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Autoimmune thyroid diseases (AITD) such as Graves' disease (GD) or Hashimoto's thyroiditis (HT) are organ-specific diseases that involve complex interactions between distinct components of thyroid tissue. Here, we use spatial transcriptomics to explore the molecular architecture, heterogeneity and location of different cells present in the thyroid tissue, including thyroid follicular cells (TFCs), stromal cells such as fibroblasts, endothelial cells, and thyroid infiltrating lymphocytes. We identify damaged antigen-presenting TFCs with upregulated CD74 and MIF expression in thyroid samples from AITD patients. Furthermore, we discern two main fibroblast subpopulations in the connective tissue including ADIRF + myofibroblasts, mainly enriched in GD, and inflammatory fibroblasts, enriched in HT patients. We also demonstrate an increase of fenestrated PLVAP + vessels in AITD, especially in GD. Our data unveil stromal and thyroid epithelial cell subpopulations that could play a role in the pathogenesis of AITD., (© 2024. The Author(s).)

Patients with autoimmune polyendocrinopathy syndrome type 1 (APS-1) caused by autosomal recessive AIRE deficiency produce autoantibodies that neutralize type I interferons (IFNs) 1,2 , conferring a predisposition to life-threatening COVID-19 pneumonia 3 . Here we report that patients with autosomal recessive NIK or RELB deficiency, or a specific type of autosomal-dominant NF-κB2 deficiency, also have neutralizing autoantibodies against type I IFNs and are at higher risk of getting life-threatening COVID-19 pneumonia. In patients with autosomal-dominant NF-κB2 deficiency, these autoantibodies are found only in individuals who are heterozygous for variants associated with both transcription (p52 activity) loss of function (LOF) due to impaired p100 processing to generate p52, and regulatory (IκBδ activity) gain of function (GOF) due to the accumulation of unprocessed p100, therefore increasing the inhibitory activity of IκBδ (hereafter, p52 LOF /IκBδ GOF ). By contrast, neutralizing autoantibodies against type I IFNs are not found in individuals who are heterozygous for NFKB2 variants causing haploinsufficiency of p100 and p52 (hereafter, p52 LOF /IκBδ LOF ) or gain-of-function of p52 (hereafter, p52 GOF /IκBδ LOF ). In contrast to patients with APS-1, patients with disorders of NIK, RELB or NF-κB2 have very few tissue-specific autoantibodies. However, their thymuses have an abnormal structure, with few AIRE-expressing medullary thymic epithelial cells. Human inborn errors of the alternative NF-κB pathway impair the development of AIRE-expressing medullary thymic epithelial cells, thereby underlying the production of autoantibodies against type I IFNs and predisposition to viral diseases., (© 2023. The Author(s).)

The genomic simplicity of differentiated cancers derived from thyroid follicular cells offers unique insights into how oncogenic drivers impact tumour phenotype. Essentially, the main oncoproteins in thyroid cancer activate nodes in the receptor tyrosine kinase-RAS-BRAF pathway, which constitutively induces MAPK signalling to varying degrees consistent with their specific biochemical mechanisms of action. The magnitude of the flux through the MAPK signalling pathway determines key elements of thyroid cancer biology, including differentiation state, invasive properties and the cellular composition of the tumour microenvironment. Progression of disease results from genomic lesions that drive immortalization, disrupt chromatin accessibility and cause cell cycle checkpoint dysfunction, in conjunction with a tumour microenvironment characterized by progressive immunosuppression. This Review charts the genomic trajectories of these common endocrine tumours, while connecting them to the biological states that they confer., (© 2023. Springer Nature Limited.)

Hashimoto thyroiditis (HT) is a pathology that often causes a gradual thyroid insufficiency in affected patients due to the autoimmune destruction of this gland. The cellular immune response mediated by T helper lymphocytes TH1 and TH17 can induce the HT disease. In this pathologic condition, there is an imbalance between the TH17 and Treg lymphocytes as well as a gut microbiota dysfunction. The objective of this work was to describe the interactions of the cell subpopulations that participate in HT. To achieve this goal, we generated a mathematical model that allowed the simulation of different scenarios for the dynamic interaction between thyroid cells, the immune system, and the gut microbiota. We used a hypothetical-deductive design of mathematical modeling based on a system of ordinary differential equations, where the state variables are the TH1, TH17, and Treg lymphocytes, the thyrocytes, and the bacteria from gut microbiota. This work generated a compartmental model of the cellular immune response occurring in the thyroid gland. It was observed that TH1 and TH17 lymphocytes could increase the immune cells' activity, as well as activate effector cells directly and trigger the apoptosis and inflammation processes of healthy thyrocytes indirectly. Likewise, the model showed that a reduction in Treg lymphocytes could increase the activity of TH17 lymphocytes when an imbalance of the gut microbiota composition occurred. The numerical results highlight the TH1, TH17, and bacterial balance of the gut microbiota activities as important factors for the development of HT disease., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society.)

Stromal interaction molecule 1 (STIM1) and the ORAI1 calcium channel mediate store-operated calcium entry (SOCE) and regulate a multitude of cellular functions. The identity and function of these proteins in thyroid cancer remain elusive. We show that STIM1 and ORAI1 expression is elevated in thyroid cancer cell lines, compared to primary thyroid cells. Knock-down of STIM1 or ORAI1 attenuated SOCE, reduced invasion, and the expression of promigratory sphingosine 1-phosphate and vascular endothelial growth factor-2 receptors in thyroid cancer ML-1 cells. Cell proliferation was attenuated in these knock-down cells due to increased G1 phase of the cell cycle and enhanced expression of cyclin-dependent kinase inhibitory proteins p21 and p27. STIM1 protein was upregulated in thyroid cancer tissue, compared to normal tissue. Downregulation of STIM1 restored expression of thyroid stimulating hormone receptor, thyroid specific proteins and increased iodine uptake. STIM1 knockdown ML-1 cells were more susceptible to chemotherapeutic drugs, and significantly reduced tumor growth in Zebrafish. Furthermore, STIM1-siRNA-loaded mesoporous polydopamine nanoparticles attenuated invasion and proliferation of ML-1 cells. Taken together, our data suggest that STIM1 is a potential diagnostic and therapeutic target for treatment of thyroid cancer., (© 2021. The Author(s).)

Background: Galectin-3 ( LGALS3 ) is an important glycoprotein involved in the malignant transformation of thyrocytes acting in the extracellular matrix, cytoplasm, and nucleus where it regulates TTF-1 and TCF4 transcription factors. Within LGALS3 gene, a common single-nucleotide polymorphism (SNP) (c.191C>A, p.Pro64His; rs4644) encoding for the variant Proline to Histidine at codon 64 has been extensively studied. However, data on rs4644 in the context of thyroid cancer are lacking. Thus, the aim of the present work was to evaluate the role of the rs4644 SNP as risk factor for differentiated thyroid cancer (DTC) and to determine the effect on the transcriptome in thyrocytes. Methods: A case/control association study in 1223 controls and 1142 unrelated consecutive DTC patients was carried out to evaluate the association between rs4644-P64H and the risk of DTC. We used the nonmalignant cell line Nthy-Ori (rs4644-C/A) and the CRISPR/Cas9 technique to generate isogenic cells carrying either the rs4644-A/A or rs4644-C/C homozygosis. Then, the transcriptome of the derivative and unmodified parental cells was analyzed by RNA-seq. Genes differentially expressed were validated by quantitative reverse transcription PCR and further tested in the parental Nthy-Ori cells after LGALS3 gene silencing, to investigate whether the expression of target genes was dependent on galectin-3 levels. Results: rs4644 AA genotype was associated with a reduced risk of DTC (compared with CC, OR adj  = 0.66; 95% confidence interval = 0.46-0.93; P ass  = 0.02). We found that rs4644 affects galectin-3 as a transcriptional coregulator. Among 34 genes affected by rs4644, HES1 , HSPA6 , SPC24 , and NHS were of particular interest since their expression was rs4644-dependent (CC>AA for the first and AA>CC for the others), also in 574 thyroid tissues of Genotype-Tissue Expression (GTEx) biobank. Moreover, the expression of these genes was regulated by LGALS3 -silencing. Using the proximity ligation assay in Nthy-Ori cells, we found that the TTF-1 interaction was genotype dependent. Conclusions: Our data show that in thyroid, rs4644 is a trans-expression quantitative trait locus that can modify the transcriptional expression of downstream genes, through the modulation of TTF-1.

