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Background: Choristoma is a well-defined benign lesion formed by histologically normal tissue in an unusual location. Diagnosis is confirmed after surgical removal of the mass. To our knowledge, to date there has been only one case of thyroid choristoma described in the literature., Patient Findings: A 70-year-old man with a history of non-Hodgkin lymphoma presented with sudden cervical enlargement. Cervical CT scan showed a 47mm hypodense nodule on the right thyroid lobe. Fine-needle aspiration revealed follicular lesion of undetermined significance. During the following weeks there was noticeable thyroid enlargement. Reassessment with thyroid ultrasound showed a 73mm nodule. The patient underwent total thyroidectomy. Histopathological examination revealed a choristoma composed of squamous epithelium lined cysts, smooth muscle, adipose tissue, connective tissue, foci of ossification and extramedullary hematopoiesis. No cytological atypia or tumoral necrosis were found. Thyroid choristomas are an exceedingly rare cause of a thyroid nodule., (© 2024. The Author(s), under exclusive licence to Hellenic Endocrine Society.)

Introduction: It is suggested to wait at least 3 months to repeat a fine needle aspiration cytology (FNAC) to avoid possible inflammatory cytological changes induced by a previous procedure. This study evaluated the influence of the interval between 2 FNACs in a cohort with a previous non-diagnostic (ND) FNAC. We analysed the occurrence of ND or atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS) results in the second FNAC, based on the intervals between procedures., Patients and Methods: Retrospective study (2017-2020) including thyroid nodules with a ND result, subjected to another FNAC. Demographic, clinical and echographic data, interval between FNACs and their results were collected. We considered the intervals: ≤/>3 months and ≤/>6 months. Second FNAC results were classified as ND, AUS/FLUS or diagnostic (including the other Bethesda categories)., Results: Included 190 nodules (190 patients - 82.1% women, mean age 60±13.7 years) with a first ND FNAC. The second FNAC results were: ND in 63 cases, AUS/FLUS in 9 and diagnostic in 118 cases. There were no statistical differences in FNAC results performed≤3 months (13 ND, 2 AUS/FLUS, 19 diagnostic) vs >3 months (50 ND, 7 AUS/FLUS, 99 diagnostic; p=0.71). Similarly, there were no statistical differences considering a longer time interval: ≤6 months (32 ND, 3 AUS/FLUS, 59 diagnostic) vs >6 months (31 ND, 6 AUS/FLUS, 59 diagnostic; p=0.61)., Conclusions: Time interval between FNACs was not relevant to the final cytological result. Early FNAC repetition did not increase the cases of ND or AUS/FLUS., (Copyright © 2024 SEEN and SED. Published by Elsevier España, S.L.U. All rights reserved.)

Introduction: The repertoire of microRNAs (miRNAs) in thyroid carcinomas starts to be elucidated. Among differentiated thyroid carcinomas (DTCs), papillary thyroid carcinoma (PTC) is the most frequent. The assessment of miRNAs expression may contribute to refine the pre-surgical diagnosis in order to obtain a personalized and more effective treatment for patients., Aims: This study aims to evaluate (1) the miRNAs in a series of DTCs, and their association with the presence of selected genetic mutations in order to improve diagnosis and predict the biologic behavior of DTC/PTC. (2) The reliability of molecular tests in Ultrasound-guided Fine Needle Aspiration Cytology (US-FNAC) for a more precise preoperative diagnosis., Material and Methods: This series includes 176 samples (98 cytology and 78 histology samples) obtained from 106 patients submitted to surgery, including 13 benign lesions (controls) and 93 DTCs (cases). The microRNA expression was assessed for miR-146b, miR-221, miR-222, and miR-15a through quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). The results were analyzed by the 2 -ΔΔCT method, using miR16 as an endogenous control. Regarding PTC diagnosis, the discriminative ability of miRNAs expression was assessed by the area under the Receiver Operating Characteristic Curve (AUC). In PTCs, the association of miRNAs expression, clinicopathological features, and genetic mutations ( BRAF , RAS, and TERTp ) was evaluated., Results/discussion: All the analyzed miRNAs presented a tendency to be overexpressed in DTCs/PTCs when compared with benign lesions, both in cytology and histology samples. In cytology, miRNAs expression levels were higher in malignant tumors than in benign tumors. In histology, the discriminative abilities regarding PTC diagnosis were as follows: miR-146b (AUC 0.94, 95% CI 0.87-1), miR-221 (AUC 0.79, 95% CI 0.68-0.9), miR-222 (AUC 0.76, 95% CI 0.63-0.89), and miR-15a (AUC 0.85, 95% CI 0.74-0.97). miR-146b showed 89% sensitivity (se) and 87% specificity (sp); miR-221 se = 68.4, sp = 90; miR-222 se = 73, sp = 70; and mi-R15a se = 72, sp = 80. MicroRNAs were associated with worst-prognosis clinicopathological characteristics in PTCs ( p < 0.05), particularly for miR-222. Our data reveal a significant association between higher expression levels of miR-146b, miR-221, and miR-222 in the presence of the BRAF mutation ( p < 0.001) and miR-146b ( p = 0.016) and miR-221 ( p = 0.010) with the RAS mutation, suggesting an interplay of these mutations with miRNAs expression. Despite this study having a relatively small sample size, overexpression of miRNAs in cytology may contribute to a more precise preoperative diagnosis. The miRNAs presented a good discriminative ability in PTC diagnosis. The association between the miRNAs expression profile and genetic alterations can be advantageous for an accurate diagnosis of DTCs/PTCs in FNAC.

Introduction: Molecular tests can contribute to improve the preoperative diagnosis of thyroid nodules. Tests available are expensive and not adapted to different populations., Aim: This study aimed to compare the cyto-histological genetic profile and to evaluate the reliability of molecular tests using ultrasound-guided fine needle aspiration cytology (US-FNAC) in accurately diagnosing differentiated thyroid carcinomas (DTCs) and predicting biologic behavior of papillary thyroid carcinomas (PTCs)., Materials and Methods: The series included 259 patients with paired cyto-histological samples totaling 518 samples. The genetic alterations were analyzed via PCR/Sanger sequencing. The association with clinicopathologic features was evaluated in PTCs., Results/discussion: From the 259 patients included, histologies were 50 (19.3%) benign controls and 209 (80.7%) DTC cases, from which 182 were PTCs; cytologies were 5.8% non-diagnostic, 18.2% benign, 39% indeterminate, and 37.1% malignant. In histology, indeterminate nodules ( n = 101) were 22.8% benign and 77.2% malignant. Mutation frequencies in cytology and histology specimens were, respectively, TERTp : 3.7% vs. 7.9%; BRAF : 19.5% vs. 25.1%; and RAS : 11% vs. 17.5%. The overall cyto-histological agreement of the genetic mutations was 94.9%, with Cohen's k = 0.67, and in indeterminate nodules agreement was 95.7%, k = 0.64. The identified mutations exhibited a discriminative ability in diagnosing DTC with a specificity of 100% for TERTp and BRAF , and of 94% for RAS , albeit with low sensitivity. TERTp and BRAF mutations were associated with aggressive clinicopathological features and tumor progression in PTCs ( p < 0.001). The obtained good cyto-histological agreement suggests that molecular analysis via US-FNAC may anticipate the genetic profile and the behavior of thyroid tumors, confirming malignancy and contributing to referring patients to surgery.

