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Background: Risk stratification in patients with papillary thyroid carcinoma (PTC) currently relies on postoperative parameters. Testing for BRAF mutations preoperatively may serve as a novel tool for identifying PTC patients at risk of persistence/recurrence after surgery., Methods: The study involved 185 consecutive patients with a histological diagnosis of PTC and BRAF analysis performed on thyroid fine-needle aspiration biopsy (FNAB). We assessed BRAF status in FNAB specimens obtained before thyroidectomy for PTC, and examined its association with the clinicopathological characteristics identified postoperatively, and with outcome after a mean 55±15 months of follow-up., Results: One hundred and fifteen of 185 (62%) PTCs carried a BRAF mutation. Univariate analysis showed that BRAF status correlated with the histological variant of PTC, cancer size, and stage at diagnosis, but not with gender, age, multifocality, or lymph node involvement. BRAF-mutated cases had a higher prevalence of persistent/recurrent disease by the end of the follow-up (11% vs. 8%), but this difference was not statistically significant. The Kaplan-Meier curve shows that among the patients with persistent/recurrent disease, BRAF-mutated patients needed a second treatment earlier than patients with BRAF wild-type, although the difference did not completely reach the statistical significance., Conclusions: Our study confirmed that preoperatively-identified BRAF mutation are associated with certain pathological features of PTC that correlate with prognosis. We speculate that it has a role in identifying PTCs that would generally be considered low-risk but that may reveal an aggressive behavior during their follow-up.

The three members of the Aurora kinase family, Aurora-A, -B and -C, regulate several aspects of the mitotic process, and their aberrant expression and/or function causes mitotic abnormalities leading either to cell death or aneuploidy. They are found overexpressed in several human malignancies, including the papillary thyroid carcinoma (PTC). In the present study, we sought to establish whether Aurora kinase inhibition could be of any therapeutic value in the treatment of aggressive forms of PTC, enduring to radioactive iodide (RAI) ablation. To this end, the effects of selective inhibitors of Aurora-A (MLN8237) and Aurora-B (AZD1152) were analyzed on 3 human PTC cell lines expressing either wild-type (K1 and TPC1) or mutant p53 (BCPAP). The two inhibitors were capable of reducing cell proliferation in a time- and dose-dependent manner, with IC₅₀ comprised between 65.4 and 114.9 nM for MLN8237, and between 26.6 and 484.6 nM for AZD1152. Immunofluorescence experiments confirmed that AZD1152 inhibited Aurora-B phosphorylation of histone H3 on Ser10, however, it did not affect Aurora-A autophosphorylation. MLN8237 inhibited Aurora-A autophosphorylation as expected, but at concentrations required to achieve the maximum antiproliferative effects it also abolished H3 (Ser10) phosphorylation. Time-lapse videomicroscopy evidenced that both inhibitors prevented the completion of cytokinesis, and cytofluorimetric analysis showed accumulation of cells in G2/M phase and/or polyploidy. Apoptosis was induced in all the cells by both inhibitors independently from the p53 status. In conclusion, in the present preclinical study MLN8237 and AZD1152 have emerged as promising drug candidates for RAI-insensitive PTC.

Small Ubiquitin–like MOdifier (SUMO) proteins are small protein modifiers capable of regulating cellular localization and function of target proteins. Over the last few years, a relevant role has been demonstrated for sumoylation in the modulation of important cellular processes, including gene transcription, DNA repair, cell-cycle regulation and apoptosis. Components of the sumoylation machinery have been found deregulated in different human cancers, and are thought to significantly affect cancer cell progression. In the present study we sought to analyze the expression of all the components of the sumoylation machinery in a case study comprising 77 papillary thyroid cancers (PTC) and normal matched tissues. In particular, we evaluated the expression of the SENP1 to SENP8 (SENtrin-specific proteases), SAE1 (SUMO1 activating enzyme subunit 1), UBA2 (UBiquitin-like modifier activating enzyme 2), UBC9 (UBiquitin conjugating enzyme 9), RanBP2 (RAN binding protein 2), MSMCE2 (Non- SMC element 2), CBX4 (ChromoBoX homolog 4), PIAS1 to PIAS4 (protein inhibitor of activated STAT), ZMIZ1 (zinc finger, MIZ-type containing 1) and ZMIZ2 (Zinc finger, MIZ-type containing 2) by means of quantitative RT-PCR. In most of the PTC examined we observed a significant alteration in the mRNAs of SENP8, ZMIZ1, SAE1, PIAS1 and PIAS2. These tended to be reduced in about 50 to 66% of cases, and unchanged or increased in the remaining ones. Univariate and Kaplan-Mayer analyses documented the lack of association between the expression of the above 5 genes and clinicopathological parameters. Only SAE1 was significantly higher in female PTC tissues, in respect to male PTC tissues (p=0.021), and SENP8 was significantly lower in TNM stages III-V, with respect to stages I-II (p=0.047). In conclusion, we demonstrated that the expression of SENP8, SAE1, PIAS1, PIAS2 and ZMIZ1 is deregulated in the majority of PTC tissues, likely contributing to the PTC phenotype. However, differently from other human cancers, their mRNA level does not represent a prognostic biomarker in PTC patients.

