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Background: There is some controversy on the potential relationship between autoimmune processes and clinicopathologic features as well as prognosis of differentiated thyroid cancer (DTC), and the evidence is limited by its largely retrospective nature. We examined the relationship between the presence of autoimmune thyroiditis (AT) and 1-year thyroid cancer treatment outcomes in a large multicenter study using prospectively collected data. Methods: We included data from consecutive DTC patients enrolled in the Italian Thyroid Cancer Observatory (ITCO) database (NCT04031339). We divided the groups according to the presence (AT) or absence (no autoimmune thyroiditis [noAT]) of associated AT. We used propensity score matching to compare the clinical features and outcomes between the two groups at 1-year follow-up. Results: We included data from 4233 DTC patients, including 3172 (75%) females. The American Thyroid Association (ATA) risk levels were as follows: 51% (2160/4233) low risk, 41.3% (1750/4233) intermediate risk, and 7.6% (323/4233) high risk. There were 1552 patients (36.7%) who had AT. Before propensity score matching, AT patients were significantly younger and had a smaller and bilateral tumor ( p  < 0.0001). Patients with AT more frequently fell into the low- and intermediate-risk categories, while the ATA high risk was more frequent among noAT patients ( p  = 0.004). After propensity score matching, patients with AT more frequently showed evidence of disease (structural/biochemical incomplete response) versus excellent/indeterminate response, compared with patients without AT (7.3% vs. 4.5%, p  = 0.001), with an odds ratio of 1.86 ([confidence interval: 1.3-2.6], p  = 0.0001). However, when considering only structural persistence as the outcome, no statistically significant differences were observed between patients with or without AT (3.4% vs. 2.7%, p  = 0.35). The elevated risk associated with the ATA intermediate and high risk at diagnosis remained consistently statistically significant. Conclusions: In this large prospective series, biochemical persistence was more frequent, at 1-year follow-up, in AT patients. However, there was no significant association between the presence of AT and structural persistence of disease. These findings may be explained by the presence of a residual thyroid tissue.

Background: The actual rates of suspicious thyroid nodules (TNs) and confirmed thyroid cancer (TC) in putatively "at-risk" selected populations (e.g., individuals with family history of TC) are still uncertain. Methods: Our aim was to explore the prevalence of TC and TN in a cross-sectional study of a consenting population of unaffected individuals (10 years of age or older) with a first-degree relative known to have non-medullary TC (NMTC). Enrolled subjects underwent ultrasonographic studies of the neck between 2009 and 2018. Nodules considered suspicious according to current guidelines were subjected to fine-needle aspiration biopsy (FNAB) for cytology. Results: The screenee population comprised 1176 individuals (median age 42 [26-56] years, 650 females, 55.3%) from 473 kindreds (346 with 1 established NMTC diagnosis at entry, 103 with 2 established NMTC diagnoses, and 24 with 3 or more established NMTC diagnoses at entry). Screening revealed TNs in 500 screenees (42.5%; confidence interval [CI] 39.7-45.4%). Ninety-seven of these (19.4%; CI 16.2-23.1%) underwent FNAB. Only 11 cases of TC were diagnosed in the whole population (0.9%; CI 0.5-1.7%). The prevalence of TC in screenees from kindreds with ≥3 cases (3/24, 12.5%) was higher than that for kindreds with one affected member (6/346, 1.7%; p  = 0.01, odds ratio [OR] 7.99; CI 1.21-40.75) and for those with two affected members (2/103, 1.9%; p  = 0.05, OR 7.05; CI 0.76-89.44). The prevalence of TNs was 61.8% (CI 56.6-66.8%), 75.7% (CI 66.6-83%), and 66.7% (CI 46.7-82%) in the kindreds with 1, 2, and ≥3 cases, respectively ( p  = 0.03). Conclusions : On the whole, ultrasound-based screening of unaffected relatives of individuals with established diagnoses of NMTC is likely to reveal a high prevalence of TN and a low prevalence of TC. However, a significantly higher prevalence of TC may be found among screenees from kindreds with at least three established NMTC diagnoses before screening, suggesting that closer surveillance may be warranted in kindreds with this level of familiality.