Complete absence of thyroid hormone is incompatible with life in vertebrates. Thyroxine is synthesized within thyroid follicles upon iodination of thyroglobulin conveyed from the endoplasmic reticulum (ER), via the Golgi complex, to the extracellular follicular lumen. In congenital hypothyroidism from biallelic thyroglobulin mutation, thyroglobulin is misfolded and cannot advance from the ER, eliminating its secretion and triggering ER stress. Nevertheless, untreated patients somehow continue to synthesize sufficient thyroxine to yield measurable serum levels that sustain life. Here, we demonstrate that TGW2346R/W2346R humans, TGcog/cog mice, and TGrdw/rdw rats exhibited no detectable ER export of thyroglobulin, accompanied by severe thyroidal ER stress and thyroid cell death. Nevertheless, thyroxine was synthesized, and brief treatment of TGrdw/rdw rats with antithyroid drug was lethal to the animals. When untreated, remarkably, thyroxine was synthesized on the mutant thyroglobulin protein, delivered via dead thyrocytes that decompose within the follicle lumen, where they were iodinated and cannibalized by surrounding live thyrocytes. As the animals continued to grow goiters, circulating thyroxine increased. However, when TGrdw/rdw rats age, they cannot sustain goiter growth that provided the dying cells needed for ongoing thyroxine synthesis, resulting in profound hypothyroidism. These results establish a disease mechanism wherein dead thyrocytes support organismal survival.

The microenvironment of an early-stage tumor, in which a small number of cancer cells is surrounded by a normal counterpart milieu, plays a crucial role in determining the fate of initiated cells. Here, we examined cell competition between anaplastic thyroid cancer cells and normal thyroid follicular cells using co-culture method. Cancer cells were grown until they formed small clusters, to which normal cells were added to create high-density co-culture condition. We found that co-culture with normal cells significantly suppressed the growth of cancer cell clusters through the activation of Akt-Skp2 pathway. In turn, cancer cells triggered apoptosis in the neighboring normal cells through local activation of ERK1/2. A bi-directional cell competition provides a suppressive mechanism of anaplastic thyroid cancer progression. Since the competitive effect was negated by terminal growth arrest caused by radiation exposure to normal cells, modulation of reciprocal stress response in vivo could be an intrinsic mechanism associated with tumor initiation, propagation, and metastasis., Competing Interests: The authors have declared that no competing inetrests exist.

Amiodarone (AMD) is a class III antiarrhythmic drug whose chronic or high dosage administration alters the tests of thyroid function. AMD is also associated with hypothyroidism or thyrotoxicosis. Total thyroidectomy is an efficient treatment of AMD-induced thyrotoxicosis in cases resistant to medical therapy, worsening of cardiac function and/or severe thyrotoxicosis. Although AMD is a widely used drug, its pathological consequences are not well known. We describe the pathological findings in the thyroid gland of a patient who underwent total thyroidectomy due to AMD-induced thyrotoxicosis. The surgical specimen was macroscopically normal, but histologically showed multiple follicles totally or partially invaded by clear vacuolated (foamy) histiocytes, sometimes multinucleated. Loss of thyrocytes, breaks in the follicular basal membrane and stromal fibrosis could also be appreciated but no lymphocytic infiltrates were found. An awareness of these histopathological features is particularly important for surgical pathologists, especially as there are very few published reports describing these alterations., (Copyright © 2019 Sociedad Española de Anatomía Patológica. Publicado por Elsevier España, S.L.U. All rights reserved.)

Background: Atypia of undetermined significance (AUS) or follicular lesion of undetermined significance (FLUS) is the most controversial category of the Bethesda System. The present study was conducted to compare the histological findings in a series of thyroid nodules diagnosed with AUS/FLUS after single or repeat fine needle aspiration (FNA) cytology., Methods: Retrospective analysis of our institution's series of 514 patients with an initial diagnosis of AUS/FLUS between 11/2011 and 02/2020., Results: Of 4887 FNA samples, 11.8% were classified as AUS/FLUS. Of patients with an initial AUS/FLUS diagnosis, 11.5% (59/514) underwent surgery after a single FNA, 55.4% (285/514) had a repeat FNA, and 32.7% (168/514) were either observed or lost to follow-up. Surgical pathology was available in 123 cases (23.9%), and malignancy was confirmed in 32.5% (40/123) cases, with similar rates in the single 32.2% (19/59) and repeat FNA 32.8% (21/64) groups. Repeat FNA reclassified 78.9% of the AUS/FLUS cases to a different category: 57.2% were reclassified as benign, 10.5% as follicular neoplasm, and 5.6% as suspicious for malignancy or malignant. The rates of nonneoplastic benign lesions were 52.5% (31/59) and 31.2% (20/64) in the single and repeat FNA groups, respectively (P = .018). The rates of follicular adenomas were higher when repeat FNA was performed (23/64, 35.9%) compared with a single FNA (9/59; 15.2%) (P = .013)., Conclusion: In this series, a repeat FNA in cases of AUS/FLUS increased detection of follicular adenomas but not the detection of malignancy. Repeat FNA reduced the rate of benign nonneoplastic lesions by 40% in the surgical samples., (© 2020 Wiley Periodicals LLC.)

The primary cilium is well-preserved in human differentiated thyroid cancers such as papillary and follicular carcinoma. Specific thyroid cancers such as Hürthle cell carcinoma, oncocytic variant of papillary thyroid carcinoma (PTC), and PTC with Hashimoto's thyroiditis show reduced biogenesis of primary cilia; these cancers are often associated the abnormalities in mitochondrial function. Here, we examined the association between primary cilia and the mitochondria-dependent apoptosis pathway. Tg-Cre;Ift88 flox/flox mice (in which thyroid follicles lacked primary cilia) showed irregularly dilated follicles and increased apoptosis of thyrocytes. Defective ciliogenesis caused by deleting the IFT88 and KIF3A genes from thyroid cancer cell lines increased VDAC1 oligomerization following VDAC1 overexpression, thereby facilitating upregulation of mitochondria-dependent apoptosis. Furthermore, VDAC1 localized with the basal bodies of primary cilia in thyroid cancer cells. These results demonstrate that loss-of-function of primary cilia results in apoptogenic stimuli, which are responsible for mitochondrial-dependent apoptotic cell death in differentiated thyroid cancers. Therefore, regulating primary ciliogenesis might be a therapeutic approach to targeting differentiated thyroid cancers.