Competing Interests: The authors declare that they have no conflict of interest.

Introduction: Results in fine-needle aspiration cytology (FNAC) of thyroid nodules may be non-diagnostic (ND). In these cases, it is recommended to repeat the FNAC. The aim of our study was to evaluate the influence of demographic, clinical and ultrasound (US) characteristics in the recurrence of an ND result in thyroid nodule FNAC., Methods: A retrospective study of ND thyroid nodule FNAC was performed for the period 2017-2020. Demographic and clinical data (age, gender, cervical radiotherapy, presence of Hashimoto's thyroiditis, and TSH value) and US characteristics (nodule size, echogenicity, composition and microcalcifications) were collected at first ND FNAC., Results: Out of 230 nodules with first ND FNAC (83% women; mean age 60.2±14.1 years), 195 (84.8%) underwent a second FNAC: 121 benign, 63 non-diagnostic, 9 indeterminate and 2 malignant. Nine (3.9%) underwent surgery, only 1 of which showed malignant histology and 26 (11.3%) remained under US monitoring. Demographically, patients with second ND FNAC were older (63.4±14 vs. 59±14 years; P=0.032). Females had lower risk of second ND FNAC (OR, 0.4, 0.2-0.9; P=0.016); risk of second ND FNAC was higher in patients treated with anticoagulant/antiplatelet drugs (OR, 2.2, 1.1-4.7; P=0.03). Previous cervical radiotherapy, family history of thyroid cancer, Hashimoto's thyroiditis and TSH value did not influence the risk of second ND FNAC. On US, nodule echogenicity differed significantly between the ND and diagnostic FNAC, with greater risk of an ND result in hypoechogenic nodules. Microcalcification increased the risk of ND FNAC (OR 2.2, 1.1-4.5; P=0.03). Nodule composition and size did not significantly differ according to ND or diagnostic second FNAC., Conclusion: Male gender, advanced age, anticoagulant/antiplatelet drug therapy, hypoechogenic nodules and microcalcified nodules are likely factors for second ND FNAC. Nodules with two ND FNACs were rarely malignant, and a more conservative approach in these cases is not unsafe., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

Purpose: A clinicopathological classification has been designed to predict recurrence/progression in patients with pituitary adenomas (PAs). We aimed to study its usefulness in predicting PAs that will have a challenging disease course and may require more often complex multimodal and multiple therapeutic approaches., Methods: Retrospective analysis of 129 patients with PAs operated in our institution between 2001 and 2020 (84 non-clinically functioning PAs, 32 acromegaly, 9 Cushing's disease, 2 prolactinomas and 2 thyrotropinomas). Grading was based on invasion and proliferation: 1a (non-invasive, non-proliferative; n = 59), 1b (non-invasive, proliferative; n = 17), 2a (invasive, non-proliferative; n = 38), and 2b (invasive, proliferative; n = 15)., Results: Of the 129 patients, 68 (52.7%) were females, and the mean age at diagnosis was 53.7 ± 15.4 years. The mean follow-up duration was 93.1 ± 61.8 months. Grade 2b PAs when compared to other grades (2b-2a-1b-1a) had significantly higher rates of persistent tumor remnant within 1-year after operation (93-78-18-30%; p < 0.001), active disease at last follow-up (40-27-12-10%; p = 0.004), re-operation (27-16-0-5%; p = 0.023), irradiation (53-38-12-7%; p < 0.001), multimodal treatment (67-49-18-25%; p = 0.003), multiple treatment (33-27-6-9%; p = 0.017). Patients with grade 2b PAs also required a higher mean number of treatments (2.6-2.1-1.2-1.4; p < 0.001)., Conclusions: This clinicopathological classification appears to be a useful grading system to identify PAs that may be more refractory and more often require complex multimodal and multiple therapeutic approaches. Invasive PAs, especially grade 2b tumors, may be more likely to need complex treatment approach, including radiotherapy, and may display higher rates of active disease at last follow-up, despite receiving higher number of treatments., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Introduction According to the 2014 Endocrine Society Clinical Practice Guideline on acromegaly, the confirmation of acromegaly diagnosis is established by finding a lack of suppression of growth hormone (GH) to < 1 ug/L following documented hyperglycemia during an oral glucose tolerance test. However, in this setting, the concept of hyperglycemia has never been clearly defined. Objective This study aimed to define the hyperglycemic threshold required to induce GH suppression. Methods We retrieved the glycemia profile of 44 individuals after a standard 2-h 75g oral glucose tolerance test prescribed to assess GH suppression and performed a comprehensive analysis of two subgroups of individuals (28 reaching GH suppression and 16 in whom GH suppression was not observed). All of the data were analyzed with the program Graph Pad Prism. Differences between means were assessed by Student's unpaired t-test or Mann-Whitney U test as deemed appropriate. Fisher's exact test was used for categorical variables. Results Individuals in G1 and G2 were different only for the median basal GH and median IGF-1. No significant differences in terms of the prevalence of diabetes and prediabetes were found. The glucose peak was achieved earlier in the group that reached GH suppression. The median of the highest glucose values of both subgroups was not different. A correlation between peak and baseline glucose value was found only among those in whom GH suppression was reached. Among these, the median glucose peak (P50) was 177 mg/dl, whereas the 75 th percentile (P75) and 25 th percentile (P25) were 199 mg/dl and 120 mg/dl, respectively. Conclusion Considering that 75% of those in whom GH suppression was observed after an oral glucose overload test reached blood glucose values above 120 mg/dl, we propose to use this value as the blood glucose threshold for inducing GH suppression. In light of our results, whenever GH suppression is not observed; and the highest glycemic value is below 120 mg/dl, it might be useful to repeat the test prior to any conclusion., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2023, Bugalho et al.)

