Thyroid nodules represent a very common pathology. Using modern high-resolution ultrasound, nodules could be found in up to 68 % of patients. The most important task is the diagnosis of thyroid cancer which represents only about 5-15 % of nodules, however the incidence is still growing. Even with the use of a fine needle aspiration biopsy, it is not always possible to decide on the biological nature of the nodule. A significant proportion of such patients have to undergo thyroid surgery for diagnostic reason. Thyroid surgery is associated with risks to the patient and financial costs to the health-care system. In recent decades, the efforts to improve non-invasive diagnostics of thyroid nodules have been made. The thyroid elastography and thyroid autoimmunity are among the examined risk parameters. Using real-time strain elastography, thyroid carcinomas elasticity has been significantly reduced compared to benign thyroid nodules in our group of patients. The elastography of thyroid nodules can be used as a suitable complement to conventional sonographic examination. In our work, the combination of both methods (conventional ultrasound and elastography) increased the negative predictive value compared to both methods individually. The results of our work further indicate that, in case of absence of...

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Θεωρητικό υπόβαθρο: Το οξειδωτικό στρες (ΟΣ) ορίζεται ως το παθολογικό αποτέλεσμα που προκύπτει από τη διαταραχή της ισορροπίας των κυτταρικών συγκεντρώσεων των οξειδωτικών, δραστικών ενώσεων και των αντιοξειδωτικών μορίων. Εκτός από τη βλάβη που υπόκεινται οι πρωτεΐνες και τα λιπίδια, το ΟΣ μπορεί επίσης να προκαλέσει μεταλλάξεις και επιγενετικές μεταβολές καταστρέφοντας τόσο το DNA όσο και τις πρωτεΐνες που τροποποιούν τη χρωματίνη. Παρ' όλα αυτά, στα θυρεοειδικά θυλακικά κύτταρα παράγονται σε καθημερινή βάση υψηλές ποσότητες υπεροξειδίου του υδρογόνου (H2O2), οξειδωτικής ουσίας απαραίτητης για την πραγματοποίηση της θυρεοειδικής ορμονογένεσης. Δεδομένου ότι ένα ελάχιστο ποσό οξειδωτικού φορτίου αποτελεί προϋπόθεση αφ’ενός για τη φυσιολογική λειτουργία των θυλακικών κυττάρων και αφ’ετέρου για την ανάπτυξη του θυρεοειδούς αδένα, πρόσφατα αποδείχτηκε ότι ο θυρεοειδής αδένας παρουσιάζει αυξημένη αμυντική ανταπόκριση έναντι του ΟΣ. Ωστόσο, οι ακριβείς μηχανισμοί με τους οποίους τα θυλακικά κύτταρα αντιλαμβάνονται και απαντούν στο ΟΣ παραμένουν ασαφείς. Ο NFE2-related factor 2 (Nrf2), ο οποίος κωδικοποιείται από το γονίδιο NFE2L2, είναι ένας μεταγραφικός παράγοντας ο οποίος απαντά σε σήματα κυτταρικού στρες και ανταποκρίνεται επιδρώντας στη μεταγραφή γονιδίων σε διάφορους τύπους ιστών. Σε βασικές συνθήκες, ο Nrf2 οδηγείται σε πρωτεασωματική αποικοδόμηση μέσω του κυτταροπλασματικού του αναστολέα, Keap1, ενώ σε συνθήκες ΟΣ, η αποικοδόμηση του Nrf2 δεν είναι δυνατή και ο Nrf2 εισέρχεται στον πυρήνα ώστε να ενεργοποιήσει τη μεταγραφή αντιοξειδωτικών γονιδίων όπως του γονιδίου Nqo1. Καθώς η οξειδοαναγωγική ομοιοστασία κατέχει κεντρικό ρόλο στην φυσιολογία του θυρεοειδούς αδένα και ο Nrf2 πρόσφατα χαρακτηρίσθηκε ως μεσολαβητής στην αντίσταση θυρεοειδικών καρκινικών κυτταρικών σειρών σε πρωτεασωμικούς αναστολείς, το αντιοξειδωτικό μονοπάτι Nrf2 μπορεί να θεωρηθεί ως εξαιρετικός υποψήφιος της διαμεσολάβησης της απόκρισης του θυρεοειδούς αδένα στο ΟΣ. Παρ 'όλα αυτά, ο ρόλος του μονοπατιού Nrf2 στον ανθρώπινο θυρεοειδικό καρκίνο παραμένει άγνωστος. Στόχος: Στόχοι της παρούσας μελέτης ήταν η εκτίμηση της δραστηριότητας του μονοπατιού Νrf2 στο διαφοροποιημένο καρκίνωμα του θυρεοειδούς