Introduction: The detection of rosette-like clusters (RLC) of follicular cells in thyroid carcinoma has been reported mostly in the columnar cell variant of papillary thyroid carcinoma (PTC). Despite the fact that diagnosing variants of PTC is no longer encouraged by The Bethesda System for Reporting Thyroid Cytopathology, the identification of cytomorphological features such as RLC linked with these tumours might help reduce possible misinterpretation in thyroid fine needle aspiration (FNA) cytology. We accordingly investigated the potential correlation of architectural patterns including RLC with PTC variants., Methods: We analysed 225 thyroid FNA cytology cases diagnosed as suspicious for malignancy (SFM) and positive for malignancy (M) over a 1-year time where all samples had corresponding histology. We also included 150 benign lesions from the same period. The presence of RLC vs similar appearing solid clusters, papillary structures and microfollicles were evaluated. We also performed immunocytochemistry and molecular testing for BRAFV600E., Results: We included 100 (44.4%) SFM favouring PTC and 125 (55.6%) M cases with cyto-histological correlation. On histology, all SFM and M cases showed malignancy including 140 (62.2%) classic PTC and 85 (37.8%) PTC variants. The cytomorphological patterns in all FNA samples included solid (74%), papillary (89%), microfollicular (70%), and pseudo-RLC morphology (25.7%). We identified only pseudo-RLC in 33 FNA specimens from PTC variant cases that included tall cell variant (42.4%), hobnail variant (21.2%) and miscellaneous variants (36.3%) of PTC. No definitive RLC were detected in our series. Immunocytochemistry and BRAFV600E were not specifically linked with an RLC pattern., Conclusions: These findings demonstrate that in our dataset the architectural pattern of RLC was not recognised within PTC variants. However, we did identify a pseudo-RLC pattern that was observed in association with tall cell variant and hobnail variant cases of PTC., (© 2020 John Wiley & Sons Ltd.)

Background: This study compares survival in patients with the rare subtypes of follicular (FTmC) and Hurthle cell (HCmC) microcarcinoma compared to that of papillary thyroid (PTmC) microcarcinoma., Methods: Patients with FTmC and HCmC were selected from the National Cancer Database 2004-2015 and compared with PTmC. Patient clinicopathological characteristics and overall survival (OS) were analyzed. Multivariable logistic and Cox regression analysis evaluated binary outcomes and predictors of survival. A propensity score matched analysis using age, gender, race, extrathyroidal extension (ETE), nodal status, distant metastasis, radiation, and operation was performed to evaluate the difference in OS with FTmC, HCmC, and PTmC., Results: We identified 858 FTmC, 476 HCmC, and 82,056 PTmC. FTmC was less likely to have macroscopic ETE compared to PTmC (2.6% vs. 3.1% p = 0.03), but more likely to have distant metastasis (2.3% vs. 0.2%, p < 0.01). FTmC and HCmC were less likely to have nodal metastasis (2.7%, 2.5% vs. 10.9%, p < 0.01). Ten-year OS was decreased in patients with FTmC (91.4%, p = 0.04) and HCmC (89.8%, p < 0.01) compared to PTmC (93.5%). On multivariable analysis, histology was not associated with OS. With HCmC, older age (OR 1.13, p < 0.01) and male gender (OR 2.72, p = 0.03) were associated with decreased OS. In propensity matched analysis, there was no difference in 10-year OS with FTmC and PTmC (91.4% vs. 93.7%, p = 0.54), but HCmC had decreased OS compared to PTmC (89.8% vs. 94.3%, p = 0.04)., Conclusions: Patients with FTmC have comparable OS to those with PTmC, but HCmC has decreased OS especially in older and male patients.

Background: Loss of the expression of thyroid differentiation markers such as sodium iodide symporter (NIS) and, consequently, radioiodine refractoriness is observed in aggressive papillary thyroid cancer and anaplastic thyroid cancer (ATC) that may harbor the BRAF V600E mutation. Activation of the BRAF V600E oncogene in thyroid follicular cells induces the expression of the miR-17-92 cluster that comprises seven mature microRNAs (miRNAs). miRNAs are a class of endogenous small RNAs (∼22 nt) that regulate gene expression post-transcriptionally. Indeed, miR-17-92 is overexpressed in ATC, and in silico prediction shows the potential targeting of thyroid transcription factors and tumor suppressor pathways. In this study, we aimed to investigate the role of the miR-17-92 cluster in thyroid cell differentiation and function. Methods: miR-17-92 silencing was performed using CRISPR/Cas9n-guided genomic editing of the miR-17-92 gene in the KTC2 ATC cell line, and miR-17-92 cluster or individual miRNAs were overexpressed in PCCl3 thyroid cells to evaluate the influence in thyroid cell differentiation and cell function. Results: In this study, we demonstrate that CRISPR/Cas9n gene editing of the miR-17-92 cluster results in promotion of thyroid follicular cell differentiation (NIS, thyroperoxidase, thyroglobulin, PAX8, and NKX2-1 expression) in the KTC2 ATC cell line and inhibits cell migration and proliferation by restoring transforming growth factor beta (TGF-β) signaling pathway responsiveness. Moreover, induction of the miR-17-92 cluster in normal thyroid follicular cells strongly impairs thyroid differentiation and induces a pro-oncogenic effect by blocking TGF-β signaling and increasing cell migration. Conclusions: miR-17-92 is a potent regulator of thyroid follicular cell differentiation, and CRISPR/Cas9n-mediated editing of the miR-17-92 gene efficiently blocks miR-17-92 expression in the KTC2 ATC cell line, resulting in improvement of thyroid differentiation. Thus, targeting miR-17-92 could provide a potential molecular approach to restoring thyroid cell differentiation and NIS expression in aggressive thyroid cancer.

Background: An altered immune microenvironment may contribute to papillary thyroid cancer development, as immune infiltrates are identified postoperatively in many papillary thyroid cancer cases with or without diagnosed thyroiditis. Oxygen radicals, endogenous or inflammation-induced, can generate DNA damage, which causes mutations when repaired incorrectly. We hypothesized that infiltrating immune cells might promote aberrant DNA repair, predisposing thyrocytes to papillary thyroid cancer., Methods: Quantitative reverse-transcriptase polymerase chain reaction assays measured gene expression in fresh-frozen samples (n = 55). RNA-seq data was obtained for papillary thyroid cancer and normal thyroid samples from the Cancer Genome Atlas (n = 564), and Hashimoto's-affected and normal thyroids from the Genotype-Tissue Expression project (n = 279). Immune cell marker expression levels were compared to histological estimates and to selected DNA repair genes. Immunohistochemistry localized gene expression to specific cell types., Results: DNA polymerase theta expression by quantitative reverse-transcriptase Polymerase chain reaction was higher in papillary thyroid cancer and papillary thyroid cancer-adjacent samples than in benign normal thyroid (P < .001). Immune markers including CD4 correlated with DNA polymerase theta expression (r = 0.50) but not other DNA repair genes examined. Benign tissue with Hashimoto's exhibited increased DNA polymerase theta (P < .0001) and CD3E (P < .0001) expression. DNA polymerase theta localized to thyrocytes, not lymphocytes., Conclusion: We identified a strong correlation between immune cell infiltrate and dysregulated thyrocyte DNA repair genes, likely reflecting a pathway to papillary thyroid cancer development., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Transforming growth factor (TGF)-β1 plays a crucial role in the epithelial-to-mesenchymal transition (EMT) in many cancer types and in thyroid cancers. Epigallocatechin-3-gallate (EGCG), the most important ingredient in the green tea, has been reported to possess antioxidant and anticancer activities. However, the cellular and molecular mechanisms explaining its action have not been completely understood. In this study, we found that EGCG significantly suppresses EMT, invasion and migration in anaplastic thyroid carcinoma (ATC) 8505C cells in vitro by regulating the TGF-β/Smad signaling pathways. EGCG significantly inhibited TGF-β1-induced expression of EMT markers (E-cadherin reduction and vimentin induction) in 8505C cells in vitro . Treatment with EGCG completely blocked the phosphorylation of Smad2/3, translocation of Smad4. Taken together, these results suggest that EGCG suppresses EMT and invasion and migration by blocking TGFβ/Smad signaling pathways.