While most cases of differentiated thyroid carcinoma (DTC) are associated with a good prognosis, a significant number progress to advanced disease exhibiting aggressive clinical characteristics and often becoming refractory to radioactive iodine (RAI) treatment, the current gold-standard therapeutic option for metastatic disease. RAI-refractoriness is caused by defective functional expression of the sodium-iodide symporter (NIS), which is responsible for the active transport of iodide across the plasma membrane (PM) into thyroid follicles. NIS deficiency in these tumors often reflects a transcriptional impairment, but also its defective targeting and retention at the cells' PM. Using proteomics, we previously characterized an intracellular signaling pathway derived from SRC kinase that acts through the small GTPase RAC1 to recruit and bind the actin-anchoring adaptor EZRIN to NIS, regulating its retention at the PM of both non-transformed and cancer thyroid cells. Here, we describe how by reanalyzing the proteomics data, we identified cell-cell adhesion as the molecular event upstream the pathway involved in the anchoring and retention at the PM. We show that by interacting with NIS at the PM, adherens junction (AJ)-associated P120-catenin recruits and is phosphorylated by SRC, allowing it to recruit RAC1 to the complex. This enables SRC-phosphorylated VAV2 exchange factor to activate RAC1 GTPase, inducing NIS retention at the PM, thus increasing its abundance and function at the surface of thyroid cells. Our findings indicate that the loss of epithelial cell-cell adhesion may contribute to RAI refractoriness, indicating that in addition to stimulating NIS expression, successful resensitization therapies might require the employment of agents that improve cell-cell adhesion and NIS PM retention in refractory TC cells.

Autoantibodies are produced within germinal centers (GC), in a process regulated by interactions between B, T follicular helper (Tfh), and T follicular regulatory (Tfr) cells. The GC dysregulation in human autoimmunity has been inferred from circulating cells, albeit with conflicting results due to diverse experimental approaches. We applied a consistent approach to compare circulating Tfr and Tfh subsets in patients with different autoimmune diseases. We recruited 97 participants, including 72 patients with Hashimoto's thyroiditis (HT, n = 18), rheumatoid arthritis (RA, n = 16), or systemic lupus erythematosus (SLE, n = 32), and 31 matched healthy donors (HD). We found that the frequency of circulating T follicular subsets differed across diseases. Patients with HT had an increased frequency of blood Tfh cells (p = 0.0215) and a reduced Tfr/Tfh ratio (p = 0.0338) when compared with HD. This was not observed in patients with systemic autoimmune rheumatic diseases (RA, SLE), who had a reduction in both Tfh (p = 0.0494 and p = 0.0392, respectively) and Tfr (p = 0.0003 and p = 0.0001, respectively) cells, resulting in an unchanged Tfr/Tfh ratio. Activated PD-1 + ICOS + Tfh and CD4 + PD-1 + CXCR5 - Tph cells were raised only in patients with SLE (p = 0.0022 and p = 0.0054), without association with disease activity. Our data suggest that GC dysregulation, assessed by T follicular subsets, is not uniform in human autoimmunity. Specific patterns of dysregulation may become potential biomarkers for disease and patient stratification., (© 2022. The Author(s).)

The majority of pituitary adenomas occur in a sporadic context, and in the absence of known genetic predisposition. Three common variants at the NEBL (rs2359536), PCDH15 (rs10763170) and CDK8 (rs17083838) loci were previously associated with sporadic pituitary adenomas in the Han Chinese population, but these findings have not yet been replicated in any other population. The aim of this case-control study was to assess if these variants are associated with susceptibility to sporadic pituitary adenomas in the Portuguese population. Genotype and allele frequencies were determined in 570 cases and in 546 controls. The CDK8 rs17083838 minor allele (A allele) was significantly associated with sporadic pituitary adenomas, under an additive (odds ratio (OR) 1.73, 95% confidence interval (CI) 1.19-2.50, p = 0.004) and dominant (OR 1.82, 95% CI 1.24-2.68, p = 0.002) inheritance model. The NEBL rs2359536 and PCDH15 rs10763170 variants were not associated with the overall risk for the disease, although a borderline significant association was observed between the PCDH15 rs10763170 minor allele (T allele) and somatotrophinomas (dominant model, OR 1.55, 95% CI 1.02-2.35, p = 0.035). These findings suggest that the CDK8 rs17083838 variant, and possibly the PCDH15 rs10763170 variant, may increase susceptibility to sporadic pituitary adenomas in the Portuguese population.

The microenvironment of pituitary adenomas (PAs) includes a range of non-tumoral cells, such as immune and stromal cells, as well as cell signaling molecules such as cytokines, chemokines and growth factors, which surround pituitary tumor cells and may modulate tumor initiation, progression, invasion, angiogenesis and other tumorigenic processes. The microenvironment of PAs has been actively investigated over the last years, with several immune and stromal cell populations, as well as different cytokines, chemokines and growth factors being recently characterized in PAs. Moreover, key microenvironment-related genes as well as immune-related molecules and pathways have been investigated, with immune check point regulators emerging as promising targets for immunotherapy. Understanding the microenvironment of PAs will contribute to a deeper knowledge of the complex biology of PAs, as well as will provide developments in terms of diagnosis, clinical management and ultimately treatment of patients with aggressive and/or refractory PAs., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The Sodium/Iodide Symporter (NIS) is responsible for the active transport of iodide into thyroid follicular cells. Differentiated thyroid carcinomas (DTCs) usually preserve the functional expression of NIS, allowing the use of radioactive iodine (RAI) as the treatment of choice for metastatic disease. However, a significant proportion of patients with advanced forms of TC become refractory to RAI therapy and no effective therapeutic alternatives are available. Impaired iodide uptake is mainly caused by the defective functional expression of NIS, and this has been associated with several pathways linked to malignant transformation. MAPK signaling has emerged as one of the main pathways implicated in thyroid tumorigenesis, and its overactivation has been associated with the downregulation of NIS expression. Thus, several strategies have been developed to target the MAPK pathway attempting to increase iodide uptake in refractory DTC. However, MAPK inhibitors have had only partial success in restoring NIS expression and, in most cases, it remained insufficient to allow effective treatment with RAI. In a previous work, we have shown that the activity of the small GTPase RAC1 has a positive impact on TSH-induced NIS expression and iodide uptake in thyroid cells. RAC1 is a downstream effector of NRAS, but not of BRAF. Therefore, we hypothesized that the positive regulation induced by RAC1 on NIS could be a relevant signaling cue in the mechanism underlying the differential response to MEK inhibitors, observed between NRAS- and BRAF-mutant tumors. In the present study, we found that the recovery of NIS expression induced through MAPK pathway inhibition can be enhanced by potentiating RAC1 activity in thyroid cell systems. The negative impact on NIS expression induced by the MAPK-activating alterations, NRAS Q61R and BRAF V600E, was partially reversed by the presence of the MEK 1/2 inhibitors AZD6244 and CH5126766. Notably, the inhibition of RAC1 signaling partially blocked the positive impact of MEK inhibition on NIS expression in NRAS Q61R cells. Conversely, the presence of active RAC1 considerably improved the rescue of NIS expression in BRAF V600E thyroid cells treated with MEK inhibitors. Overall, our data support an important role for RAC1 signaling in enhancing MAPK inhibition in the context of RAI therapy in DTC, opening new opportunities for therapeutic intervention.