Background: The role of minimal extrathyroidal extension (mETE) as a risk factor for persistent papillary thyroid carcinoma (PTC) is still debated. The aims of this study were to assess the clinical impact of mETE as a predictor of worse initial treatment response in PTC patients and to verify the impact of radioiodine therapy after surgery in patients with mETE. Methods: We reviewed all records in the Italian Thyroid Cancer Observatory database and selected 2237 consecutive patients with PTC who satisfied the inclusion criteria (PTC with no lymph node metastases and at least 1 year of follow-up). For each case, we considered initial surgery, histological variant of PTC, tumor diameter, recurrence risk class according to the American Thyroid Association (ATA) risk stratification system, use of radioiodine therapy, and initial therapy response, as suggested by ATA guidelines. Results: At 1-year follow-up, 1831 patients (81.8%) had an excellent response, 296 (13.2%) had an indeterminate response, 55 (2.5%) had a biochemical incomplete response, and 55 (2.5%) had a structural incomplete response. Statistical analysis suggested that mETE (odds ratio [OR] 1.16, p  = 0.65), tumor size >2 cm (OR 1.45, p  = 0.34), aggressive PTC histology (OR 0.55, p  = 0.15), and age at diagnosis (OR 0.90, p  = 0.32) were not significant risk factors for a worse initial therapy response. When evaluating the combination of mETE, tumor size, and aggressive PTC histology, the presence of mETE with a >2 cm tumor was significantly associated with a worse outcome (OR 5.27 [95% confidence interval], p  = 0.014). The role of radioiodine ablation in patients with mETE was also evaluated. When considering radioiodine treatment, propensity score-based matching was performed, and no significant differences were found between treated and nontreated patients ( p  = 0.24). Conclusions: This study failed to show the prognostic value of mETE in predicting initial therapy response in a large cohort of PTC patients without lymph node metastases. The study suggests that the combination of tumor diameter and mETE can be used as a reliable prognostic factor for persistence and could be easily applied in clinical practice to manage PTC patients with low-to-intermediate risk of recurrent/persistent disease.

Background: One of the most widely used risk stratification systems for estimating individual patients' risk of persistent or recurrent differentiated thyroid cancer (DTC) is the American Thyroid Association (ATA) guidelines. The 2015 ATA version, which has increased the number of patients considered at low or intermediate risk, has been validated in several retrospective, single-center studies. The aims of this study were to evaluate the real-world performance of the 2015 ATA risk stratification system in predicting the response to treatment 12 months after the initial treatment and to determine the extent to which this performance is affected by the treatment center in which it is used. Methods: A prospective cohort of DTC patients collected by the Italian Thyroid Cancer Observatory web-based database was analyzed. We reviewed all records present in the database and selected consecutive cases that satisfied inclusion criteria: (i) histological diagnosis of DTC, with the exclusion of noninvasive follicular thyroid neoplasm with papillary-like nuclear features; (ii) complete data of the initial treatment and pathological features; and (iii) results of 1-year follow-up visit (6-18 months after the initial treatment), including all data needed to classify the estimated response to treatment. Results: The final cohort was composed of 2071 patients from 40 centers. The ATA risk of persistent/recurrent disease was classified as low in 1109 patients (53.6%), intermediate in 796 (38.4%), and high in 166 (8.0%). Structural incomplete responses were documented in only 86 (4.2%) patients: 1.5% in the low-risk, 5.7% in the intermediate-risk, and 14.5% in the high-risk group. The baseline ATA risk class proved to be a significant predictor of structural persistent disease, both for intermediate-risk (odds ratio [OR] 4.67; 95% confidence interval [CI] 2.59-8.43) and high-risk groups (OR 16.48; CI 7.87-34.5). Individual center did not significantly influence the prediction of the 1-year disease status. Conclusions: The ATA risk stratification system is a reliable predictor of short-term outcomes in patients with DTC in real-world clinical settings characterized by center heterogeneity in terms of size, location, level of care, local management strategies, and resource availability.

Background: Over 50% of newly diagnosed thyroid nodules are either cytologically benign or presumed to be benign on the basis of low-suspicion sonographic findings. The strategies used for their long-term surveillance are based mainly on the estimated residual risk of malignancy calculated with various ultrasonographic classification systems (e.g., Thyroid Image Reporting and Data Systems [TIRADS]). We conducted a longitudinal study to evaluate the temporal stability of the initial risk estimates computed with five widely used systems and to determine whether risk class increases during follow-up are indeed predictive of malignancy., Methods: We re-analyzed data prospectively collected at a single academic referral center on 232 patients (age: 54.1 ± 13.7 years) with 432 asymptomatic, sonographically or cytologically benign thyroid nodules at baseline (T0) and 122 new nodules that were present five years later (T5). At both time points, the sonographically estimated risk of malignancy was calculated as recommended by the American Association of Clinical Endocrinologists/American College of Endocrinology/Associazione Medici Endocrinologi, the American College of Radiologists' TIRADS, the American Thyroid Association's 2015 practice guidelines, the European Thyroid Association's TIRADS (EU-TIRADS), and the TIRADS of the Korean Society of Thyroid Radiology (K-TIRADS)., Results: For 57 to 127 (13.2-29.4%) of the original nodules, depending on the system used, the estimated malignancy risk increased over the 5-year interval. Of the nodules whose baseline risk had not warranted cytological assessment, very few (6.3-8.3%) met the criteria for cytology at the 5-year evaluation. Biopsy was indicated for only 4 to 8 (3.3-6.6%) of the new nodules based on T5 risk estimates. Despite these changes, none of the 232 patients was ever diagnosed with a cancer., Conclusions: Ultrasound-based risk classes of presumably benign thyroid nodules remain fairly stable over time, and changes warranting biopsy are rare indeed. The appearance of new nodules is a frequent event, but very few (<5%) are classified as high risk, and only the 3-7% meet the criteria for cytological assessment. Collectively, these findings support the view that patients with presumably benign thyroid nodules can be safely followed with less intensive protocols.