The aim of the work was to carry out an immunocytochemical (ICC) study of thyroid peroxidase (TPO) and thyroglobulin (Tg) expression in the punctates of the radioiodine (RI) refractory and RI uptake metastases of thyroid papillary carcinoma (PTC), on the basis of which it is possible to develop new methods of preoperative prediction of RI resistance and RI therapy efficiency for thyroid papillary carcinoma metastases., Materials and Methods: The ICC research was carried out on a fine needle aspiration biopsy material of 104 metastases that were found after thyroidectomy and RI therapy, i.e. in postoperative period (79 - radioiodine-refractory metastases, 25 - radioiodine-uptake metastases). The ICC analysis of TPO and Tg expression in PTC was performed with the use of monoclonal antibodies against TPO and Tg., Results: RI-refractory (RIRM) and RI-uptake metastases of PTC significantly differ by the percentage of cells expressing TPO and Tg (p < 0.05, and p < 0.05 respectively). The effectiveness of RI therapy was different for patients with different percentages of TPO-positive cells., Conclusion: A statistically significant difference was demonstrated in the expression of TPO in a punctates of RI-resistant and RI-sensitive metastatic TPC, based on which a new method for preoperative prediction of RI resistance and RI therapy efficiency was developed. It was shown that ICC determination of Tg expression in metastases is effective in preoperative monitoring of RI resistance of PTC if the part of Tg-positive cells in punctates is lower than 56%. The comprehensive study of the ICC profile of thyrocytes in RI-uptake metastases punctates allows us to develop a personified approach to prediction, monitoring and therapy of patients with PTC.

An 18-y-old female llama ( Lama glama ) in Saskatoon, Saskatchewan was examined during a routine herd check, and a mass was detected in the ventral cervical area just below the angle of the jaw. No clinical signs were evident except for the mass and chronic loss of body condition. Postmortem examination revealed bilateral enlargement of the thyroid gland with multiple cysts. Histopathology of the thyroid gland revealed follicular compact-cellular carcinoma lesions, with infiltration of neoplastic thyroid follicular cells in regional lymph nodes.

Background: Noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) is a newly described entity with an indolent clinical course as compared to the follicular variant of papillary thyroid carcinoma (FVPTC). As the cytologic features of NIFTP overlap those of the FVPTC, distinction between the two on FNA cytology poses diagnostic difficulties. This study analyzes the presence of multinucleated giant cells (MGCs)., Methods: Twenty histologically confirmed NIFTP cases and 17 FVPTC cases each with a prior FNA and performed over a 2-year period were reviewed, the cytologic diagnoses compared and the number of MGCs counted., Results: Of the 20 NIFTP cases, 55% were diagnoses as FLUS on FNA, 25% as FN/SFN and 10% as SPTC. Of the 17 FVPTC cases, 12% were diagnosed as FLUS, 53% as FN/SFN and 35% as SPTC. Fifteen (88%) FVTPC cases contained MGCs, whereas none of the 20 NIFTP cases contained MGCs., Conclusion: The presence or absence of MGCs may serve as an additional finding to differentiate NIFTP cases from FVTPC cases., (© 2019 Wiley Periodicals, Inc.)

Autophagy is a catabolic process that involves the degradation of cellular components through the lysosomal machinery, relocating nutrients from unnecessary processes to more pivotal processes required for survival. It has been reported that systemic disruption of the Atg5 or Atg7 gene, a component of autophagy, is lethal and that its tissue-specific disruption causes tissue degeneration in several organs. However, the functional significance of autophagy in the thyroid glands remains unknown. Our preliminary data imply the possible involvement of dysfunctional autophagy in radiation-induced thyroid carcinogenesis. Therefore, we evaluated the effect of Atg5 gene knockout (KO) on thyroid morphology and function. To this end, Atg5flox/flox mice were crossed with TPO-Cre mice, yielding the thyroid follicular epithelial cell (thyrocyte)‒specific ATG5-deficient mice (Atg5thyr-KO/KO). Atg5 gene KO was confirmed by a lack of ATG5 expression, and disruption of autophagy was demonstrated by a decrease in microtubule-associated protein 1 light chain 3-II puncta and an increase in p62. Atg5thyr-KO/KO mice were born normally, and thyroid morphology, thyroid weights, and serum T4 and TSH levels were almost normal at 4 months. However, at 8 and 12 months, a decrease in the number of thyrocytes and an increase in TUNEL+-thyrocytes were observed in Atg5thyr-KO/KO mice even though thyroid function was still normal. The number of irregularly shaped (gourd-shaped) follicles was also increased. Excess oxidative stress was indicated by increased 8-hydroxy-2'-deoxyguanosine and 53BP1 foci in Atg5thyr-KO/KO mice. These data demonstrate that thyrocytes gradually undergo degradation/cell death in the absence of basal levels of autophagy, indicating that autophagy is critical for the quality control of thyrocytes., (Copyright © 2019 Endocrine Society.)

The biologic and clinical significance of reactive C cell hyperplasia (CCH), adjacent to differentiated thyroid cancers, remains unknown. Our aim was to investigate the presence of CCH in thyroidectomy specimens with papillary thyroid carcinomas (PTC) and discuss its epidemiology and histology. In total, 413 patients were prospectively included in the study (189 benign goiters, 224 PTC). Reactive CCH was observed in 9.8% of PTC cases (32% males, 68% females, mean age 48.3 ± 16.4 years) and usually ipsilateral to the primary tumor (91%). Histologically, CCH was either focal (91%) or diffuse (9%) and almost always (92%) found in the middle or upper thirds of the thyroid lobes. Patients with PTC/CCH were generally younger than patients with benign goiters (0.027). On the other hand, patients with PTC and with PTC/CCH did not differ in terms of age, gender, basal calcitonin levels, primary tumor size, multifocality, extrathyroidal invasion, or lymph node metastasis. Thyroiditis, however, was more frequent in cases with PTC/CCH compared to PTC alone. Reactive CCH is considered a physiological response of the C cells to various stimuli, differentiated thyroid cancer among others. It bears no malignant potential and requires no additional treatment, following thyroidectomy.

Thyroid cancer incidence is increased in volcanic areas where environment pollution biocontaminates residents. Tungsten (W) is the most increased heavy metal in drinking water of Mount Etna volcanic area where it exceeds the normal range in the urine of 27% inhabitants. The possible connection between increased tungsten and thyroid cancer has never been studied. We investigated in vitro the effect tungsten on both human thyrocytes in primary culture, thyrospheres (aggregates of stem/precursor thyroid cells) and thyrocytes differentiated from tungsten-exposed thyrospheres. Chronic exposure to low-dose (nanomolar range, as in the urines of volcanic area residents) soluble tungsten had major biological effects on thyroid stem/precursor cells, promoting growth with a biphasic (hormetic) dose-response and reducing apoptosis. No such effects were observed in mature thyrocytes. In addition, tungsten-exposed thyrospheres had abnormal expression of genes commonly altered also in thyroid cancer and increased activation of the DNA-repair proteins H2AX and 53BP1. Moreover, exposure to tungsten decreased thyrosphere differentiation, as indicated by the reduced expression of thyroid-specific genes in derived thyrocytes that also showed preneoplastic changes such as increased anchorage-independent growth, clonogenic growth and migration capacity. The mechanism of action of tungsten on thyroid stem/precursor cells is unclear but involves membrane G-proteins and activation of the ERK signaling pathway. These data indicate that chronic exposure to slightly increased tungsten, harmless for mature thyrocytes, importantly affects the biology of stem/precursor thyroid cells and of their progeny, inducing characteristics of preneoplastic transformation.