αδένα και η διερεύνηση σωματικών μεταλλάξεων των γονιδίων NFE2L2 και Keap1. Yλικά και Μέθοδοι Ασθενείς: Στη μελέτη συμμετείχαν 90 περιστατικά εκ των οποίων τα 42 αφορούσαν θηλώδη καρκινώματα (papillary thyroid carcinomas, PTCs), τα 6 θυλακιώδη καρκινώματα (follicular thyroid carcinomas, FTCs) και τα υπόλοιπα 42 καλοήθεις όγκους (24 αδενώματα και 18 οζώδης υπερπλασία). Κυτταρικές σειρές: Στα πλαίσια της παρούσας μελέτης χρησιμοποιήθηκαν κυτταρικές σειρές PTC (K1, TPC-1, XTC-1), κυτταρική σειρά φτωχά διαφοροποιημένου PTC (T243), κυτταρικές σειρές αδιαφοροποίητου καρκινώματος (C643, 8505C, Hth74) και τέλος κυτταρικές σειρές αναπλαστικού καρκινώματος (T235 , T241, T238). Μέθοδοι: Αναδρομική ανοσοϊστοχημική ανάλυση δειγμάτων PTC και FTC, παρακείμενου φυσιολογικού ιστού και καλοηθών βλαβών. Ανάλυση αλληλουχίας DNA των κυτταρικών σειρών και PTC δειγμάτων. Κύριες μετρήσεις και υπολογισμοί: Αξιολογήθηκε η ένταση της ανοσοαντίδρασης των δειγμάτων των ιστών σε αντισώματα για τα Nrf2, Nqo1, Keap1 και 4-HNE. Μελετήθηκε η αλληλουχία του εξονίου 2 του γονιδίου NFE2L2 καθώς και του γονιδίου Keap1. Αποτελέσματα: O μεταγραφικός παράγοντας Nrf2 καθώς και ο στόχος του, η πρωτεΐνη Nqo1 ήταν μη ανιχνεύσιμα σε φυσιολογικό ιστό θυρεοειδούς αδένα. Τα επίπεδά τους ήταν σημαντικά υψηλότερα στα PTC δείγματα από ό,τι στα δείγματα καλοηθών βλαβών. Η έκφραση του Keap1 εμφάνισε διακύμανση στα δείγματα PTC με τα επίπεδά του να μην εμφανίζουν συσχέτιση με τα αντίστοιχα του Nrf2, ενάντια στη θεωρία πως τα μειωμένα επίπεδα του Κeap1 συνιστούν μηχανισμό ενεργοποίησης του Nrf2. Ο δείκτης ΟΣ, 4-HNE βρέθηκε αυξημένος στη πλειοψηφία των δειγμάτων PTC σε σχέση με το φυσιολογικό ιστό αναδεικνύοντας την ύπαρξη αυξημένου ΟΣ στο PTC. Επιπλέον, όσον αφορά τα δείγματα FTC, ο μεταγραφικός παράγοντας Nrf2 και η πρωτεΐνη Nqo1 ήταν ανιχνεύσιμα σε όλα τα δείγματα, ενώ τα επίπεδα του 4-ΗΝΕ ήταν αυξημένα. Όσον αφορά την ανάλυση αλληλουχίας DNA στις καρκινικές σειρές και σε 11 δείγματα PTC με υψηλή έκφραση Nrf2, καμία μετάλλαξη δεν ανευρέθηκε στο εξόνιο 2 του γονιδίου NFE2L2 και στο γονίδιο Keap1. Συμπεράσματα: Τα αποτελέσματα της μελέτης μας σε συνδυασμό με περαιτέρω μελέτες από το εργαστήριο Ενδοκρινολογίας και Ανατομικής του Πανεπιστημίου Πατρών καθώς και από το BC κέντρο έρευνας καρκίνου (Vancouver, Canada) αποδεικνύουν ότι το μονοπάτι Nrf2 ενεργοποιείται σε PTC και κατέχει ρυθμιστικό ρόλο στην αντιοξειδωτική απόκριση και τη βιωσιμότητα των θυρεοειδικών καρκινικών κυττάρων. Συνεπώς, αναδεικνύεται το Nrf2 μονοπάτι ως νέο “σήμα κατατεθέν” του PTC. Παρά το γεγονός ότι δεν ήταν δυνατή η πραγματοποίηση στατιστικών συσχετίσεων στη μελέτη του FTC λόγω του περιορισμένου αριθμού δειγμάτων, το μονοπάτι Nrf2 φαίνεται να ενεργοποιείται επίσης στο FTC. Η σταθερή ενεργοποίηση του Nrf2 στο PTC και ενδεχομένως στο FTC δίνει το έναυσμα για περαιτέρω διερεύνηση του μονοπατιού αυτού σε όλα τα είδη θυρεοειδικού καρκίνου καθώς και της πιθανής διαγνωστικής, προγνωστικής, και/ή θεραπευτικής χρησιμότητας του μονοπατιού στο διαφοροποιημένο καρκίνο του θυρεοειδούς αδένα. / Scientific background: Oxidative stress (ΟS) is experienced by cells when pro-oxidant and electrophilic reactive species overwhelm the cell’s antioxidant and detoxification proteins. In addition to causing protein and lipid damage, oxidative stress can cause mutations and epigenetic perturbation by damaging DNA and proteins that modify chromatin. Nevertheless, in thyrocytes a daily basis high amounts of the oxidant hydrogen hyperoxide (H2O2) was generated due to the fact that H2O2 is a reactive oxygen species required for