Objectives: The goal of evidence-based practice guidelines is to optimize the management of emerging diseases, such as differentiated thyroid cancer (DTC). The aim of this study was to assess therapeutic approaches for DTC in Italy and to see how closely these practices conformed to those recommended in the 2009 American Thyroid Association (ATA) guidelines., Methods: The Italian Thyroid Cancer Observatory was established to collect data prospectively on thyroid cancers consecutively diagnosed in participating centers (uniformly distributed across the nation). Data on the initial treatment of all pathologically confirmed DTC cases present in the database from January 1, 2013 (database creation) to January 31, 2016, were analyzed., Results: A total of 1748 patients (77.2% females; median age 48.1 years [range 10-85 years]) were enrolled in the study. Most (n = 1640; 93.8%) were papillary carcinomas (including 84 poorly differentiated/aggressive variants); 6.2% (n = 108) were follicular and Hürthle cell carcinomas. The median tumor diameter was 11 mm (range 1-93 mm). Tumors were multifocal in 613 (35%) and presented extrathyroidal extension in 492 (28%) cases. Initial treatments included total thyroidectomy (involving one or two procedures; n = 726; 98.8%) and lobectomy (n = 22; 1.2%). A quarter of the patients who underwent total thyroidectomy had unifocal, intrathyroidal tumors ≤1 cm (n = 408; 23.6%). Neck dissection was performed in 40.4% of the patients (29.5% had central compartment dissection). Radioiodine remnant ablation (RRA) was performed in 1057 (61.2%) of the 1726 patients who underwent total thyroidectomy: 460 (41.2%) of the 983 classified by 2009 ATA guideline criteria as low-risk, 570 (87.1%) of the 655 as intermediate-risk, and 82 (93.1%) of the 88 as high-risk patients (p < 0.001). RRA was performed in 44% of the cases involving multifocal DTCs measuring ≤1 cm., Conclusions: The treatment approaches for DTCs used in Italy display areas of inconsistency with those recommended by the 2009 ATA guidelines. Italian practices were characterized by underuse of thyroid lobectomy in intrathyroidal, unifocal DTCs ≤1 cm. The use of RRA was generally consistent with risk-stratified recommendations. However, its frequent use in small DTCs (≤1 cm) that are multifocal persists, despite the lack of evidence of benefit. These data provide a baseline for future assessments of the impact of international guidelines on DTC management in Italy. These findings also illustrate that the dissemination and implementation of guideline recommendations, and the change in practice patterns, require ongoing education and time.

Background: The term "nodular goiter" has long been used to refer to a nodular thyroid gland, based on the assumption that nodule growth may be associated with hyperplasia of the surrounding non-nodular tissue. The aim of this prospective, multicenter, observational study was to determine whether nodule growth is accompanied by growth in the non-nodular tissue., Methods: Eight Italian thyroid-disease referral centers enrolled 992 consecutive patients with one to four benign nodules. Nodular and non-nodular thyroid tissue volumes were assessed for five years with annual ultrasound examinations., Results: In participants whose nodules remained stable (n = 839), thyroid volumes did not change (baseline 15.0 mL [confidence interval (CI) 14.5-15.6]; five-year evaluation 15.1 mL [CI 14.5-15.7]). In participants with significant growth of one or more nodule (n = 153), thyroid volumes increased and by year 5 were significantly greater than those of the former group (17.4 mL [CI 16-18.7]). In 76 individuals with unilateral nodules that grew, the mean nodular lobe volume significantly exceeded that of the contralateral lobe (8.6 mL [CI 7.4-9.8] vs. 6.7 mL [CI 6-7.4]). The unaffected lobe volumes remained stable over time, while nodular lobes grew steadily and were significantly greater at the end of follow-up (10.1 mL [CI 8.9-11.3]). Excluding the volume of the largest growing nodule in these cases, the remaining volume of the affected lobe remained virtually unchanged with respect to its baseline value. Furthermore, there was no significant difference in the non-nodular tissue volume between the unaffected lobe and the affected lobe (with the largest growing nodule volume subtracted), both at baseline and at the end of follow-up., Conclusions: The growth of thyroid nodules is a local process, not associated with growth of the surrounding non-nodular tissue. Therefore, a normal-sized thyroid containing nodules should be referred to as a "uni- or multinodular thyroid gland" and considered a distinct entity from "uni- or multinodular goiter."