Microgravity in space or simulated by special ground-based devices provides an unusual but unique environment to study and influence tumour cell processes. By investigating thyroid cancer cells in microgravity for nearly 20 years, researchers got insights into tumour biology that had not been possible under normal laboratory conditions: adherently growing cancer cells detach from their surface and form three-dimensional structures. The cells included in these multicellular spheroids (MCS) were not only altered but behave also differently to those grown in flat sheets in normal gravity, more closely mimicking the conditions in the human body. Therefore, MCS became an invaluable model for studying metastasis and developing new cancer treatment strategies via drug targeting. Microgravity intervenes deeply in processes such as apoptosis and in structural changes involving the cytoskeleton and the extracellular matrix, which influence cell growth. Most interestingly, follicular thyroid cancer cells grown under microgravity conditions were shifted towards a less-malignant phenotype. Results from microgravity research can be used to rethink conventional cancer research and may help to pinpoint the cellular changes that cause cancer. This in turn could lead to novel therapies that will enhance the quality of life for patients or potentially develop new preventive countermeasures.

Background: Thyroid carcinoma includes several variants characterized by different biological and clinical features: from indolent microcarcinoma to undifferentiated and aggressive anaplastic carcinoma. Inflammation plays a critical role in thyroid tumors. Conditions predisposing to cancer, as well as oncogene activity, contribute to the construction of an inflammatory microenvironment that facilitates thyroid tumor progression. Moreover, oncogene-induced senescence, a mechanism tightly connected with inflammation, and able to restrain or promote cancer progression, is involved in thyroid cancer. The interactions between thyroid tumor cells and the microenvironment are not completely clarified., Methods: We characterize in vitro the interplay between macrophages and senescent thyrocytes and tumor-derived cell lines, modeling early and late thyroid tumor stages, respectively. Purified peripheral blood-derived human monocytes were exposed to thyroid cell-derived conditioned medium (CM) and assessed for phenotype by flow cytometry. The factors secreted by thyroid cells and macrophages were identified by gene expression analysis and ELISA. The protumoral effect of macrophages was assessed by wound healing assay on K1 thyroid tumor cells. The expression of PTGS2 and M2 markers in thyroid tumors was investigated in publicly available datasets., Results: Human monocytes exposed to CM from senescent thyrocytes and thyroid tumor cell lines undergo M2-like polarization, showing high CD206 and low MHC II markers, and upregulation of CCL17 secretion. The obtained M2-like macrophages displayed tumor-promoting activity. Among genes overexpressed in polarizing cells, we identified the prostaglandin-endoperoxide synthase enzyme (PTGS2/COX-2), which is involved in the production of prostaglandin E2 (PGE2). By using COX-2 inhibitors we demonstrated that the M2-like polarization ability of thyroid cells is related to the production of PGE2. Co-expression of PTGS2 and M2 markers is observed a significant fraction of human thyroid tumors., Conclusions: Our results demonstrate that both senescent thyrocytes and thyroid tumor cell lines trigger M2-like macrophage polarization that is related to PGE2 secretion. This suggests that the interaction with the microenvironment occurs at both early and late thyroid tumor stages, and favors tumor progression. The co-expression of PTGS2 gene and M2 markers in human thyroid carcinoma highlights the possibility to counteract tumor growth through COX-2 inhibition.

Background: Primary cilia (PC) are conserved structures in the adult thyroid gland of different mammals. It was recently described that in humans, PC are usually present as a single copy per follicular cell emerging from the follicular cell apex into the follicular lumen., Methods: To understand the role developed by PC in thyroid hormonogenesis better, their changes in different human functional thyroid diseases (diffuse toxic hyperplasia/Graves' disease [GD] and nodular hyperplasia [NH]/nodular goiter), in comparison to normal thyroid tissue, were investigated using immunofluorescence, morphometry, and electron microscopy analyses., Results: Significantly decreased ciliary frequencies were found in both NH (51.16 ± 11.69%) and GD (44.43 ± 23.70%) compared to normal thyroid tissue (76.09 ± 7.31%). Similarly, PC lengths were also significantly decreased in both NH (2.02 ± 0.35 μm) and GD (2.4 ± 0.48 μm) compared to normal glands (3.93 ± 0.90 μm). Moreover, in GD patients, hyperactive-follicle foci always showed diminished ciliary frequency and length compared to any other thyroid follicle pattern, independent of their thyroid status. Finally, in GD, the percentage of thyrocytes exhibiting PC in the "normal-appearance areas" was significantly lower in correspondence with the subsistence of signs of thyroid biosynthetic hyperactivity after long-term antithyroid drug treatment., Conclusions: The results suggest a direct relationship between ciliogenesis and both follicle activity and tissue heterogeneity in the functional pathology of the thyroid gland.

Communications at the interface between the apical membrane of follicular cells and the follicular lumen are critical for the homeostasis of thyroid gland. Primary cilia at the apical membrane of thyroid follicular cells may sense follicular luminal environment and regulate follicular homeostasis, although their role in vivo remains to be determined. Here, mice devoid of primary cilia were generated by thyroid follicular epithelial cell-specific deletion of the gene encoding intraflagellar transport protein 88 ( Ift88 ). Thyroid follicular cell-specific Ift88 -deficient mice showed normal folliculogenesis and hormonogenesis; however, those older than 7 weeks showed irregularly dilated and destroyed follicles in the thyroid gland. With increasing age, follicular cells with malignant properties showing the characteristic nuclear features of human thyroid carcinomas formed papillary and solid proliferative nodules from degenerated thyroid follicles. Furthermore, malignant tumor cells manifested as tumor emboli in thyroid vessels. These findings suggest that loss-of-function of Ift88 /primary cilia results in malignant transformation from degenerated thyroid follicles.

Lithium (Li) is used for the treatment and prophylaxis of mental disorders, associated with many serious hazards. Resveratrol (RSV) has various beneficial therapeutic effects. Platelet-rich plasma (PRP) is a new promising curative tool. This study aimed to assess the impacts of RSV versus PRP on lithium-induced thyroid follicular cell toxicity in adult male rats. Forty-nine adult male rats were divided into four groups: group I: control rats; group II: lithium-treated rats; group III: lithium- and resveratrol-treated rats; group IV: lithium and PRP-treated rats. Thyroid specimens were taken and processed for histological and immunohistochemical methods. Morphometrical studies and statistical analysis were done. Group II showed distorted thyroid follicles, significantly increased collagen fibers, and highly positive P53 immunostaining (P < 0.01). Ultrastructural examination showed dilated rough endoplasmic reticulum and damaged mitochondria. Groups III and IV exhibited significant amelioration of the histological and electron microscopic changes mentioned previously. PRP remedy was more effective than RSV for treatment of Li-induced thyroid follicular cell toxicity.

Insulin and insulin-like growth factor (IGF)-1 signaling in the thyroid are thought to be permissive for the coordinated regulation by thyroid-stimulating hormone (TSH) of thyrocyte proliferation and hormone production. However, the integrated role of insulin receptor (IR) and IGF-1 receptor (IGF-1R) in thyroid development and function has not been explored. Here, we generated thyrocyte-specific IR and IGF-1R double knockout (DTIRKO) mice to precisely evaluate the coordinated functions of these receptors in the thyroid of neonates and adults. Neonatal DTIRKO mice displayed smaller thyroids, paralleling defective folliculogenesis associated with repression of the thyroid-specific transcription factor Foxe1. By contrast, at postnatal day 14, absence of IR and IGF-1R paradoxically induced thyrocyte proliferation, which was mediated by mTOR-dependent signaling pathways. Furthermore, we found elevated production of TSH during the development of follicular hyperplasia at 8 weeks of age. By 50 weeks, all DTIRKO mice developed papillary thyroid carcinoma (PTC)-like lesions that correlated with induction of the ErbB pathway. Taken together, these data define a critical role for IR and IGF-1R in neonatal thyroid folliculogenesis. They also reveal an important reciprocal relationship between IR/IGF-1R and TSH/ErbB signaling in the pathogenesis of thyroid follicular hyperplasia and, possibly, of papillary carcinoma., (© 2018 UICC.)

