impact the circulating levels of Tg. Based on these investigations, we were triggered (i) to investigate whether common SNPs in the Nrf2 pathway correlate with circulating Tg levels; and (ii) to examine whether the rare SNP in one of the TG regulatory AREs may affect gene expression. To address the first question, we analyzed GWAS data from a general population and its two subpopulations, one with thyroid disease and/or abnormal thyroid function tests and the other without, in which circulating Tg levels had been measured. Statistically significant associations with Tg levels were observed in the genes encoding Nrf2 and Keap1, including, notably, a known functional SNP in the promoter of the gene encoding Nrf2. Regarding the rare SNP (rs778940395) in the proximal ARE of the TG enhancer, luciferase reporter gene expression studies in PCCL3 rat thyroid follicular cells showed that this SNP abrogated the basal and sulforaphane- or TSH-induced luciferase activity, behaving as a complete loss-of-function mutation. Thus, both rare and common genetic variation in the Keap1/Nrf2 pathway can impact TG expression and Tg circulating levels, respectively.

Цитологічні особливості багаторазово метастазуючих тиреоїдних папілярних карцином

Мета — комплексне цитологічне та імуноцитохімічне дослідження тиреоцитів пунктатів поопераційних реґіонарних метастазів папілярних тиреоїдних карцином із багаторазовим метастазуванням і порівняльний аналіз цитологічних характеристик первинних і повторних поопераційних метастазів у кожного пацієнта. Матеріал і методи. Цитоморфологічні та імуноцитохімічні дослідження з використанням моноклональних антитіл проти тиреоїдної пероксидази, тиреоглобуліну, антигену Кі‑67 проводили на матеріалі тонкоголкових аспіраційних пункційних біопсій 26 метастазів, виявлених у поопераційний період (після проведення тиреоїдектомії та радіойодтерапії), отриманих від 12 хворих на папілярний тиреоїдний рак із багаторазовим метастазуванням. Результати. За результатами дослідження фенотипових характеристик тиреоцитів пунктатів метастазів рецидивуючих папілярних карцином щитоподібної залози з повторним метастазуванням виявлено зміни цитологічних та імуноцитохімічних характеристик тиреоцитів у пунктатах повторних метастазів порівняно з такими первинних метастазів. Показано вірогідне зниження експресії тиреоїдної пероксидази та тиреоглобуліну та появу фенотипової гетерогенності популяції тиреоцитів (світлі клітини, клітини з макулами, оксифільні клітини) повторних метастазів порівняно з первинними. Висновок. У клітинах метастазів рецидивуючих папілярних карцином із повторним метастазуванням виявлено втрату ознак диференціації — вірогідне зниження експресії тиреоїдної пероксидази та тиреоглобуліну та появу фенотипової гетерогенності популяції тиреоцитів.

Follicular Adenoma of the Thyroid With Spindle Cell Metaplasia Mimicking Insular Thyroid Carcinoma and Lacking Thyroglobulin Expression

Abstract We report a case of a well-encapsulated thyroid adenoma with areas of spindle cell metaplasia mimicking insular or poorly differentiated growth pattern. Emphasis on overall features, especially encapsulation and lack of invasion, is critical in the evaluation of thyroid neoplasms. In this example, there was loss of thyroglobulin expression, which to date has not been reported in spindled metaplasia but was not considered diagnostically relevant in the face of low proliferative activity and lack of mitotic figures.

Cervical Lymph Node Fine-Needle Aspiration and Needle-Wash Thyroglobulin Reflex Test for Papillary Thyroid Carcinoma.

Fine-needle aspiration (FNA) cytology coupled with needle-wash thyroglobulin (FNA-Tg) testing is recommended for cervical lymph node (LN) biopsies in patients with a history of papillary thyroid carcinoma (PTC). However, the procedure has not been standardized with the assay for FNA-Tg testing. A standard operating procedure (SOP) has been generated at our facility for cervical LN FNAs with Tg reflex testing on patients with a history of PTC. The procedure requires FNA cytology to be reviewed first, and all cases not positive for PTC are reflexed for FNA-Tg testing with the Beckman Access thyroglobulin assay. The thyroglobulin cutoff value is ≤ 1.0ng/mL. From 2016 to 2017, 117 patients, including 71 women and 46 men, were identified as having a history of PTC. Patients' clinical characteristics were collected from medical records. A total of 143 LN biopsies were investigated for these patients. The results show that four out of 11 (36.4%) non-diagnostic LNs and five out of five (100%) atypical/suspicious LNs tested positive for FNA-Tg. Among these nine patients with positive thyroglobulin testing, LN metastases were proven histologically for all nine patients, and two patients were treated with LN ablation. Out of 68 LNs positive for PTC, three had FNA-Tg results. FNA-Tg testing was ordered for unknown reasons on two positive LNs (> 5000ng/mL thyroglobulin) from one patient. The third LN was tested due to non-classic morphology, and the result was less than the cutoff value. Three patients with negative LN biopsies were tested to have elevated (> 1.0ng/mL) thyroglobulin levels. One patient (FNA-Tg ng/mL) was proven to have multiple metastatic LNs through follow-up surgery. However, no positive LN was identified for the other two patients who had FNA-Tg level of 4.1ng/mL and 37ng/mL respectively. This is likely due to contamination, as these two patients had intact thyroids. In our practice, the FNA-Tg test is a very useful adjunct test to LN FNA specimens with a non-positive diagnosis in patients with a history of PTC. Furthermore, FNA-Tg testing increases diagnostic sensitivity among non-diagnostic and atypical/suspicious LNs. However, FNA-Tg testing should not substitute conventional cytology due to the following reasons: (1) false-negative thyroglobulin lab results; (2) PTC with loss of thyroglobulin expression; (3) LN metastasis from other origins; and (4) false-positive thyroglobulin testing due to blood contamination in patients who are not completely athyrotic.