Background: Current surgical standard of care in sporadic medullary thyroid carcinoma (sMTC) consists of a minimum of total thyroidectomy with central neck dissection. Some have suggested thyroid lobectomy with isthmusectomy and central neck dissection for patients with sMTC, given their lower frequency of bilateral disease, although this topic has not been thoroughly studied. This study assessed the prevalence of multifocality in sMTC via a large international multi-institutional retrospective review to quantify this prevalence, including the impact of geography, to assess more accurately the risks associated with alternative surgical approaches., Methods: A retrospective chart review of sMTC patients from 11 institutions over 29 years (1983-2011) was undertaken. Data regarding focality, extent of disease, RET germline analysis plus family and clinical history for multiple endocrine neoplasia type 2 (MEN2), and demographic data were collected and analyzed., Results: Patients from four continents and seven countries were included in the sample. Data for 313 patients with documented sMTC were collected. Of these, 81.2% were confirmed with negative RET germline testing, while the remaining 18.8% demonstrated a negative family history and no manifestations of MEN2 syndromes other than MTC. Bilateral disease was identified in 17/306 (5.6%) patients, while multifocal disease was noted in 50/312 (16.0%) sMTC patients. When only accounting for germline negative patients, these rates were not significantly different (5.6% and 17%, respectively). Among them, when disease was unifocal in the ipsilateral lobe and isthmus, bilateral disease was present in 6/212 (2.8%) cases. When disease was multifocal in the ipsilateral lobe or isthmus, then bilateral disease was present in 8/37 (21.6%) cases (p < 0.001). No geographic differences in focality were identified., Conclusions: The 5.6% prevalence of bilateral foci in sMTC suggests that total thyroidectomy should remain the standard of care for initial surgery, as less complete thyroid surgery may fail to address fully the primary site of disease. Whether ipsilateral tumor focality should be an independent factor determining the need for completion thyroidectomy when sMTC is diagnosed after hemithyroidectomy remains to be determined.

Background: Small papillary thyroid carcinomas have contributed to the worldwide increased incidence of differentiated thyroid cancer observed over the past decades. However, the mortality rate has not changed over the same period of time, raising questions about the possibility that thyroid cancer patients, especially those with small tumors, are overdiagnosed and overtreated. Molecular prognostic marker able to discriminate aggressive thyroid cancers from those with an indolent course would be of great relevance to tailor the therapeutic approach and reduce overtreatment. Mutations in the TERT promoter were recently reported to correlate strongly with aggressiveness in advanced forms of thyroid cancer, holding promise for a possible clinical application. The occurrence and potential clinical relevance of TERT mutations in papillary thyroid microcarcinomas (mPTCs) is currently unknown. This study aimed to analyze the occurrence of two TERT promoter mutations (-124C>T and -146C>T) and their potential association with unfavorable clinical features in a large cohort of mPTCs., Methods: A total of 431 mPTCs cases were collected from six Italian institutions, and TERT promoter mutational status was assessed by a next-generation sequencing approach., Results: TERT promoter mutations were found in 4.7% of the analyzed mPTCs, showing that even microcarcinomas carry mutations in this gene. Correlation analysis showed that TERT promoter mutations are not associated with aggressive features or clinical outcome in the cohort analyzed., Conclusions: TERT mutations are present but uncommon in mPTCs. Apparently, in mPTCs, the occurrence of TERT mutations is not correlated with unfavorable clinical features.

Objective: The association between papillary thyroid cancer (PTC) and Hashimoto's thyroiditis is widely recognized, but less is known about the possible link between circulating anti-thyroglobulin antibody (TgAb) titers and PTC aggressiveness. To shed light on this issue, we retrospectively examined a large series of PTC patients with and without positive TgAb., Methods: Data on 220 TgAb-positive PTC patients (study cohort) were retrospectively collected in 10 hospital-based referral centers. All the patients had undergone near-total thyroidectomy with or without radioiodine remnant ablation. Tumor characteristics and long-term outcomes (follow-up range: 2.5-24.8 years) were compared with those recently reported in 1020 TgAb-negative PTC patients with similar demographic characteristics. We also assessed the impact on clinical outcome of early titer disappearance in the TgAb-positive group., Results: At baseline, the study cohort (mean age 45.9 years, range 12.5-84.1 years; 85% female) had a significantly higher prevalence of high-risk patients (6.9% vs. 3.2%, p<0.05) and extrathyroidal tumor extension (28.2% vs. 24%; p<0.0001) than TgAb-negative controls. Study cohort patients were also more likely than controls to have persistent disease at the 1-year visit (13.6% vs. 7.0%, p=0.001) or recurrence during subsequent follow-up (5.8% vs. 1.4%, p=0.0001). At the final follow-up visit, the percentage of patients with either persistent or recurrent disease in the two cohorts was significantly different (6.4% of TgAb-positive patients vs. 1.7% in the TgAb-negative group, p<0.0001). At the 1-year visit, titer normalization was observed in 85 of the 220 TgAb-positive individuals. These patients had a significantly lower rate of persistent disease than those who were still TgAb positive (8.2% vs. 17.3%. p=0.05), and no relapses were observed among patients with no evidence of disease during subsequent follow-up., Conclusions: PTC patients with positive serum TgAb titer during the first year after primary treatment were more likely to have persistent/recurrent disease than those who were consistently TgAb-negative. Negative titers at 1 year may be associated with more favorable outcomes.