The functional expression of the sodium-iodide symporter (NIS) at the membrane of differentiated thyroid cancer (DTC) cells is the cornerstone for the use of radioiodine (RAI) therapy in these malignancies. However, NIS gene expression is frequently downregulated in malignant thyroid tissue, and 30% to 50% of metastatic DTCs become refractory to RAI treatment, which dramatically decreases patient survival. Several strategies have been attempted to increase the NIS mRNA levels in refractory DTC cells, so as to re-sensitize refractory tumors to RAI. However, there are many RAI-refractory DTCs in which the NIS mRNA and protein levels are relatively abundant but only reduced levels of iodide uptake are detected, suggesting a posttranslational failure in the delivery of NIS to the plasma membrane (PM), or an impaired residency at the PM. Because little is known about the molecules and pathways regulating NIS delivery to, and residency at, the PM of thyroid cells, we here employed an intact-cell labeling/immunoprecipitation methodology to selectively purify NIS-containing macromolecular complexes from the PM. Using mass spectrometry, we characterized and compared the composition of NIS PM complexes to that of NIS complexes isolated from whole cell (WC) lysates. Applying gene ontology analysis to the obtained MS data, we found that while both the PM-NIS and WC-NIS datasets had in common a considerable number of proteins involved in vesicle transport and protein trafficking, the NIS PM complexes were particularly enriched in proteins associated with the regulation of the actin cytoskeleton. Through a systematic validation of the detected interactions by co-immunoprecipitation and Western blot, followed by the biochemical and functional characterization of the contribution of each interactor to NIS PM residency and iodide uptake, we were able to identify a pathway by which the PM localization and function of NIS depends on its binding to SRC kinase, which leads to the recruitment and activation of the small GTPase RAC1. RAC1 signals through PAK1 and PIP5K to promote ARP2/3-mediated actin polymerization, and the recruitment and binding of the actin anchoring protein EZRIN to NIS, promoting its residency and function at the PM of normal and TC cells. Besides providing novel insights into the regulation of NIS localization and function at the PM of TC cells, our results open new venues for therapeutic intervention in TC, namely the possibility of modulating abnormal SRC signaling in refractory TC from a proliferative/invasive effect to the re-sensitization of these tumors to RAI therapy by inducing NIS retention at the PM.

Adrenocortical carcinoma (ACC) is a rare and aggressive malignancy. For stage I and II tumors, surgery is a curative option, but even in these cases recurrence is frequent. Practical guidelines advocate a combination of mitotane with etoposide, doxorubicin, and cisplatin as first-line therapy for metastatic adrenocortical carcinoma. However, this scheme presents limited efficacy and high toxicity. The use of Immune Checkpoint Inhibitors (ICI) and multi-Tyrosine Kinase Inhibitors (mTKI) has modified the approach of multiple malignancies. The expectation of their applicability on advanced adrenocortical carcinoma is high but the role of these new therapies persists unclear. This article provides a short summary of last years' findings targeting outcomes, limitations, and adverse effects of these new therapeutic approaches. The results of recent trials and case series pointed pembrolizumab as the most promising drug among these new therapies. It is the most often used ICI and the one presenting the best results with less related adverse effects when in comparison to the standard treatment with mitotane. Hereafter, the identification of specific molecular biomarkers or immune profiles associated with ICI or mTKI good response will facilitate the selection of candidates for these therapies. So far, microsatellite instability and Lynch Syndrome related germline mutations are suggested as predictive biomarkers of good response. Contrarywise, cortisol secretion has been associated with more aggressive ACC tumors and potentially poor responses to immunotherapy., Competing Interests: The authors declare that they have no conflict of interest., (Thieme. All rights reserved.)

Summary: Menopause is a relative hyperandrogenic state but the development of hirsutism or virilizing features should not be regarded as normal. We report the case of a 62-year-old woman with a 9-month history of progressive frontotemporal hair loss and hirsutism, particularly on her back, arms and forearms. Blood tests showed increased total testosterone of 5.20 nmol/L that remained elevated after an overnight dexamethasone suppression test. Free Androgen Index was 13.1 and DHEAS was repeatedly normal. Imaging examinations to study adrenals and ovaries were negative. The biochemical profile and the absence of imaging in favor of an adrenal tumor made us consider the ovarian origin as the most likely hypothesis. After informed consent, bilateral salpingectomy-oophorectomy and total hysterectomy were performed. Gross pathology revealed ovaries of increased volume and histology showed bilateral ovarian stromal hyperplasia. Testosterone levels normalized after surgery and hirsutism had completely subsided 8 months later., Learning Points: Menopause is a relative hyperandrogenic state Hirsutism and/or virilizing features, in a postmenopausal woman, should raise the hypothesis of a malignant cause In the absence of an identifiable ovarian or adrenal tumor, the ovarian origin remains the most likely Peripheral aromatization of excess androgen may conduct to high levels of estrogen increasing the risk of endometrial cancer Bilateral oophorectomy results in significant clinical improvement.

Adrenocortical carcinoma (ACC) is an orphan disease lacking effective systemic treatment options. The low incidence of the disease and high cost of clinical trials are major obstacles in the search for improved treatment strategies. As a novel approach, registry-based clinical trials have been introduced in clinical research, so allowing for significant cost reduction, but without compromising scientific benefit. Herein, we describe how the European Network for the Study of Adrenal Tumours (ENSAT) could transform its current registry into one fit for a clinical trial infrastructure. The rationale to perform randomized registry-based trials in ACC is outlined including an analysis of relevant limitations and challenges. We summarize a survey on this concept among ENSAT members who expressed a strong interest in the concept and rated its scientific potential as high. Legal aspects, including ethical approval of registry-based randomization were identified as potential obstacles. Finally, we describe three potential randomized registry-based clinical trials in an adjuvant setting and for advanced disease with a high potential to be executed within the framework of an advanced ENSAT registry. Thus we, therefore, provide the basis for future registry-based trials for ACC patients. This could ultimately provide proof-of-principle of how to perform more effective randomized trials for an orphan disease.

Objective: Mutations in the genes coding for succinate dehydrogenase (SDHx) are the most frequent germline alterations in pheochromocytomas and paragangliomas. Evidence for the advantages associated with presymptomatic screening for SDHx mutation carriers is scarce. This study describes a nationwide cohort of these mutation carriers and aims to compare patients with clinical manifestations of the disease and those diagnosed through genetic screening., Design: Cross-sectional study., Patients: SDHx mutation carriers (n = 118) followed through the Portuguese Oncology referral centres: 41 probands and 77 nonprobands., Measurements: All participants were subjected to biochemical and body imaging examinations for a complete assessment of the extent and spread of disease. Clinical data obtained this way were further analysed., Results: The mean age of this cohort was 44.5 ± 17.4 years, and more than half carried the same founder SDHB mutation. About 50.8% of the mutation carriers developed pheochromocytomas or paragangliomas. Compared to patients diagnosed through genetic screening, those diagnosed clinically were characterized by larger tumours (P < .001), more frequent metastases (P = .024), were more frequently subjected to surgery (P = .011) and radiotherapy (P = .013), and had worse outcomes, such as macroscopic positive margins (P = .034). Persistent and/or unresectable disease and disease-related mortality were also more frequent in symptomatic patients compared to those diagnosed through genetic screening (P = .014)., Conclusions: In this nationwide cohort study, a large proportion of mutation carriers were found to develop SDHx-related neoplasia. Genetic testing and subsequent follow-up resulted in the diagnosis of smaller and nonmetastatic tumours, fewer treatment procedures, fewer complications and greater number of disease-free patients., (© 2020 John Wiley & Sons Ltd.)