thyroid hormonogenesis. A minimal oxidative load is a prerequisite for normal thyroid cell function and development and it was recently shown that the thyroid has increased capacity for defending itself against OS. However, precise mechanisms by which thyrocytes sense and respond to OS remain obscure. NFE2-related factor 2 (Nrf2), encoded by NFE2L2 gene, is a transcription factor that integrates cellular stress signals and responds by directing transcriptional program in various tissues. In basal conditions, Nrf2 is targeted for proteasomal degradation by its cytoplasmic inhibitor, Kelch-like ECH-associated protein 1 (Keap1), while in oxidative stress Nrf2 degradation is abolished and Nrf2 accumulates in the nucleus where it transactivates protective genes such as NAD(P)H dehydrogonase quinone 1 (Nqo1). As redox homeostasis plays a principal role in thyroid gland’s physiology, and Nrf2 has recently been characterised as mediator of thyroid cancer cell lines’ resistance to proteasome inhibitors, the Nrf2 antioxidant pathway seems to be an excellent candidate for mediating the antioxidant response of the thyroid gland. Nevertheless, the activity status of the Nrf2 pathway in human thyroid cancer remains unknown. Objective: The aims of this study were to assess the activity status of the Nrf2 pathway in differentiated thyroid carcinoma and investigate somatic mutations in NFE2L2 and Keap1 genes. Μethods and Materials Patients: The study included 90 individual samples; 42 papillarz thyroid carcinomas (PTCs), 6 follicular thyroid carcinomas (FTCs) and 42 benign lesions (24 adenomas and 18 nodular hyperplasias). Cell lines: Ten thyroid cell lines are used for this study: The PTC cell lines, K1, TPC-1 and XTC-1; the poorly differentiated PTC cell line, T243; the undifferentiated carcinoma cell lines, C643, 8505C and Hth74; and the anaplastic carcinoma cell lines, T235, T241 and T238. Methods: We conducted retrospective immunohistochemical analyses of PTC and FTC specimens, adjacent normal tissue, and benign lesions; DNA sequencing in cell lines and PTC samples. Main Outcome Measures: We assessed the abundance of Nrf2, Nqo1, Keap1, and 4HNE; and the sequence of NFE2L2 gene’s exon 2 and of KEAP1 gene. Results: Nrf2 and its target Nqo1 were undetectable in normal tissue; their levels were significantly higher in PTC than in benign lesions. The Nrf2 inhibitor, Keap1 was variably abundant in PTC, and its levels did not correlate with Nrf2, arguing against decreased levels as the mechanism for Nrf2 activation. The oxidized lipid 4HNE was more abundant in PTC than normal tissue indicating oxidative stress. In addition, as far as FTC samples are concerned, Nrf2 and Nqo1 were detectable in all samples as well as the levels of 4-HNE were significantly high. No mutations were detectable in exon 2 of NFE2L2 gene and in Keap1 gene. Conclusions: Our study’s results supported by further studies in laboratories of Endocrinology and Anatomy at University of Patras and BC Cancer Research Center (Vancouver, Canada) demonstrate that the Nrf2 pathway is commonly activated in PTC and that it regulates antioxidant responses and viability of cancer cells. Thus, Nrf2 is highlighted as a new hallmark of PTC. Although, statistic correlations were not possible in FTC samples’ study because of small sample size, the Nrf2 pathway seems to be also activated in FTC. The high activity of Nrf2 in PTC and possibly in FTC warrants further exploration of this pathway’s potential diagnostic, prognostic, and/or therapeutic utility in differentiated thyroid carcinoma.