Background: Comprehension of the regulatory mechanism involved in the sodium iodide symporter (NIS) expression is of great relevance for thyroid cancer. In fact, restoration of NIS expression would be a strategy to treat undifferentiated thyroid cancer. Previous in vitro findings suggest that the cyclic AMP-response element (CRE) modulator (CREM) is involved in control of NIS expression. In this work, we examined the expression of CREM in a series of thyroid cancer tissues and its action on NIS promoter in human thyroid cancer cells., Methods: Expression of mRNA levels for CREM, PAX8 and NIS was measured by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) in 6 normal thyroid tissues, 22 papillary, 12 follicular and 4 anaplastic thyroid cancers. The effect of CREM on transcriptional activity of the NIS promoter was investigated by transient transfection of human thyroid cell lines., Results: Compared to normal tissues, NIS and PAX8 mRNA levels were significantly reduced in all types of thyroid cancer. As expected, the maximal decrease was detected in anaplastic thyroid cancer. Conversely, CREM mRNA levels were increased in all types of thyroid cancer, reaching statistical significance for follicular and anaplastic thyroid carcinoma (p=0.0157 and 0.0045, respectively). Transfection experiments showed an inhibitory effect of CREM on NIS promoter activity in various thyroid cancer cell lines., Conclusions: These data demonstrate that CREM expression is increased in thyroid cancer tissue and may play a role in the downregulation of NIS expression in thyroid cancer acting at the transcriptional level.

Background: Novel molecularly targeted drugs are undergoing preclinical and clinical testing to assess their efficacy against refractory thyroid carcinomas. The multikinase inhibitor Sunitinib has been shown to inhibit the kinase activity of the RET oncogene and reduce proliferation in differentiated thyroid cancer cells harboring the RET/PTC rearrangement. In this study, we evaluated its effects in human cell lines derived from differentiated (TPC-1) and anaplastic (8505C, CAL-62, and C643) thyroid cancers., Methods: The cells exposed to various concentrations of Sunitinib were examined for: (1) cell viability and presence of apoptosis, analyzed by cell counts, MTT assay, trypan blue exclusion assay, western blotting, and immunofluorescence; (2) expression of cyclin D1 and phosphorylated and nonphosphorylated extracellular signal-regulated kinase (ERK) and Akt proteins, analyzed by western blotting; and (3) transcription of genes encoding thyrocyte differentiation markers (thyroid-stimulating hormone receptor, sodium/iodide symporter, thyroglobulin, and thyroperoxidase) and proangiogenic factors (vascular endothelial growth factor A, platelet-derived growth factors A and B), measured by quantitative reverse transcriptase-polymerase chain reaction., Results: Exposure to nanomolar concentrations of Sunitinib significantly reduced cell viability in only TPC-1 cells, and this effect was paralleled by reduction of cyclin D1 levels. Western blotting revealed reduced phosphorylation of ERK and Akt after 3 and 6 hours of drug exposure. In contrast, the growth of 8505C, CAL-62, and C-643 cells was significantly reduced only by micromolar concentrations of Sunitinib, mainly due to induced necrotic rather than apoptotic death. In these cells, Sunitinib exerted a few significant effects on the transcription of angiogenic factors or thyrocyte differentiation markers., Conclusions: Sunitinib has little or no effect on the growth or differentiation of anaplastic thyroid cancer cells, thus suggesting that it is unlikely to be effective in the treatment of anaplastic thyroid cancer.

Background: Thyroglobulin (Tg) assay of material from fine-needle aspiration of neck masses can help distinguish neck masses of thyroid origin from other masses. We describe its utility in a patient with an unusual constellation of findings, a neck lump identified as a lymph node metastasis from a contralateral occult papillary thyroid carcinoma (PTC)., Summary: A 56-year-old woman was referred to our center for evaluation of a 15-mm right lateral cervical neck mass which was strongly hypoechoic, not homogenous and contained several microcalcifications. There was no family history of thyroid disease, the patient was euthyroid and was not taking medications for thyroid disorders. On physical examination the thyroid was slightly enlarged and was normal on ultrasound except for a 1 x 3 mm hypoechoic nodule in the middle of the left lobe. Ultrasound-guided fine-needle aspiration biopsy (FNAB) of the right lateral cervical mass was performed with the Tg concentration of the FNAB washout liquid being >300 ng/mL and the cytology showing lymphoid elements mixed with polymorphous epithelial cells with atypical nuclei, suggesting lymph node metastasis from a cancer of epithelial origin. A lymph node metastasis from a papillary thyroid microcarcinoma (micro-PTC) was the presumptive diagnosis with the preoperative staging being Tx N1b. The patient underwent total thyroidectomy and bilateral lymph node dissection. At pathology, the right cervical mass was confirmed as lymph node metastasis of a PTC, and a unifocal micro-PTC was found in the middle left lobe. The patient was readmitted for a therapeutic (131)I dose (4810 MBq). At the time of (131)I administration, the whole-body scan showed only minimal thyroid bed uptake and serum Tg was <1 ng/mL. She was maintained on l-thyroxine treatment (150 microg/d). Five year later she did not have evidence of recurrent or residual PTC., Conclusions: We describe the first case of contralateral lymph node metastasis from a unifocal micro-PTC identified by the detection of high Tg levels in the wash-out liquid of FNAB.