Aim: Acromegaly reportedly carries an increased risk of malignant and benign thyroid tumors, with a prevalence of thyroid cancer of around 3-7%. Germline mutations in the aryl-hydrocarbon receptor (AHR) interacting protein (AIP) have been identified in familial forms of acromegaly. The molecular and endocrine relationships between follicular thyroid growth and GH-secreting pituitary adenoma have yet to be fully established. Our aim was to study the prevalence of differentiated thyroid cancer (DTC) in acromegaly, focusing on the role of genetic events responsible for the onset of thyroid cancer., Methods: Germline mutations in the AIP gene were assessed in all patients; BRAF and H-N-K RAS status was analyzed by direct sequencing in thyroid specimens, while immunohistochemistry was used to analyze the protein expression of AIP and AHR. A set of PTCs unrelated to acromegaly was also studied., Results: 12 DTCs (10 papillary and 2 follicular carcinomas) were identified in a cohort of 113 acromegalic patients. No differences in GH/IGF-1 levels or disease activity emerged between patients with and without DTC, but the former were older and more often female. BRAF V600E was found in 70% of the papillary thyroid cancers; there were no RAS mutations. AIP protein expression was similar in neoplastic and normal cells, while AHR protein was expressed more in PTCs carrying BRAF mutations than in normal tissue, irrespective of acromegaly status., Conclusions: The prevalence of DTC in acromegaly is around 11% and endocrinologists should bear this in mind, especially when examining elderly female patients with uninodular goiter. The DTC risk does not seem to correlate with GH/IGF-1 levels, while it may be associated with BRAF mutations and AHR over-expression. Genetic or epigenetic events probably play a part in promoting thyroid carcinoma.

Background: BRAF(V600E) mutation, which represents the most frequent genetic mutation in papillary thyroid carcinoma (PTC), is widely considered to have an adverse outcome on PTC outcome, however its real predictive value is not still well stated. The aim of the present study was to evaluate if BRAF(V600E) mutation could be useful to identify within patients with intrathyroid ultrasound-N0 PTC those who require more aggressive treatment, by central neck node dissection (CLND) or subsequent postoperative (131)I treatment., Methods: Among the whole series of 931 consecutive PTC patients operated on at 2nd Clinical Surgery of University of Padova and at General Surgery Department of University of Trieste during a period from January 2007 to December 2012, we selected 226 patients with an intrathyroid tumor and no metastases (preoperative staging T1-T2, N0, M0). BRAF(V600E) mutation was evaluated by PCR-single-strand conformation polymorphism analysis and direct genomic sequencing. We analyzed the correlation between the presence/absence of the BRAF(V600E) mutation in the fine-needle aspiration (FNA) and the clinical-pathological features: age, gender, extension of surgery, node dissection, rate of cervical lymph node involvement, tumor size, TNM stage, variant of histotype, mono/plurifocality, association with lymphocitary chronic thyroiditis, radioactive iodine ablation doses, and outcome., Results: The BRAF(V600E) mutation was present in 104 of 226 PTC patients (47.8%). BRAF(V600E) mutation correlated with multifocality, more aggressive variants, infiltration of the tumoral capsule, and greater tumor's diameter. BRAF(V600E) mutation was the only poor prognostic factor in these patients., Discussion: In our series, BRAF(V600E) mutation demonstrated to be an adverse prognostic factor indicating aggressiveness of disease and it could be useful in the management of low-risk PTC patients, as supplementary prognostic factor to assess the preoperative risk stratification with the aim to avoid unnecessary central neck node dissection (BRAF pos.) or to perform complementary (131)I-therapy (BFAF neg.)., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Background: Papillary thyroid carcinoma (PTC) is the most common malignant tumor of the thyroid gland, accounting for 74-80% of all thyroid cancers. The 1799T>A transversion is an activating mutation of the BRAF oncogene that is common in and specific to conventional PTC. We studied the prevalence, tumorigenic role, and biochemical implications of rare BRAF variants in a large cohort of patients., Methods: A total of 2131 fine-needle aspiration biopsy samples were collected and subjected to BRAF mutation analysis. BRAF genetic variants were analyzed by Western blot, immunofluorescence, and in silico analysis., Results: BRAF mutations were found in 50% (347/700) of thyroid cancers (644 PTCs, 22 anaplastic thyroid carcinomas, 34 follicular thyroid carcinomas). They were the classic (c.1799T>A, p.V600E) mutation in 96.8% (336/347) and rare genetic variants in 3.2% (11/347). In all, five infrequent BRAF alterations were detected: (i) c.1795_1797dupACA (p.T599dup); (ii) c.1801A>G (p.K601E); (iii) c.1799_1801delTGA (p.V600_K601>E); (iv) c.1799_1814>A (p.V600_S605>D); and (v) c.1798_1810delinsA (p.V600_W604>R). The last BRAF variant has never been described in the literature. Western blot analysis and immunofluorescence both revealed a variegated reactivity pattern, again emphasizing the peculiar role of every specific BRAF genetic alteration. In silico analysis of the samples studied revealed a stabilization of the "active" geometrical conformation of the B-raf enzyme associated with the activated and productive state of the kinase domain., Conclusions: Rare BRAF variants were found in 1.6% of all thyroid malignancies, all clustered around the codon V600, in the binding pocket named A-loop, confirming its crucial role in the enzymatic activation of the B-Raf protein. These mutations were associated mainly with the activation of key effectors in the mitogen-activated protein kinase pathway, but a simultaneous stimulation of the PI3k/Akt cascade was demonstrated in some cases. The rare BRAF variants were not generally associated with an aggressive behavior of the PTC. To our knowledge, this is the largest series of thyroid cancers analyzed to identify and functionally characterize rare BRAF variants.