Context: Urine steroid metabolomics, combining mass spectrometry-based steroid profiling and machine learning, has been described as a novel diagnostic tool for detection of adrenocortical carcinoma (ACC)., Objective, Design, Setting: This proof-of-concept study evaluated the performance of urine steroid metabolomics as a tool for postoperative recurrence detection after microscopically complete (R0) resection of ACC., Patients and Methods: 135 patients from 14 clinical centers provided postoperative urine samples, which were analyzed by gas chromatography-mass spectrometry. We assessed the utility of these urine steroid profiles in detecting ACC recurrence, either when interpreted by expert clinicians or when analyzed by random forest, a machine learning-based classifier. Radiological recurrence detection served as the reference standard., Results: Imaging detected recurrent disease in 42 of 135 patients; 32 had provided pre- and post-recurrence urine samples. 39 patients remained disease-free for ≥3 years. The urine "steroid fingerprint" at recurrence resembled that observed before R0 resection in the majority of cases. Review of longitudinally collected urine steroid profiles by 3 blinded experts detected recurrence by the time of radiological diagnosis in 50% to 72% of cases, improving to 69% to 92%, if a preoperative urine steroid result was available. Recurrence detection by steroid profiling preceded detection by imaging by more than 2 months in 22% to 39% of patients. Specificities varied considerably, ranging from 61% to 97%. The computational classifier detected ACC recurrence with superior accuracy (sensitivity = specificity = 81%)., Conclusion: Urine steroid metabolomics is a promising tool for postoperative recurrence detection in ACC; availability of a preoperative urine considerably improves the ability to detect ACC recurrence., (© Endocrine Society 2019.)

The sodium-iodide symporter (NIS) mediates transport of iodide across the basolateral membrane of thyroid cells. NIS expression in thyroid cancer (TC) cells allows the use of radioactive iodine (RAI) as a diagnostic and therapeutic tool, being RAI therapy the systemic treatment of choice for metastatic disease. Still, a significant proportion of patients with advanced TC lose the ability to respond to RAI therapy and no effective alternative therapies are available. Defective NIS expression is the main reason for impaired iodide uptake in TC and NIS downregulation has been associated with several pathways linked to malignant transformation. NF-κB signaling is one of the pathways associated with TC. Interestingly, NIS expression can be negatively regulated by TNF-α, a bona fide activator of NF-κB with a central role in thyroid autoimmunity. This prompted us to clarify NF-kB's role in this process. We confirmed that TNF-α leads to downregulation of TSH-induced NIS expression in non-neoplastic thyroid follicular cell-derived models. Notably, a similar effect was observed when NF-κB activation was triggered independently of ligand-receptor specificity, using phorbol-myristate-acetate (PMA). TNF-α and PMA downregulation of NIS expression was reverted when NF-κB-dependent transcription was blocked, demonstrating the requirement for NF-kB activity. Additionally, TNF-α and PMA were shown to have a negative impact on TSH-induced iodide uptake, consistent with the observed transcriptional downregulation of NIS. Our data support the involvement of NF-κB-directed transcription in the modulation of NIS expression, where up- or down-regulation of NIS depends on the combined output to NF-κB of several converging pathways. A better understanding of the mechanisms underlying NIS expression in the context of normal thyroid physiology may guide the development of pharmacological strategies to increase the efficiency of iodide uptake. Such strategies would be extremely useful in improving the response to RAI therapy in refractory-TC., Competing Interests: The authors have declared that no competing interests exist.

Context: Metabolomic studies of pheochromocytoma and paraganglioma tissue showed a correlation between metabolomic profile and presence of SDHx mutations, especially a pronounced increase of succinate., Objective: To compare the metabolomic profile of 24-hour urine samples of SDHx mutation carriers with tumors (affected mutation carriers), without tumors (asymptomatic mutation carriers), and patients with sporadic pheochromocytomas and paragangliomas., Methods: Proton nuclear magnetic resonance spectroscopic profiling of urine samples and metabolomic analysis using pairwise comparisons were complemented by metabolite set enrichment analysis to identify meaningful patterns., Results: The urine of the affected SDHx carriers showed substantially lower levels of seven metabolites than the urine of asymptomatic mutation carriers (including, succinate and N-acetylaspartate). The urine of patients with SDHx-associated tumors presented substantially higher levels of three metabolites compared with the urine of patients without mutation; the metabolite set enrichment analysis identified gluconeogenesis, pyruvate, and aspartate metabolism as the pathways that most probably explained the differences found. N-acetylaspartate was the only metabolite the urinary levels of which were significantly different between the three groups., Conclusions: The metabolomic urine profile of the SDHx mutation carriers with tumors is different from that of asymptomatic carriers and from that of patients with sporadic neoplasms. Differences are likely to reflect the altered mitochondria energy production and pseudohypoxia signature of these tumors. The urinary levels of N-acetylaspartate and succinate contrast with those reported in tumor tissue, suggesting a defective washout process of oncometabolites in association with tumorigenesis. The role of N-acetylaspartate as a tumor marker for these tumors merits further investigation., (Copyright © 2019 Endocrine Society.)

Thyroid cancer (TC) is the most common endocrine malignancy. The sodium-iodide symporter (NIS), responsible for active transport of iodide into thyroid cells, allows the use of radioactive iodine (RAI) as the systemic treatment of choice for TC metastatic disease. Still, patients with advanced forms of TC often lose the ability to respond to RAI therapy, which results in worse survival rates. We have shown that the overexpression of RAC1b, a tumor-related RAC1 splice variant, is associated with less favorable clinical outcomes in differentiated TCs derived from the follicular epithelial (DTCs). RAC1b overexpression is also significantly associated with the presence of MAPK-activating BRAFV600E mutation, which has been previously implicated in the loss of NIS expression. Here, we show that increased RAC1b levels are associated with NIS downregulation in DTCs and demonstrate that ectopic overexpression of RAC1b in non-transformed thyroid cells is sufficient to decrease TSH-induced NIS expression, antagonizing the positive effect of the canonically spliced RAC1 GTPase. Moreover, we clearly document for the first time in thyroid cells that both NIS expression and iodide uptake are hampered by RAC1 inhibition, highlighting the role of RAC1 in promoting TSH-induced NIS expression. Our findings support a role for RAC1 and RAC1b signaling in the regulation of NIS expression in thyroid cells and suggest that RAC1b in cooperation with other cancer-associated signaling cues may be implicated in the response of DTCs to RAI therapy.