Here we describe a conditional doxycycline-dependent mouse model of RET/PTC3 (NCOA4-RET) oncogene-induced thyroid tumorigenesis. In these mice, after 10 days of doxycycline (dox) administration, RET/PTC3 expression induced mitogen activated protein kinase (MAPK) stimulation and a proliferative response which resulted in the formation of hyperplastic thyroid lesions. This was followed, after 2 months, by growth arrest accompanied by typical features of oncogene-induced senescence (OIS), including upregulation of p16INK4A and p21CIP, positivity at the Sudan black B, activation of the DNA damage response (DDR) markers γH2AX and pChk2 T68, and induction of p53 and p19ARF. After 5 months, about half of thyroid lesions escaped OIS and formed tumors that remained dependent on RET/PTC3 expression. This progression was accompanied by activation of AKT-FOXO1/3a pathway and increased serum TSH levels., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Thyroid cancer is the most common endocrine malignancy. Knowledge of the molecular pathology of thyroid tumours originating from follicular cells has greatly advanced in the past several years. Common molecular alterations, such as BRAF p.V600E, RAS point mutations, and fusion oncogenes (RET-PTC being the prototypical example), have been, respectively, associated with conventional papillary carcinoma, follicular-patterned tumours (follicular adenoma, follicular carcinoma, and the follicular variant of papillary carcinoma/non-invasive follicular thyroid neoplasm with papillary-like nuclear features), and with papillary carcinomas from young patients and arising after exposure to ionising radiation, respectively. The remarkable correlation between genotype and phenotype shows how specific, mutually exclusive molecular changes can promote tumour development and initiate a multistep tumorigenic process that is characterised by aberrant activation of mitogen-activated protein kinase and phosphoinositide 3-kinase-PTEN-AKT signalling. Molecular alterations are becoming useful biomarkers for diagnosis and risk stratification, and as potential treatment targets for aggressive forms of thyroid carcinoma. What follows is a review of the principal genetic alterations of thyroid tumours originating from follicular cells and of their clinicopathological relevance., (© 2017 John Wiley & Sons Ltd.)

Objective: to investigate morphological changes in the primary thyroid cell culture of rat infants whose parents were prenatally exposed by radioisotope iodine 131., Materials and Methods: obtaining and culturing of thyroid tissue primary cell cultures of newborn rats, cytological (receipt and analysis of cell cultures agents for optical microscopy), biophysical (flow cytometry), statistics., Results: It was shown that cells in thyroid primary culture of offspring rats prenatally exposed by radioisotopes of iodine 131 signs of destructive degenerative changes were observed mostly when animals of both sexes were irra diated. Increased number of two and three nuclear cells and induction of ring like cells is an evidence of signifi cant genotoxic violation and points to the genome instability in offspring of animals exposed by radioisotope iodine 131., Conclusions: Analysis and quantitative morphological parameters of cells in thyroid primary culture of newborn rats whose parents were exposed prenatally by radioisotopes of iodine 131 showed that upon exposure to radiation thy roid undergoes destructive changes at the cellular level and, even in the second generation of offspring, leads to disruption of its functions., (O. A. Boiko, H. Yo. Lavrenchuk, A. I. Lypska, V. V. Talko, V. S. Asmolkov.)

MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression that modulate the vast majority of cellular processes. During development, the correct timing and expression of miRNAs in the tissue differentiation is essential for organogenesis and functionality. In thyroid gland, DICER and miRNAs are necessary for accurately establishing thyroid follicles and hormone synthesis. Moreover, DICER1 mutations and miRNA deregulation observed in human goiter influence thyroid tumorigenesis. The thyroid malignant transformation by MAPK oncogenes is accompanied by global miRNA changes, with a marked reduction of "tumor-suppressor" miRNAs and activation of oncogenic miRNAs. Loss of thyroid cell differentiation/function, and consequently iodine trapping impairment, is an important clinical characteristic of radioiodine-refractory thyroid cancer. However, few studies have addressed the direct role of miRNAs in thyroid gland physiology. Here, we focus on what we have learned in the thyroid follicular cell differentiation and function as revealed by cell and animal models and miRNA modulation in thyroid tumorigenesis., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Objective: To determine the relationship between papillary thyroid carcinoma and environmental exposure to bisphenol A (BPA) or 17-β estrogen (E 2 ) by assessing the effects of these compounds on estrogen receptor expression and AKT/mTOR signaling., Methods: The effects of low levels of BPA (1mM-10nM) and 17β-estradiol (E2, 0.1mM-1nM) on ER expression and cellular proliferation were determined in human thyroid papillary cancer BHP10-3 cells. Protein and mRNA levels of estrogen nuclear receptors (ERα/ERβ) and membrane receptors (GPR30) were determined by immunofluorescence assay, Western blotting, and RT-PCR, respectively, and proliferation was assessed by CCK-8 assay., Results: The proliferative effects of BPA and E 2 were both concentration- and time-dependent. Expression of ERα/ERβ and GPR30 were enhanced by BPA and E 2. BPA and E 2 could quickly phosphorylate AKT/mTOR. Moreover, ICI suppressed ERα expression and activated GPR30 as did G-1. G-15 reversed the effects of E 2 on GPR30 and AKT/mTOR, but did not alter the effect of BPA., Conclusions: BPA influences thyroid cancer proliferation by regulating expression of ERs and GPR30, a mechanism that differs from E 2 . In addition, ICI and G-15 may have the potential to be used as anti-thyroid cancer agents., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Iodine is a key ingredient in the synthesis of thyroid hormones and also a major factor in the regulation of thyroid function. A local reduction of iodine content in follicular lumen leads to overexpression of local thyroid-stimulating hormone receptor (TSHr), which in turn excessively stimulates the regional thyroid tissue, and result in the formation of nodular goiter. In this study, we investigated the relationship between iodine content and sodium iodide symporter (NIS) expression by using the clinical specimens from patients with nodular goiter and explored the pathogenesis triggered by iodine deficiency in nodular goiter. In total, 28 patients were clinically histopathologically confirmed to have nodular goiter and the corresponding adjacent normal thyroid specimens were harvested simultaneously. Western blot and immunohistochemistry were performed to assay NIS expression and localization in thyrocytes of both nodular goiter and adjacent normal thyroid tissues. NIS expression mediated by iodine in follicular lumen was confirmed by follicular model in vitro. Meanwhile, radioscan with iodine-131were conducted on both nodular goiter and adjacent normal thyroid. Our data showed that NIS expression in nodular goiter was significantly higher than that in adjacent normal tissues, which was associated with low iodine in the follicular lumen. Abnormal localization of NIS and lower amount of radioactive iodine-131 were also found in nodular goiter. Our data implied that low iodine in the follicular lumen caused by cytoplasm mis-localization of NIS may induce nodular goiter.

The thyroid gland secretes primarily tetraiodothyronine (T 4 ), and some triiodothyronine (T 3 ). Under normal physiological circumstances, only one-fifth of circulating T 3 is directly released by the thyroid, but in states of hyperactivation of thyroid-stimulating hormone receptors (TSHRs), patients develop a syndrome of relative T 3 toxicosis. Thyroidal T 4 production results from iodination of thyroglobulin (TG) at residues Tyr 5 and Tyr 130 , whereas thyroidal T 3 production may originate in several different ways. In this study, the data demonstrate that within the carboxyl-terminal portion of mouse TG, T 3 is formed de novo independently of deiodination from T 4 We found that upon iodination in vitro , de novo T 3 formation in TG was decreased in mice lacking TSHRs. Conversely, de novo T 3 that can be formed upon iodination of TG secreted from PCCL3 (rat thyrocyte) cells was augmented from cells previously exposed to increased TSH, a TSHR agonist, a cAMP analog, or a TSHR-stimulating antibody. We present data suggesting that TSH-stimulated TG phosphorylation contributes to enhanced de novo T 3 formation. These effects were reversed within a few days after removal of the hyperstimulating conditions. Indeed, direct exposure of PCCL3 cells to human serum from two patients with Graves' disease, but not control sera, led to secretion of TG with an increased intrinsic ability to form T 3 upon in vitro iodination. Furthermore, TG secreted from human thyrocyte cultures hyperstimulated with TSH also showed an increased intrinsic ability to form T 3 Our data support the hypothesis that TG processing in the secretory pathway of TSHR-hyperstimulated thyrocytes alters the structure of the iodination substrate in a way that enhances de novo T 3 formation, contributing to the relative T 3 toxicosis of Graves' disease.