Background: Prohibitin (PHB) is a multifunctional protein that is localized in different intracellular sites. PHB may exert different roles in tumorigenesis, having either a permissive action on tumor growth or an oncosuppressor role, depending on the cellular context. The objective of this study was to evaluate PHB expression in normal thyroid tissues, thyroid follicular adenomas (FAs), and papillary thyroid carcinomas (PTCs)., Methods: PHB expression was analyzed by immunohistochemistry, Western blot, and quantitative reverse transcription polymerase chain reaction (RT-PCR). Transfections in the BCPAP and TPC-1 thyroid cancer cell lines were used to evaluate the PHB promoter activity., Results: In terms of protein and mRNA levels, normal tissues from patients with serum thyrotropin (TSH) values >0.8mU/L had PHB levels that were significantly reduced compared to specimens from patients with serum TSH values <0.5mU/L, suggesting that TSH exerts an inhibitory effect on PHB expression. Consistent with this was the finding that the presence of TSH was associated with low PHB levels in normal FRTL5 thyroid cells. Immunohistochemical analysis showed relatively low and high PHB expression in FAs and PTCs, respectively. PHB mRNA and protein overexpression, as assessed by quantitative RT-PCR and Western blot, was noted only in PTCs bearing the BRAF(V600E) mutation. Notably, cell transfection experiments suggested that presence of the BRAF(V600E) mutation may be associated to increase of the PHB promoter activity., Conclusions: PHB is overexpressed in PTCs bearing the BRAF(V600E) mutation. We postulate that the presence of the BRAF(V600E) mutation increases PHB promoter activity and therefore potentially mediates effects of this mutation on the behavior of BRAF(V600E) positive PTCs.

Background: Serum thyroglobulin (Tg) stimulation by recombinant human TSH (rhTSH), in combination with neck ultrasonography (US), is an important tool in the first follow-up of differentiated epithelial cell thyroid carcinoma (DTC) patients. The objective of this study was to investigate if a second rhTSH stimulation, performed 2-3 years later, is of clinical utility in the follow-up of these patients., Methods: One hundred and one consecutive ambulatory DTC patients were studied. The great majority of them (89/101) were low-risk patients, being stage I or II at tumor node metastasis (TNM) staging classification. All study patients had been treated by surgery and radioiodine ablation, and exhibited, at first rhTSH follow-up, either undetectable Tg (1-5 ng/mL) (rhTSH1-Tg+, n = 12 patients considered with uncertain prognosis), with no US evidence of residual disease. In all patients, serum Tg measurement after a second rhTSH stimulation and neck US were performed., Results: At the second follow-up, all 89 rhTSH1-Tg-patients showed a negative US, and Tg became low positive only in one case, whereas it remained undetectable in the other patients. The overall negative predictive value of rhTSH1-Tg- was, then, 98.9%. Out of the remaining 12 patients (i.e., rhTSH1-Tg+ patients), 2 showed disease persistence/recurrence (with a positive predictive value of rhTSH1-Tg+ of 16.7%) and 6 became Tg-., Conclusions: A second rhTSH stimulation is useless in DTC patients who were rhTSH-Tg and imaging negative at first follow-up, while it is suggested in patients with detectable, although low, rhTSH-Tg levels at first follow-up: in the absence of clinical or US evidence of disease persistence, these patients should not be retreated by radioiodine, but simply scheduled for a later rhTSH stimulation.

Background: Hepatocyte nuclear factor (HNF)-1alpha and HNF-1beta are related transcription factors that are mainly expressed in liver cells. Our previous study showed that HNF-1beta was highly expressed in papillary thyroid cancer cell lines and tumors. HNF-1alpha mRNA, however, was not detected in differentiated thyroid cancer cell lines. The objective of this study was to determine whether HNF-1alpha is expressed in dedifferentiated anaplastic thyroid cancer cells., Methods: Total RNA isolated from six anaplastic thyroid cancer cell lines and 38 surgical samples was analyzed for HNF-1alpha mRNA by conventional reverse-transcription polymerase chain reaction (RT-PCR) or real-time RT-PCR. HNF-1alpha DNA binding activity was measured by gel retardation assay and HNF-1alpha protein was identified by Western blotting., Results: HNF-1alpha mRNA was expressed in four of the six anaplastic cell lines. The presence of HNF-1alpha protein and DNA binding activity was detected in three lines with higher HNF-1alpha mRNA level. Three cell lines also expressed HNF-1beta. HNF-1alpha transcripts were also detected in five out of six anaplastic tumors, but not in the papillary tumors except one with weak PCR signal., Conclusion: HNF-1alpha mRNA was detected in high frequency in anaplastic thyroid cancer cell lines and tumors. HNF-1alpha might play a role in the pathogenesis of anaplastic thyroid cancer.