Background: The incidence of gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs) has been increasing in the last five decades, but there is no large-scale data regarding these tumours in Portugal. We conducted a cross-sectional, multicentric study in main Portuguese centers to evaluate the clinical, pathological, and therapeutic profile of GEP-NENs., Methods: From November, 2012, to July, 2014, data from 293 patients diagnosed with GEP-NENs from 15 centers in Portugal was collected and registered in an online electronic platform., Results: Median age at diagnosis was 56.5 (range: 15-87) years with a preponderance of females (54.6%). The most frequent primary sites were the pancreas (31.1%), jejunum-ileum (24.2%), stomach (13.7%), and rectum (8.5%). Data regarding hormonal status was not available in most patients (82.3%). Stratified by the tumour grade (WHO 2010 classification), we observed 64.0% of NET G1, 24.7% of NET G2, and 11.3% of NEC. Poorly differentiated tumours occurred mainly in older patients ( p = 0.017), were larger ( p < 0.001), and presented more vascular ( p = 0.004) and lymphatic ( p = 0.001) invasion. At the time of diagnosis, 44.4% of GEP-NENs presented metastatic disease. Surgery (79.6%) and somatostatin analogues (30.7%) were the most frequently used therapies of GEP-NENs with reported grading., Conclusion: In general, Portuguese patients with GEP-NENs presented similar characteristics to other populations described in the literature. This cross-sectional study represents the first step to establish a national database of GEP-NENs that may aid in understanding the clinical and epidemiological features of these tumours in Portugal., Competing Interests: The authors declare no conflict of interest.

Introduction: Calcitonin (Ctn) is a hormone secreted by thyroid "C" cells and is considered an excellent marker for medullary thyroid carcinoma (MTC). However, the use of Ctn to screen patients with nodular thyroid disease (NTD) remains controversial., Objective: The aim of this work was to define the frequency of hypercalcitoninemia among NTD patients followed at a tertiary referral hospital., Methods: A retrospective analysis was made of basal Ctn measurements and corresponding patients' records between January 2011 and December 2015. Hypercalcitoninemia was defined as > 10 pg/mL. Depending on the Ctn value, three groups were considered: G1, ≤10 pg/mL; G2, 10-100 pg/mL; G3, ≥100 pg/mL., Results: Ctn was requested in an NTD context for 1,504 patients, 69 of whom had hypercalcitoninemia. Of these, 20 underwent surgery (G2, 11; G3, 9), and a histological diagnosis of MTC was established in 12 (G2, 3/27%; G3, 9/100%). Surgery was chosen based solely on Ctn levels in 7 cases, since only 5 had a positive cytology., Conclusions: Hypercalcitoninemia was found in 4.6% of NTD patients. Ctn levels ≥100 pg/mL were associated with a greater CMT risk than values between 10 and 100 pg/mL, reinforcing results from other groups. The need for an adequate interpretation of results as well as an appropriate selection of patients to surgery stresses the importance of endocrinologists requesting and interpreting results.

Background: Multiple endocrine neoplasia type 2B is a rare syndrome caused mainly by Met918Thr germline RET mutation, and characterised by medullary thyroid carcinoma, phaeochromocytoma, and extra-endocrine features. Data are scarce on the natural history of multiple endocrine neoplasia type 2B. We aimed to advance understanding of the phenotype and natural history of multiple endocrine neoplasia type 2B, to increase awareness and improve detection., Methods: This study was a retrospective, multicentre, international study in patients carrying the Met918Thr RET variant with no age restrictions. The study was done with registry data from 48 centres globally. Data from patients followed-up from 1970 to 2016 were retrieved from May 1, 2016, to May 31, 2018. Our primary objectives were to determine overall survival, and medullary thyroid carcinoma-specific survival based on whether the patient had undergone early thyroidectomy before the age of 1 year. We also assessed remission of medullary thyroid carcinoma, incidence and treatment of phaeochromocytoma, and the penetrance of extra-endocrine features., Findings: 345 patients were included, of whom 338 (98%) had a thyroidectomy. 71 patients (21%) of the total cohort died at a median age of 25 years (range <1-59). Thyroidectomy was done before the age of 1 year in 20 patients, which led to long-term remission (ie, undetectable calcitonin level) in 15 (83%) of 18 individuals (2 patients died of causes unrelated to medullary thyroid carcinoma). Medullary thyroid carcinoma-specific survival curves did not show any significant difference between patients who had thyroidectomy before or after 1 year (comparison of survival curves by log-rank test: p=0·2; hazard ratio 0·35; 95% CI 0.07-1.74). However, there was a significant difference in remission status between patients who underwent thyroidectomy before and after the age of 1 year (p<0·0001). There was a significant difference in remission status between patients who underwent thyroidectomy before and after the age of 1 year (p<0·0001). In the other 318 patients who underwent thyroidectomy after 1 year of age, biochemical and structural remission was obtained in 47 (15%) of 318 individuals. Bilateral phaeochromocytoma was diagnosed in 156 (50%) of 313 patients by 28 years of age. Adrenal-sparing surgery was done in 31 patients: three (10%) of 31 patients had long-term recurrence, while normal adrenal function was obtained in 16 (62%) patients. All patients with available data (n=287) had at least one extra-endocrine feature, including 106 (56%) of 190 patients showing marfanoid body habitus, mucosal neuromas, and gastrointestinal signs., Interpretation: Thyroidectomy done at no later than 1 year of age is associated with a high probability of cure. The reality is that the majority of children with the syndrome will be diagnosed after this recommended age. Adrenal-sparing surgery is feasible in multiple endocrine neoplasia type 2B and affords a good chance for normal adrenal function. To improve the prognosis of such patients, it is imperative that every health-care provider be aware of the extra-endocrine signs and the natural history of this rare syndrome. The implications of this research include increasing awareness of the extra-endocrine symptoms and also recommendations for thyroidectomy before the age of 1 year., Funding: None., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Aim: To determine the incidence and associated factors of new-onset diabetes after transplantation (NODAT) in a Portuguese central hospital., Methods: This single-center retrospective study involved consecutive adult nondiabetic transplant recipients, who had undergone kidney transplantation between January 2012 and March 2016. NODAT was diagnosed according to the criteria of the American Diabetes Association. Data were collected from an institutional database of the Nephrology and Kidney Transplantation Department (Santa Maria Hospital, Lisbon, Portugal) and augmented with data of laboratorial parameters collected from the corresponding patient electronic medical records. Exclusion criteria were preexisting diabetes mellitus, missing information and follow-up period of less than 12 mo. Data on demographic and clinical characteristics as well as anthropometric and laboratorial parameters were also collected. Patients were divided into two groups: With and without NODAT - for statistical comparison., Results: A total of 156 patients received kidney transplant during the study period, 125 of who were included in our analysis. NODAT was identified in 27.2% of the patients ( n = 34; 53% female; mean age: 49.5 ± 10.8 years; median follow-up: 36.4 ± 2.5 mo). The incidence in the first year was 24.8%. The median time to diagnosis was 3.68 ± 5.7 mo after transplantation, and 76.5% of the patients developed NODAT in the first 3 mo. In the group that did not develop NODAT ( n = 91), 47% were female, with mean age of 46.4 ± 13.5 years and median follow-up of 35.5 ± 1.6 mo. In the NODAT group, the pretransplant fasting plasma glucose (FPG) levels were significantly higher [101 (96.1-105.7) mg/dL vs 92 (91.4-95.8) mg/dL, P = 0.007] and pretransplant impaired fasting glucose (IFG) was significantly more frequent (51.5% vs 27.7%, P = 0.01). Higher pretransplant FPG levels and pretransplant IFG were found to be predictive risk factors for NODAT development [odds ratio (OR): 1.059, P = 0.003; OR: 2.772, P = 0.017, respectively]., Conclusion: NODAT incidence was high in our renal transplant recipients, particularly in the first 3 mo posttransplant, and higher pretransplant FPG level and IFG were risk factors., Competing Interests: Conflict-of-interest statement: All authors declare no conflicts-of-interest in relation to this article.