Mouse Model of Poorly Differentiated Thyroid Carcinoma Driven by STRN-ALK Fusion

Chromosomal rearrangements of the ALK gene, which lead to constitutive activation of ALK tyrosine kinase, are found in various cancers. In thyroid cancers, ALK fusions, most commonly the STRN-ALK fusion, are detected in papillary thyroid cancer and with higher frequency in poorly differentiated and anaplastic thyroid cancers. Our aim was to establish a mouse model of thyroid-specific expression of STRN-ALK and to test whether this fusion drives the development of thyroid cancer with a propensity for dedifferentiation. Transgenic Tg-STRN-ALK mice with thyroglobulin-controlled expression of STRN-ALK were generated and aged with or without goitrogen treatment. Thyroids from these mice were subjected to histologic and immunohistochemical analysis. Transgenic mice with thyroid-specific expression of STRN-ALK developed poorly differentiated thyroid tumors by the age of 12 months in 22% of mice without goitrogen treatment and in 36% of mice with goitrogen treatment. Histologically and immunohistochemically, the tumors resembled poorly differentiated thyroid cancers in humans, demonstrating a solid growth pattern with sheets of round or spindle-shaped cells, decreased expression of thyroglobulin, and a tendency to lose E-cadherin. In this study, we report a novel mouse model of poorly differentiated thyroid cancer driven by the STRN-ALK oncogene with phenotypic features closely recapitulating human tumor, and with a more pronounced phenotype after additional thyroid-stimulating hormone stimulation.

Interferon-α Triggers Autoimmune Thyroid Diseases via Lysosomal-Dependent Degradation of Thyroglobulin.

Autoimmune thyroid diseases (AITDs) arise from complex interactions among genetic, epigenetic, and environmental factors. Thyroglobulin (TG) is a major susceptibility gene for both Graves disease and Hashimoto thyroiditis. Interferon-α (IFNα), a cytokine secreted during viral infections, has emerged as a key trigger of AITD. We have shown that IFNα upregulates TG transcription; however, how the upregulation of TG transcription by IFNα triggers AITD is still unknown. To evaluate how IFNα triggers AITD by testing its effects on TG processing. We exposed human thyroid cells to IFNα and evaluated its effects on TG expression and processing. Human thyroid cells exposed to INFα had increased levels of TG mRNA but reduced TG protein levels, indicating TG protein degradation. IFNα induced endoplasmic reticulum stress, but surprisingly, neither the use of chemical chaperones nor proteasome inhibitor prevented IFNα-induced TG degradation. IFNα also increased LysoTracker staining and autophagy flux measured by net light chain 3 (LC3)-II and p62 fluxes. In addition, expression of autophagy markers LC3 and autophagy-related gene 5 was higher in thyroid tissues from patients with AITD. Finally, blocking lysosomal degradation prevented IFNα-induced degradation of TG. We have shown in this study IFNα-induced lysosomal-dependent degradation of TG in human thyroid cells. Our findings suggest that during viral infections, local thyroidal IFNα production can lead to lysosomal TG degradation, releasing pathogenic TG peptides that can trigger AITD.

Anaplastic thyroid carcinoma: an epidemiologic, histologic, immunohistochemical, and molecular single-institution study

Anaplastic thyroid carcinoma (ATC) is a highly aggressive form of thyroid cancer. A single-institution thyroid cancer cohort of ATC was identified within the last 10 years at our institution. Retrospective analysis revealed that the frequency of ATC was 0.5% (11/2106 thyroid carcinomas). The average age at diagnosis of ATC was 74 years, and the female-to-male ratio was 1.2:1. ATC presented as a rapidly enlarging neck mass involving predominantly the left thyroid lobe (7/11; 64%). Cervical adenopathy was present in 7 (64%) of 11 cases. Fifty-five percent (6/11) of patients had distant metastases at the time of diagnosis. Histologically, ATC closely simulated a large variety of soft tissue sarcomas; osteoclast-like giant cell-rich tumors; squamous cell, spindle cell, and small cell carcinomas; and anaplastic/large cell lymphomas. Four tumors (4/11; 36%) showed heterologous elements, including rhabdoid and chondroid differentiation. Immunohistochemical studies showed that all ATCs lost TTF-1 and thyroglobulin expression, whereas PAX-8 expression was identified in 36% (4/11) of tumors. Intense and extensive nuclear staining of p53 (>50%) and high Ki-67 proliferative rate (>30%) were seen in all ATCs (11/11; 100%). Next-generation sequencing revealed recurrent BRAF V600E and TP53 gene mutations. Individual examples of a BRAF G469A mutation in ATC with follicular carcinoma component, EGFR, PTEN, PIK3CA, and FGFR3 mutations, were also identified, whereas 1 case of ATC showed wild-type sequencing with no identifiable alterations.