Objective: Nonfunctioning thyroid nodules (NFTNs) display a diminished iodide-concentrating ability, owing to defective expression and cell membrane targeting of the sodium-iodide symporter (NIS). Since NIS expression is primarily modulated by thyroid iodine content in vitro and in animal models, we attempted to determine whether iodine supply influences the expression and localization of human NIS (hNIS) in NFTNs., Design: Using immunohistochemistry, we analyzed cold hyperplastic nodules and nonnodular thyroid samples (controls) from patients living in iodine-sufficient (n = 19) or severely iodine-deficient (n = 15) areas., Main Outcome: Nodules from the iodine-sufficient area exhibited weak or absent hNIS immunostaining whereas almost all nodules from the iodine-deficient area were hNIS positive. Heterogeneous hNIS staining was common among the iodine-deficient samples (p = 0.028). hNIS was localized on membrane in all nodular samples from the iodine-deficient area and in less than 40% in the iodine-sufficient area., Conclusions: hNIS is adequately expressed and appropriately localized in NFTNs cell membrane from iodine-deficient areas and its expression in vivo is modulated by iodine supply.

Context: Radioiodine uptake is rarely observed in normal non-lactating breast tissue. Investigation of the in vivo regulation of iodide uptake in breast tissue may be useful for the induction of radioiodine uptake in breast cancer tissue for diagnostic and therapeutic purposes., Case Reports: We report the cases of two post-menopausal women who underwent radioiodine therapy for papillary thyroid carcinoma and in whom breast uptake of radioiodine on post-therapy whole body scan (WBS) was observed., Methods and Results: In both patients, elevated serum prolactin levels (123 ng/mL in patient 1 and 48 ng/mL in patient 2) were documented at the time when radioiodine uptake in the breast was observed. The hyperprolactinemia was due to prolonged treatment with the anti-dopaminergic neuroleptic risperidone in Case 1, and chronic renal failure in Case 2. When prolactin levels were normalized (by withdrawal of risperidone in Case 1 and with cabergoline in Case 2), breast tissue uptake was no longer evident on WBS., Conclusion: These cases provide the first documented correlation between serum levels of endogenous prolactin and radioiodine uptake by involuted breast tissue in humans.

Immunohistochemistry provides insights in the expression of functional proteins and of their localization in normal thyroid tissue and in thyroid diseases. In hyperfunctional thyroid tissues, staining for sodium/iodide symporter (NIS), pendrin, thyroid peroxidase (TPO), and thyroglobulin (Tg) is increased. In hypofunctioning thyroid tissues, NIS staining is markedly decreased; in benign hypofunctioning adenomas, the expression of the other functional proteins is unmodified or slightly decreased, whereas their expression is profoundly decreased or absent in differentiated thyroid carcinoma.

Resistance to thyroid hormone (RTH) is an inherited disease characterized by reduced tissue sensitivity to thyroid hormone. Approximately 90% of subjects with RTH have mutation in the thyroid hormone receptor beta (TRbeta) gene. Approximately 10% of subjects diagnosed as having RTH do not carry mutation in the TRbeta gene. A possible linkage was reported with the retinoid X receptor-gamma (RXR-gamma) gene in two families. The aim of this study is to search for mutation within the RXR-gamma gene in unrelated subjects with diagnosed RTH without mutations in the TRbeta gene. Four subjects with RTH were studied, and sequence variants in the RXR-gamma gene were searched by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP). Analysis of all the 10 exons of the RXR-gamma gene, including intron-exon boundaries, promoter region and 3' untranslated region (UTR) reveled two variant bands in subjects II and III. Sequencing of these variants showed two single nucleotide polymorphisms (SNPs): 447C > T in exon 3 for patients II and IVS9 + 6A > G for patient III. Both SNPs were also present at high frequency in a group of normal subjects and in nonaffected relatives of subject III. In conclusion, in patients with RTH we have found two SNPs in the RXR-gamma gene; these SNPS are common in the general population, thus excluding a role for the RXR-gamma gene in these patients.

Ten years after the first description of activating mutations in the thyroid stimulating hormone receptor (TSHR) gene in sporadic autonomous hyperfunctioning thyroid adenomas, there is general agreement in assigning a major pathogenic role of this genetic abnormality, acting via the constitutive activation of the cAMP pathway, in both the growth and functional characteristic of these tumours. From the beginning, however, the pathophysiological and clinical relevance of somatic TSHR mutations has been debated and some arguments still exist against a fully causative role of these mutations and the practical value of detecting these mutations for the diagnosis, treatment and prognosis of thyroid hot nodules. Some major issues will be examined herein, including (a) the frequency of TSHR alterations in various reports showing that the genetic abnormality underlying the pathogenesis of a substantial subset of thyroid tumours has yet to be identified; (b) the limitations of the present experimental models, which suggest greater caution in the interpretation of in vitro results; (c) the still unresolved question of absence of genotype-phenotype correlation. Clarification of these issues may hopefully provide new and useful tools for improving the clinical management of this disease.