Determination of the specific type of thyroid cancer is crucial for the prognosis and selection of treatment of this malignancy. However, in some cases appropriate classification is not possible based on histopathological features only, and it might be supported by molecular biomarkers. Here we aimed to characterize molecular profiles of different thyroid malignancies using mass spectrometry imaging (MSI) which enables the direct annotation of molecular features with morphological pictures of an analyzed tissue. Fifteen formalin-fixed paraffin-embedded tissue specimens corresponding to five major types of thyroid cancer were analyzed by MALDI-MSI after in-situ trypsin digestion, and the possibility of classification based on the results of unsupervised segmentation of MALDI images was tested. Novel method of semi-supervised detection of the cancer region of interest (ROI) was implemented. We found strong separation of medullary cancer from malignancies derived from thyroid epithelium, and separation of anaplastic cancer from differentiated cancers. Reliable classification of medullary and anaplastic cancers using an approach based on automated detection of cancer ROI was validated with independent samples. Moreover, extraction of spectra from tumor areas allowed the detection of molecular components that differentiated follicular cancer and two variants of papillary cancer (classical and follicular). We concluded that MALDI-MSI approach is a promising strategy in the search for biomarkers supporting classification of thyroid malignant tumors. This article is part of a Special Issue entitled: MALDI Imaging, edited by Dr. Corinna Henkel and Prof. Peter Hoffmann., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Overexpression of tumor-associated RAC1b has been recently highlighted as one of the most promising targets for therapeutic intervention in colon, breast, lung and pancreatic cancer. RAC1b is a hyperactive variant of the small GTPase RAC1 and has been recently shown to be overexpressed in a subset of papillary thyroid carcinomas associated with unfavorable outcome. Using the K1 PTC derived cell line as an in vitro model, we observed that both RAC1 and RAC1b were able to induce a significant increase on NF-kB and cyclin D1 reporter activity. A clear p65 nuclear localization was found in cells transfected with RAC1b-WT, confirming NF-kB canonical pathway activation. Consistently, we observed a RAC1b-mediated decrease in IκBα (NF-kB inhibitor) protein levels. Moreover, we show that RAC1b overexpression stimulates G1/S progression and protects thyroid cells against induced apoptosis, the latter through a process involving the NF-kB pathway. Present data support previous findings suggesting an important role for RAC1b in the development of follicular cell-derived thyroid malignancies and point out NF-kB activation as one of the molecular mechanisms associated with the pro-tumorigenic advantage of RAC1b overexpression in thyroid carcinomas.

The detection of thyroid nodules, consisting of different diseases, represents a common finding in population. Their evaluation and diagnosis are mostly achieved with fine-needle aspiration cytology (FNAC). Even though the majority of thyroid nodules are correctly diagnosed, a total of 25% to 30% of them are classified "indeterminate" comprising lesions with varying risk of malignancy and different types of management. Although the number of thyroid FNACs, including small lesions, is increasing due to the reliance upon sonographic and cytologic interpretations, there are issues concerning cytomorphologic interpretation and interobserver reproducibility. Different classification systems have tried to better define the criteria for inclusion in specific categories and to therefore reduce the rate of indeterminate diagnoses such as atypia of undetermined significance, follicular neoplasms, and suspicious for malignancy. However, the support of ancillary techniques (eg, immunocytochemistry and molecular analysis) are reshaping morphologic diagnoses made on materials obtained from FNAC.

The Epidermal Growth Factor Receoptor (EGFR) family member human epidermal growth factor receptor 2 (HER2) is overexpressed in many human epithelial malignancies, representing a molecular target for specific anti-neoplastic drugs. Few data are available on HER2 status in differentiated thyroid cancer (DTC). The present study was aimed to investigate HER2 status in sporadic cancers of follicular cell origin to better clarify the role of this receptor in the stratification of thyroid cancer. By immunohistochemistry and fluorescence in-situ hybridization, HER2 expression was investigated in formalin-fixed paraffin-embedded surgical specimens from 90 DTC patients, 45 follicular (FTC) and 45 papillary (PTC) histotypes. No HER2 immunostaining was recorded in background thyroid tissue. By contrast, overall HER2 overexpression was found in 20/45 (44%) FTC and 8/45 (18%) PTC, with a significant difference between the two histotypes ( p = 0.046). Five of the six patients who developed metastatic disease during a median nine-year follow-up had a HER2-positive tumor. Therefore, we suggest that HER2 expression may represent an additional aid to identify a subset of patients who are characterized by a worse prognosis and are potentially eligible for targeted therapy., Competing Interests: There is no potential conflict of interest, and the authors have nothing to disclose. This work was not supported by any grant.

Signal transducer and activator of transcription 6 (STAT6) is involved in epithelial cell growth. However, little is known regarding the STAT6 phosphorylation status in Graves' disease (GD) and its role in thyroid epithelial cells (TECs). In this study, we found that STAT6 phosphorylation (p-STAT6) was significantly increased in TECs from both GD patients and experimental autoimmune Graves' disease mice and that STAT6 deficiency ameliorated GD symptoms. Autocrine IL-4 signalling in TECs activated the phosphorylation of STAT6 via IL-4 R engagement, and the downstream targets of STAT6 were Bcl-xL and cyclin D1. Thus, the IL-4-STAT6-Bcl-xL/cyclin D1 pathway is crucial for TEC hyperplasia, which aggravates GD. More importantly, in vitro and in vivo experiments demonstrated that STAT6 phosphorylation inhibited by AS1517499 decreased TEC hyperplasia, thereby reducing serum T3 and T4 and ameliorating GD. Thus, our study reveals that in addition to the traditional pathogenesis of GD, in which autoantibody TRAb stimulates thyroid-stimulating hormone receptors and consequently produces T3, T4, TRAb could also trigger TECs producing IL-4, and IL-4 then acts in an autocrine manner to activate p-STAT6 signalling and stimulate unrestricted cell growth, thus aggravating GD. These findings suggest that STAT6 inhibitors could be potent therapeutics for treating GD.

Objectives: To investigate whether repeat ultrasound-guided fine-needle aspiration (US-FNA) in initial atypia of undetermined significance or follicular lesion of undetermined significance (AUS/FLUS) results could be performed 6 months after or more., Methods: A total of 221 AUS/FLUS ≥10 mm with any follow-up were grouped according to the first follow-up interval at less than 6 months (group 1, n = 87) and 6 months or more (group 2, n = 134). Clinical features, final assessment of ultrasound (US) or the Thyroid Imaging Reporting and Data System (TIRADS), tumour size, extrathyroidal extension and lymph node metastasis in malignancies were compared., Results: Thirty-four (15.4 %) were malignant. Age, gender, size, final assessment, TIRADS and malignancy rate were not significantly different between the two groups (p = 0.660, 0.691, 0.502, 0.237, 0.819 and 0.420). Tumour size, extrathyroidal extension and lymph node metastasis were not significantly different between the two malignancy groups (p = 0.770, 0.611 and 0.068). Two of 10 nodules with increased size were malignancies found at 7.1 and 25.0 months. None of 33 nodules (14.9 %) with decreased size at a median 10 months were malignant., Conclusions: Repeat US-FNA performed on nodules ≥10 mm at 6 months or more after initial AUS/FLUS results can reduce unnecessary repeat US-FNAs without progression of malignancy., Key Points: • Follow-up intervals of AUS/FLUS did not affect the malignancy rate • Tumour stage was not different according to the follow-up intervals • None of the nodules with decreased size were malignant • Repeat US-FNA can be performed at ≥6 months after initial AUS/FLUS.