Thyroid cancer is not very common, accounting for 1-2% of all cancers, with a population incidence of about 0.004%. Currently, the ability to discriminate between follicular adenoma and carcinoma represents the major challenge in preclinical diagnosis of thyroid proliferative lesions. Better discrimination between the two would help avoid unnecessary thyroidectomy and save valuable resources. Over the years, galectin-3 (Gal-3) has been proposed as a diagnostic marker with varied success. In this paper, we used Environmental Scanning Electron Microscopy Immunogold Labelling (ESEM-IGL) to investigate the expression of Gal-3 on Thin-Prep fine needle aspiration cytology (FNAC). We optimized the ESEM-IGL method on thyroid cell lines (RO-82 and FTC-133) comparing our membrane Gal-3 labeling data with Western blot. We evaluated 183 thyroid FNAC from Italian patients with a uncertain pre-surgical diagnosis. ESEM-IGL method marker sensitivity is 71.2%, while specificity is 53.3% and diagnostic efficacy is 61.2%. Our results confirmed that Gal-3 expression is associated with situations of hypertrophy and/or cellular hyperproliferation, pathophysiological situations common both to adenomas and to thyroid carcinomas. The innovation of thyroid FNAC Thin-Prep ESEM-IGL shows the levels of Gal-3 immunolabeling clearly, even through the individual cells of a thyroid nodule. However, Gal-3 alone, as a molecular marker of thyroid cancer, can still have a limited application in pre-surgery diagnosis., (Copyright © 2012 Wiley Periodicals, Inc.)

Context: It has been suggested that patients with papillary thyroid cancer (PTC) harbouring the BRAF(V600E) mutation have a worse prognosis. We showed in PTC that high levels of urokinase plasminogen activator (uPA) and its cognate receptor (uPAR) inversely correlate with disease-free interval (DFI)., Objectives: To investigate the effects of BRAF(V600E) on the expression of uPA and uPAR and to evaluate the prognostic relevance of BRAF(V600E) alone or in combination with uPA and uPAR. DESIGN/SETTING/PATIENTS/INTERVENTION: The case study included 91 patients with PTC. All patients underwent thyroidectomy and radioiodine therapy. Follow-up was available for 75 patients., Main Outcome Measures: The BRAF(V600E) mutation was analysed by sequencing and mutant allele-specific PCR amplification; uPA and uPAR expression by quantitative RT-PCR., Results: BRAF(V600E) was found in 44 of the 91 patients and associated with older age, but not with high-risk clinicopathological features. Urokinase PA and uPAR mRNA levels were higher in tumour tissues by 9·51 ± 1·30 and 4·64 ± 0·44 fold, respectively, compared to normal matched tissues, being significantly higher in BRAF(V600E) -positive patients. In vitro induction of BRAF(V600E) in PCCL3 cells caused a significant increase in both uPA and uPAR mRNAs. Higher levels of uPA and uPAR correlated with lymph node metastases, TNM stage and disease recurrences. Kaplan-Meier and multivariate analyses demonstrated that uPA and uPAR were associated with shorter DFI, while the BRAF(V600E) was not., Conclusion: In PTC, BRAF(V600E) induces uPA and uPAR expression. The latter, but not BRAF(V600E) , associates with advanced stages and shorter DFI. If confirmed in larger case studies, they may represent reliable prognostic markers for more accurate risk stratification and postoperative decision-making in patients with PTC., (© 2012 Blackwell Publishing Ltd.)