Radioactive iodine (131I) is routinely used for the treatment of differentiated thyroid cancers. Attempts have been made to enlarge this therapeutic strategy to nonthyroid tumors by coupling radioactive iodide administration with transfer of the sodium iodide symporter (NIS) gene into target cells, for example with an adenoviral vector (AdNIS). Although efficient iodide uptake was achieved in the tumors treated with AdNIS, no therapeutic effect could be observed with 131I, most probably because the iodide retention time in the target cells was short. To circumvent this problem, we propose to organify the iodide taken up, as it occurs in the thyroid. We constructed a recombinant adenovirus encoding the human thyroperoxidase (TPO) gene under the control of the cytomegalovirus early promoter (AdTPO). Infection of nonthyroid tumor cells with this virus led to production of an enzymatically active protein. A significant increase in iodide organification could be observed in cells coinfected with both AdNIS and AdTPO in the presence of exogenous hydrogen peroxide. However, the levels of iodide organification obtained were too low to significantly increase the iodide retention time in the target cells.

Duox2, and probably Duox1 are glycoflavoproteins involved in the thyroid H2O2 generator functionally associated to thyroperoxidase (TPO). We investigated both DUOX1 and DUOX2 gene expressions using quantitative reverse transcription-polymerase chain reaction (RT-PCR) in 47 thyroid carcinomas, including 10 paired normal/tumoral tissues. In carcinomas, variations of DUOX1 and DUOX2 mRNA levels were parallel, indicating that control mechanisms of both gene expressions operate in tumors as well as in normal thyroid tissues; DUOX1 expression was in the normal range in 20, was decreased up to 50-fold in 8, and increased up to 7-fold in 19 samples. DUOX2 expression was in the normal range in 15, was decreased up to 200-fold in 10, and increased up to 5-fold in 22 samples. In the 10 paired samples, variations of DUOX and TPO gene expressions were not correlated. We analyzed Duoxl/2 protein expression in 86 tumor samples using an antipeptide antiserum reacting with both Duox proteins. In normal tissue, Duox proteins are localized at the apical pole of thyrocytes, with 40% to 60% of thyrocytes being stained. In the 86 cancer tissues, immunostaining was absent in 19 samples, was low in 32, and normal or even slightly increased in the other 35 samples. The expression of Duox proteins was related to tumor differentiation, being more frequently found in neoplastic tissues that were able to pick up radioiodine, and in those with a detectable expression of sodium iodide symporter (NIS), pendrin and TPO.

Decrease or loss of the sodium iodide (Na+/I-) symporter (NIS) activity influences the suitability of using radioiodine to detect and treat metastatic thyroid tissues. In previous studies, the presence of the NIS transcript, albeit at lower expression levels, has been shown in most thyroid differentiated carcinomas. In this study we searched for point mutations or other genetic alterations that may be responsible for an altered function of the NIS protein in tumors that still express NIS transcripts. Tumoral cDNAs derived from seven differentiated thyroid carcinomas (DTC), five papillary and two follicular, were analyzed by direct sequencing after polymerase chain reaction (PCR) amplification of the structural gene of the Na+/I- symporter. Neither mutations nor other genetic abnormalities were detected in any tumor sample examined. The data indicate that mutations or other genetic alterations of the NIS structural gene are not a major cause of the reduced iodide uptake in DTC.

Hot nodules are rarely found to be carcinomas. We report a case of a nonmetastatic follicular carcinoma that presented as a hot nodule that was causing hyperthyroidism. A base substitution (ATC for TTC) was found in codon 486 of the TSH receptor gene and this resulted in the substitution of an isoleucine for a phenylalanine in the first extracellular loop of the receptor. This was absent in the deoxyribonucleic acid from the surrounding normal thyroid tissue indicating its somatic origin. This mutation, which was previously reported to activate both cyclic adenosine monophosphate and the inositol phosphate-diacylglycerol cascades, may have been responsible for the constitutive activation of the thyrotropin receptor and resulting hyperfunction of this follicular carcinoma.

Thyroid nodules presenting as hot at 131I-scintigraphy are usually benign follicular adenomas. We report a 42-year-old female patient with an autonomously functioning Hürthle cell thyroid carcinoma causing thyrotoxicosis. Genetic analysis of her thyroid tumoral DNA revealed a heterozygotic activating mutation of the thyrotropin receptor (TSHR) gene that was located downstream to all of the other genetic alterations currently identified, and is due to a base substitution at codon 677 (normal cytosine replaced by guanine, CTG for GTG causing leucine substitution by valine in the seventh transmembrane domain of the receptor). This mutation was detected in the tumor, but not in the leucocytes from the same patient. The Val 677-TSHR mutant showed constitutive activity, in terms of cyclic adenosine monophosphate (cAMP) production, when permanently transfected in Chinese hamster ovary (CHO) cells. Gsp and ras oncogenes and the p53 tumor suppressor gene were not present in the Hürthle cell cancer. The TSHR mutation in this Hürthle cell carcinoma may be responsible for maintaining differentiated thyroid function and hyperthyroidism.