Fine needle aspiration cytology (FNAC) plays an essential role in the evaluation of thyroid nodules especially for the category of follicular neoplasms (FN) representing 25 % of all thyroid cases including different neoplastic entities. Hence, one of the most promising areas is the application of molecular tests to FNAC. Among them, microRNAs (miRNA),identified as negative (post-transcriptional) gene expression regulators involved in tumor development, are likely to discriminate among FNs. Limited data explored the use of miRNAs on FNAC as well as their role in the malignant risk stratification. We aimed to define whether liquid-based cytology (LBC) is a valid method for miRNA evaluation. From June 2014 to March 2015, we enrolled 27FNs with histological follow-up. In the same reference period, 13 benign nodules (BN) and 20 positive for malignancy (PM) were selected as controls. Histologically, FNs resulted in 14 malignancies (3 papillary thyroid carcinoma-PTC and 11 follicular variant of PTC-FVPC) and 13 follicular adenomas (FA). The 20 PMs included two FVPC, 16 PTC and two medullary thyroid carcinoma (MTC). Five miRNAs (10b, 92a, 221/222 cluster, and 375) were studied on LBC and quantified by real-time PCR. Only miR-375 was over-expressed in the FNs diagnosed as carcinomas and in the PMs. A cut-off of 12 miR-375/U6 relative ratio recognized all BNs and 95 % PMs. Specifically, in each category, FVPCs and PTCs did not show any difference while MTCs had the highest value. miR-375 shows 97.1 % sensitivity, 100 % specificity, 96.3 % negative predictive value (NPV), 100 % positive predictive value (PPV), and 98.3 % diagnostic accuracy. LBC is suitable for miRNAs evaluation. miR-375 resulted over-expressed in all malignant FNs and 95 % PMs. It may represent a valid aid in ruling out BNs and supporting PTCs and/or FVPCs.

Primary cilia are found in the apical membrane of thyrocytes, where they may play a role in the maintenance of follicular homeostasis. In this study, we examined the distribution of primary cilia in the human thyroid cancer to address the involvement of abnormal ciliogenesis in different thyroid cancers. We examined 92 human thyroid tissues, including nodular hyperplasia, Hashimoto's thyroiditis, follicular tumor, Hürthle cell tumor, and papillary carcinoma to observe the distribution of primary cilia. The distribution and length of primary cilia facing the follicular lumen were uniform across variable-sized follicles in the normal thyroid gland. However, most Hürthle cells found in benign and malignant thyroid diseases were devoid of primary cilia. Conventional variant of papillary carcinoma (PTC) displayed longer primary cilia than those of healthy tissue, whereas both the frequency and length of primary cilia were decreased in oncocytic variant of PTC. In addition, ciliogenesis was markedly defective in primary Hürthle cell tumors, including Hürthle cell adenomas and carcinomas, which showed higher level of autophagosome biogenesis. Remarkably, inhibition of autophagosome formation by Atg5 silencing or treatment with pharmacological inhibitors of autophagosome formation restored ciliogenesis in the Hürthle cell carcinoma cell line XTC.UC1 which exhibits a high basal autophagic flux. Moreover, the inhibition of autophagy promoted the accumulation of two factors critical for ciliogenesis, IFT88 and ARL13B. These results suggest that abnormal ciliogenesis, a common feature of Hürthle cells in diseased thyroid glands, is associated with increased basal autophagy.

Dedifferentiation of follicular cells is a central event in resistance to radioactive iodine and patient mortality in papillary thyroid carcinoma (PTC). We reveal that platelet derived growth factor receptor alpha (PDGFRα) specifically drives dedifferentiation in PTC by disrupting the transcriptional activity of thyroid transcription factor-1 (TTF1). PDGFRα activation dephosphorylates TTF1 consequently shifting the localization of this transcription factor from the nucleus to the cytoplasm. TTF1 is required for follicular cell development and disrupting its function abrogates thyroglobulin production and sodium iodide transport. PDGFRα also promotes a more invasive and migratory cell phenotype with a dramatic increase in xenograft tumor formation. In patient tumors we confirm that nuclear TTF1 expression is inversely proportional to PDGFRα levels. Patients exhibiting PDGFRα at time of diagnosis are three times more likely to exhibit nodal metastases and are 18 times more likely to recur within 5years than those patients lacking PDGFRα expression. Moreover, high levels of PDGFRα and low levels of nuclear TTF1 predict resistance to radioactive iodine therapy. We demonstrate in SCID xenografts that focused PDGFRα blockade restores iodide transport and decreases tumor burden by >50%. Focused PDGFRα inhibitors, combined with radioactive iodine, represent an additional avenue for treating patients with aggressive variants of PTC., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Three paratracheal lymph nodes of a 20-year-old patient were submitted for examination, of which one showed numerous thyrocytes with large void nuclei and was suspected of being metastatic papillary thyroid carcinoma. The simultaneously resected thyroid gland, which was subsequently submitted showed findings consistent with Hashimoto's autoimmune thyroiditis (AIT). In the context of the resected goiter tissue, the suspected lymph node metastasis was identified as a hyperplastic ectopic (so-called parasitic) goiter nodule with thyrocytic changes typically seen in Hashimoto's AIT, such as oxyphilic cell alterations and a high plasma cell content. The re-examination of the suspicious lymph node revealed complete lack of a marginal sinus, thus excluding the diagnosis of a lymph node as well as the diagnosis of thyroid carcinoma metastasis.

FOXO transcription factors are key regulators of DNA damage repair, proliferation and apoptosis in thyrocytes. Thyroid malignancies show impaired FOXO function. In this study, we investigated the transcriptional regulation of FOXO isoforms in thyroid epithelial cells. mRNA expression of FOXO isoforms (FOXO1, 3 and 4) was determined in FRTL-5 cells stimulated with different growth factors and H2O2. Furthermore, the impact of PI3K/AKT signalling on FOXO transcription was investigated in PI3K p110α mutant FRTL-5 cells and regulatory dependence of FOXO transcription on FOXO was studied in FRTL-5 cells with hFOXO3 overexpression. Finally, mRNA expression levels of FOXO isoforms were determined in human epithelial thyroid tumours. Growth factor deprivation induced transcription of FOXO1, 3 and 4, whereas insulin stimulation decreased FOXO1 and FOXO4 transcription in FRTL-5 cells. Inhibition of the PI3K/AKT cascade amplified FOXO1 and FOXO4 expression. In contrast, H2O2 and TSH did not influence FOXO transcription in thyrocytes. Overexpression of PI3K p110α inhibited FOXO3 and induced FOXO4 transcription. In human thyroid tumours, FOXO1 and FOXO3 mRNA levels were significantly downregulated in papillary thyroid carcinoma when compared to normal tissues. In contrast, follicular thyroid carcinomas showed significant upregulation of FOXO4 mRNA.In this paper, we demonstrate an influence of PI3K signalling on FOXO transcription in thyrocytes. Moreover, we show that thyroid cancers exhibit alterations in FOXO transcription besides the previously reported alterations in posttranslational FOXO3 regulation. These findings may add to the concept of targeting the PI3K pathway in advanced thyroid cancers., (© Georg Thieme Verlag KG Stuttgart · New York.)