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.

Introduction: Metastases to central nervous system (M 1 -CNS) are rarely reported in thyroid cancer (TC) patients. We aimed to characterize patients with M 1 -CNS from TC followed in our department., Methods: Review of the medical records of 27 patients with TC-related M 1 -CNS., Results: Mean age at TC diagnosis was 56.9 ± 19.1 years. Papillary TC (55.6%) was the commonest histological type, followed by poorly differentiated (18.5%), medullary (11.1%), follicular (7.4%) and Hürthle cell (7.4%) carcinomas. Angioinvasion and extrathyroidal extension were observed in a high number of patients. At M 1 -CNS diagnosis, other distant metastases were already present in 77.8% of the patients. Treatment directed to M 1 -CNS was offered to 20 (74%) patients: 1 was submitted to surgery, 18 to radiotherapy (either whole-brain radiotherapy or stereotaxic radiosurgery or both) and 4 to surgery and radiotherapy. Four patients received cytotoxic chemotherapy and one was submitted to 131 I. Median survival since M 1 -CNS detection was 5.0 months. The only factor associated with better survival was surgery to brain metastases (P = 0.012)., Conclusions: The management of these patients is very challenging given the inexistence of effective treatments, except for brain surgery in selected cases., (© 2016 The authors.)

pT3 papillary thyroid carcinoma (PTC) can be divided into three subgroups according to the largest diameter >4 cm or evidence of minimal extrathyroidal extension (ETE): A >4 cm, no ETE; B ≤4 cm, with ETE; and C >4 cm, with ETE. The aim of this study was to analyze whether these subgroups are clinically different. A retrospective analysis of clinicopathological data of patients with pT3 PTC, with a minimum follow-up of 2 years, at a referral center was conducted. Outcome was evaluated after primary treatment and after 2 and 5 years of follow-up. Patients were classified as no evidence of disease (NED), biochemical evidence of disease (BED), and structural evidence of disease (SED) either locoregional (SED-L) or at distance (SED-D). The study patients were classified into three groups as follows: Group A (n = 91), Group B (n = 101), and Group C (n = 23). Most patients were female (80.0 %); mean age at diagnosis was 49.9 ± 16.5 years. 214 patients underwent total thyroidectomy; 208 patients were treated with (131)I. Median follow-up was 6.0 years. After primary treatment, the condition of NED was significantly different between the groups (A-87.9 %, B-68.3 %, C-43.5 %; p < 0.001). Recurrence rate, either biochemical or structural, was 8.8, 7.2, and 30.0 % in groups A, B, and C, respectively. Clinical status after 2 years anticipated clinical status after 5 years, except for Group B. ETE and tumor size were found to be predictors of disease status after primary treatment and after 2 years. ETE appeared as the strongest predictor of persistence of disease after primary treatment as well as of evidence of disease, either biochemical or structural, after 2 years of follow-up.

Familial cases of medullary thyroid carcinoma (MTC) may be diagnosed by genetic screening, while in sporadic tumors the diagnosis relies mainly on fine-needle aspiration cytology. The aim of the present study was to determine the demographic, clinical and pathological characteristics of MTC patients followed-up at the Portuguese Institute of Oncology Francisco Gentil (Lisbon, Portugal). For that purpose, a retrospective analysis of 140 MTC patients diagnosed between 1990 and 2010 was performed. The results indicated that patients with hereditary MTC (11.4%) were significantly younger than patients with sporadic MTC. Of the latter, 34.3% had no clinical suspicion of MTC prior to surgery. The sensitivity of cytology and calcitonin (CT) assay in diagnosing MTC were 51.3 and 98.7%, respectively. All familial index cases and 69.0% of sporadic cases presented with advanced stage disease at the time of diagnosis, while 73.0% of familial MTC detected by genetic/pentagastrin screening were diagnosed at the early stage of the disease. Biochemical cure (BC) was achieved in 39.7% of patients and, of these, only 6.5% relapsed. The 5 and 10-year survival rates were 79.3 and 73.6%, respectively. Age >45 years (P=0.026), advanced stage at diagnosis (P<0.001) and absence of BC (P<0.001) were predictors of a worse prognosis on univariate analysis. However, when the patients detected by genetic/pentagastrin screening were excluded from the analysis, age was no longer a prognostic factor, although disease stage remained a significant prognostic factor. On multivariate analysis, BC was the only factor with a significant impact on prognosis (P=0.031). In addition, the present study confirmed that the majority of patients were diagnosed at advanced stages, and CT determination was observed to be more sensitive than cytology to diagnose MTC. Patients at early stages were more prone to achieve BC, which was a favorable prognostic factor. To the best of our knowledge, the present study reports for the first time that age at diagnosis is not a predictor of survival for patients with MTC when cases diagnosed by genetic/pentagastrin screening (who are usually young patients at the initial stages of the disease), are excluded from the analysis.

We present results obtained in five patients with advanced thyroid cancer, derived from the follicular epithelium, treated with Sorafenib used off-label. The median age at the time Sorafenib was started was 61 years. Only one patient tolerated the standard dose of 400 mg twice daily. The most severe adverse events were.

Multiple endocrine neoplasia type 4 (MEN 4) is a novel form of multiple endocrine neoplasia caused by mutations in the CDKN1B gene. Its clinical presentation includes MEN 1-related tumours such as parathyroid and anterior pituitary tumours in possible association with gonadal, adrenal, renal and thyroid tumours as well as facial angiofibromas, colagenomas and meningiomas. We describe the case of a patient with meningioma, papillary thyroid carcinoma, parathyroid adenoma and, additionally, Hürthle cell adenoma, cholesteatoma and uterine leiomyomas. Considering that this association could represent a MEN 4-like phenotype, we looked for germline mutations in the CDKN1B gene. A rare heterozygous single nucleotide substitution c.397C>A was identified. Its role as a susceptibility factor remains to be established., (2016 BMJ Publishing Group Ltd.)