Doenças tireoideanas são bastante comuns, sendo sua maioria benigna. A relação entre os diversos tipos de doenças tireoideanas, bem como seus aspectos moleculares, são pouco conhecidos. O bócio (hiperplasia), por exemplo, é descrito por alguns como relacionado com carcinoma (tumor maligno) papilífero, enquanto que outros afirmam não haver relação causal entre as duas doenças. A questão mais desafiante, porém, refere-se à distinção entre adenoma (tumor benigno) e carcinoma folicular, que atualmente é feita apenas após à cirurgia, não permitindo tratamento diferenciado para os dois tipos de tumor. Este trabalho buscou identificar genes diferencialmente expressos entre tecido tireoideano normal, bócio, adenoma e carcinoma papilífero utilizando microarrays. Carcinomas foliculares não foram incluídos devido ao número e tamanho reduzidos das amostras. Dois tipos de array foram utilizados: arrays em membranas de nylon, contendo 213 clones obtidos por DDRT-PCR de amostras de tireóide, e arrays em vidro, contendo 3800 clones ORESTES. Experimentos utilizando o primeiro tipo de array identificaram três genes diferencialmente expressos, cuja expressão foi analisada por RT-PCR em 10 amostras de cada tipo de tecido. Dois deles foram capazes de diferenciar carcinomas papilíferos de tecido normal e bócio com 89% de precisão para o tumor maligno e 80% para os tecidos não malignos. Os arrays em vidro foram utilizados para avaliar o perfil de expressão de aproximadamente 10 amostras de cada tipo de tecido tireoideano. Foram identificados 160 clones diferencialmente expressos entre quaisquer dois tipos de tecido, cujas seqüências foram determinadas e comparadas com as dos bancos de dados. Dentre os genes mais interessantes destacam-se o correspondente à ATPase Na/K, cuja expressão está reduzida nos carcinomas em relação a tecidos normais e adenomas, o da proteína PDCD4, envolvida em morte celular programada, mais expresso em adenomas e tecidos normais em comparação com carcinomas e bócios, e os da calgizzarin (S100A11) e da α1-anti-tripsina, ambos mais ativos nos carcinomas do que nos demais tecidos. Todos esses genes já foram descritos como diferencialmente expressos em algum tipo de tumor. Este trabalho levou à padronização da metodologia de microarray em lâminas de vidro em nosso laboratório, bem como à identificação de genes que podem elucidar as alterações envolvidas na formação do bócio, adenoma e carcinoma papilífero. A implantação da técnica de amplificação de mRNA em nosso laboratório viabilizou a utilização de 10 amostras de carcinoma folicular, cuja massa de RNA total era insuficiente para as hibridizações. Essas amostras serão hibridizadas, juntamente com 10 amostras de adenoma, com microarrays contendo 4800 genes humanos conhecidos para a busca de genes diferencialmente expressos, de grande interesse diagnóstico. / Thyroid diseases are very common and are usually benign. The causal relationships among the different types of disease, as well as their molecular aspects, are not well understood. The goiter (hyperplasia), for instance, is described by some as related to papillary carcinoma (a malignant tumor), while others say there is no causal relationship between the two diseases. The most defying question, however, concerns the distinction between adenoma (benign tumor) and follicular carcinoma, which is currently made only after surgery, not allowing distinct treatments for the two kinds of tumor. This work aimed to identify differentially expressed genes among normal thyroid tissue, goiter, adenoma and papillary carcinoma using microarrays. Follicular carcinomas were not included due to the reduced number and size of the samples. Two kinds of array were used: arrays in nylon membranes, with 213 clones isolated from thyroid samples by differential display (DDRT-PCR); and glass slide arrays containing 3800 ORESTES clones.Experiments using the first type of array identified three differentially expressed genes, whose expression was analyzed by RT-PCR in 10 samples of each kind of tissue. Two of these genes were able to differentiate papillary carcinomas from goiters and normal tissues with precisions of 89% for the malignant tumor and 80% for the non-malignant tissues. Glass slide arrays were used to evaluate gene expression profile of approximately 10 samples of each type of thyroid tissue. 160 clones differentially expressed between any two tissues were identified, and their sequences were determined and compared with databases. Among the most interesting genes are Na/K ATPase gene, whose expression is reduced in carcinomas compared to normal tissues and adenomas, the gene corresponding to PDCD4 protein, involved in program cell death, with elevated expression in adenomas and normal tissues than carcinomas and goiters, and the genes of calgizzarin (S100A11) and α1-antitrypsin, both more active in carcinomas than the other tissues. All these genes have already been described as differentially expressed in at least one type of human cancer. This work led to the standardization of glass slide microarray technology in our laboratory, and to the identification of genes that may clarify the alterations involved in the formation of goiter, adenoma and follicular carcinoma. The implementation of mRNA amplification technique in our laboratory allowed the utilization of 10 samples of follicular carcinoma, whose mass was insufficient for microarray hybridizations. These samples will be hybridized along with 10 samples of adenomas, with microarrays containing 4800 known human genes to search for differentially expressed genes, of great diagnostic interest.