RAC1b is a hyperactive variant of the small GTPase RAC1 known to be a relevant molecular player in different cancers. Previous studies from our group lead to the evidence that its overexpression in papillary thyroid carcinoma (PTC) is associated with an unfavorable prognosis. In the present study, we intended to extend the analysis of RAC1b expression to thyroid follicular neoplasms and to seek for clinical correlations. RAC1b expression levels were determined by RT-qPCR in thyroid follicular tumor samples comprising 23 follicular thyroid carcinomas (FTCs) and 33 follicular thyroid adenomas (FTAs). RAC1b was found to be overexpressed in 33% of carcinomas while no RAC1b overexpression was documented among follicular adenomas. Patients with a diagnosis of FTC were divided into two groups based on longitudinal evolution and final outcome. RAC1b overexpression was significantly associated with both the presence of distant metastases (P = 0.01) and poorer clinical outcome (P = 0.01) suggesting that, similarly to that previously found in PTCs, RAC1b overexpression in FTCs is also associated with worse outcomes. Furthermore, the absence of RAC1b overexpression in follicular adenomas hints its potential as a molecular marker likely to contribute, in conjunction with other putative markers, to the preoperative differential diagnosis of thyroid follicular lesions.

The paraganglioma (PGL)/pheochromocytoma (PHEO)-papillary thyroid carcinoma (PTC) dyad has been reported rarely. Whether the association is coincidental or results from an underlying genetic predisposition is difficult to ascertain. We analyzed clinical and molecular data on four unrelated patients identified and treated by one of us (MJB) at a tertiary center. Patients were screened for germline variants in a panel of candidate genes: RET, VHL, SDHB, SDHC, SDHD, SDHAF2, TMEM127, MAX, PTEN, CDKN1B. All patients were female; median age at diagnosis of PGL/PHEO was 45 years and at diagnosis of PTC was 49.5 years. Only one patient had family history of thyroid cancer. PTC was multifocal in 2 cases, of the classical type in 2 cases and of the follicular type in 2 cases. Two patients harbored heterozygous germline variants of uncertain significance in the SDHB gene: Ser163Pro and Ala3Gly. The -79T>C polymorphism in the CDKN1B gene was present in all patients (3 in homozygous and 1 in heterozygous state). Results deriving from a comprehensive analysis of a panel of genes suggest that there is no single explanation for the association PGL/PHEO-PTC. It may occur through different mechanisms such as the combinatorial effect of different genetic variants, be a coincidental association or, alternatively, result from genetic variants in genes still awaiting identification.

Thyroid-stimulating hormone-receptor autoantibodies normally causes hyperthyroidism. However, they might have blocking activity causing hypothyroidism. A 11-year-old girl followed due to type 1 diabetes mellitus, celiac disease and euthyroid lymphocytic thyroiditis at diagnosis. Two years after the initial evaluation, thyroid-stimulating hormone was suppressed with normal free T4; nine months later, a biochemical evolution to hypothyroidism with thyroid-stimulating hormone-receptor autoantibodies elevation was seen; the patient remained always asymptomatic. Chinese hamster ovary cells were transfected with the recombinant human thyroid-stimulating hormone -receptor, and then exposed to the patient's serum; it was estimated a 'moderate' blocking activity of these thyroid-stimulating hormone-receptor autoantibodies, and concomitantly excluded stimulating action. In this case, the acknowledgment of the blocking activity of the serum thyroid-stimulating hormone-receptor autoantibodies, supported the hypothesis of a multifactorial aetiology of the hypothyroidism, which in the absence of the in vitro tests, we would consider only as a consequence of the destructive process associated to lymphocytic thyroiditis.

Activating germline mutations in the RET proto-oncogene are responsible for about 98 % of the familial forms of medullary thyroid carcinoma (MTC), which represent 25 % of all MTC cases. The search for germline mutations in this gene is important for the recognition of hereditary forms of MTC and further identification of at-risk relatives who may benefit from early clinical intervention. Genotype-phenotype correlations are well established for most disease-causing RET mutations, allowing risk stratification. The association of a new RET variant with the MTC phenotype and familial predisposition requires the assessment of its functional and clinical significance. The aim of this study was to evaluate the oncogenic potential of two newly identified RET germline variants associated with late-onset MTC. In vitro functional assays were designed to address the transforming potential of novel RET variants, through their expression in non-transformed cells, and comparing their effect with wild-type RET. The new variants were identified in codons 515 (p.C515W) and 636 (p.T636M) located, respectively, in exons 8 and 11, thus resulting in amino acid substitutions in the extracellular region of the tyrosine kinase receptor RET. Through functional assays, we observed increased cell growth and proliferation, loss of contact inhibition, and a stimulation of cell migration, suggesting that these new RET variants hold some relevant transforming potential. The transforming potential of these novel RET variants was of low-grade, when compared to that of RET MEN2A-causing mutation p.C634R, probably explaining the mild phenotype characterized by late onset and low clinical aggressiveness.

The familial forms of non-medullary thyroid carcinoma (FNMTC) represent approximately 5 % of thyroid neoplasms. Nine FNMTC susceptibility loci have been mapped; however, only the DICER1 and SRGAP1 susceptibility genes have been identified. The transcription factors NKX2-1, FOXE1, PAX8, and HHEX are involved in the morphogenesis and differentiation of the thyroid. Recent studies have identified NKX2-1 germline mutations in FNMTC families. However, the role of high-penetrant FOXE1 variants in FNMTC etiology remains unclear. The aim of this study was to investigate the role of FOXE1 germline mutations in the pathogenesis of FNMTC. We searched for molecular changes in the FOXE1 gene in the probands from 60 Portuguese families with FNMTC. In this series, we identified nine polymorphisms and one variant (c.743C>G, p.A248G) which was not previously described. This variant, which involved an amino acid residue conserved in evolution, segregated with disease in one family, and was also detected in an apparently unrelated case of sporadic NMTC. Functional studies were performed using rat normal thyroid cells (PCCL3) clones and human papillary thyroid carcinoma cell line (TPC-1) pools, expressing the wild type and mutant (p.A248G) forms of FOXE1. In these experiments, we observed that the p.A248G variant promoted cell proliferation and migration, suggesting that it may be involved in thyroid tumorigenesis. Additionally, somatic p.V600E BRAF mutations were also detected in the thyroid tumors of two members of the family carrying the p.A248G variant. This study represents the first evidence of involvement of a germline FOXE1 rare variant in FNMTC etiology and suggests that mutations in MAPK pathway-related genes may contribute to tumor development in these familial cases.