STUDIO DELL'ATTIVITa DELLA GAMMA-GLUTAMILTRANSFERASI (GGT) SUL NITROSOGLUTATIONE (GSNO)

RIASSUNTO Il monossido d’azoto (NO•) è una specie chimica reattiva di natura radicalica. L’NO è una molecola prodotta a partire dall’amminoacido L-arginina ed O2, in una reazione catalizzata da una famiglia di enzimi chiamata NO-sintetasi (NOS). Nei sistemi biologici, l’NO agisce come messaggero intra ed inter-cellulare regolando numerosissime funzioni. All’inizio si pensava all’NO come ormone paracrino e recentemente è stato dimostrato che questa molecola è in grado di esplicare la sua azione in siti più distanti da quello di produzione. Pertanto, l’NO, in quanto molecola molto reattiva, per svolgere la sua azione a distanza deve essere trasportato nel sangue da molecole che lo stabilizzano e che possano, quindi, rilasciarlo in siti lontani da quelli in cui è stato prodotto. Per molti anni in terapie cardiovascolari sono state usati farmaci NO-donatori che vengono decomposti nell’organismo per liberare NO, in particolare la nitroglicerina, per la prevenzione e il trattamento di angina pectoris e il sodio nitroprussiato per il trattamento dell’ipertensione. Fra gli NO-donatori, gli S-nitrosotioli (RSNO) sono molecole che possono sostituire i farmci di sintesi con vantaggio per gli organismi, in quanto, sono molecole biologiche e non estranee all’organismo. Fra i nitrosotioli a basso peso molecolare di particolare interesse è il Nitrosoglutatione (GSNO). Il GSNO è un addotto del glutatione con l’NO che si forma normalmente nelle cellule in condizioni fisiologiche e patologiche, in particolare, durante ischemia. Il nostro laboratorio è particolarmente interessato al metabolismo del glutatione e dell’NO, pertanto è stato importante mettere a punto un metodo di facile esecuzione per il dosaggio del GSNO in campioni biologici. E’ stato ipotizzato che il trasporto dell’NO avvenga in vivo tramite il GSNO che viene decomposto dalla γ-glutamiltranspeptidasi (γ-GT o GGT) secondo la reazione: GSNO + accettore → CysGlyNO + γ-Glu-accettore La GGT è un enzima localizzato sulla membrana plasmatica di molti tipi cellulari che catalizza il primo passaggio del processo di idrolisi del glutatione (GSH) attraverso la scissione del legame γ-glutamilico. Il substrato naturale della GGT è il GSH. Per l’analogia fra il GSH e il GSNO è stato supposto , in diversi lavori, che quest’ultimo possa essere substrato della GGT e pertanto, che l’enzima GGT possa avere un ruolo nel rilascio di NO dal GSNO. Noi ci siamo occupati di verificare che il GSNO potesse essere un buon substrato della GGT. In letteratura non esistono metodi in grado di dosare l’attività della GGT sul GSNO. In questo lavoro di tesi e’ stato messo a punto un metodo per dosare il GSNO tramite la reazione con la GGT in presenza della glicilglicina (GlyGly) come substrarto accettore. GSNO + GlyGly → CysGlyNO + γ-GluGlyGly (1) CysGlyNO → NO + CysGlyox (2) Poiche’ sia il GSNO che la CysGlyNO presentano il medesimo assorbimento a λ=334 nm è stato necessario accoppiare una reazione ancillare (2) che rilasciasse un prodotto il quale non avesse un picco di assorbimento a 334 nm. La reazione 2 si adegua perfettamente a questa necessità in quanto il prodotto CysGly non assorbe a quella lunghezza d’onda. D’altra parte si sa dalla letteratura che la CysGlyNO viene rapidamente decomposta da Cu(II) a CysGlyox e NO, mentre il GSNO e’ stabile in presenza di Cu(II). La reazione 2 è, dunque, quella che ci ha permesso di visualizzare la decomposizione del GSNO allo spettrofotometro seguendo la diminuzione di assorbanza del nitrosotiolo ad una lunghezza d’onda λ=334 nm. Attraverso questo studio cinetico ci è stato possibile calcolare la KmGSNO = 0.374 ± 0.024 mM (a 37°C) e KmGG = 0.687 ± 0.063 mM. Questo dato, fino ad ora non riportato in letteratura, e’ risultato paragonabile con la Km del substrato naturale GSH (0.4 mM) già esistente in letteratura. Pertanto, abbiamo dimostrato, in questo lavoro, che il GSNO e’ un substrato della GGT con affinità paragonabile a quella del GSH e che il GSNO tramite la GGT rilascia NO in presenza di CU++, il quale si ritrova nel sangue in concentrazioni fisiologiche sufficienti (dell’ordine di micromolare) a produrre la decomposizione della CysGlyNO

L’ANALISI GENETICA DI CARCINOMI PAPILLARI E MIDOLLARI DELLA TIROIDE PRESENTI CONTEMPORANEAMENTE RIVELA LA LORO NATURA CASUALE

Il Carcinoma Papillare (PTC) ed il Carcinoma Midollare (MTC) della tiroide originano rispettivamente dalle cellule follicolari e parafollicolari della ghiandola. Abbastanza frequentemente, essi possono però presentarsi contemporaneamente. Diversi studi hanno tentato di trovare una causa patogenetica comune ma i dati a disposizione sono ancora controversi. Scopo di questo studio è quello di fornire un’analisi genetica dei tumori (PTC e MTC) che si presentano contemporaneamente per capire l’origine di questo fenomeno. Allo scopo abbiamo studiato 24 pazienti (6 maschi, 18 femmine) con presenza simultanea di PTC e MTC. DNA genomico è stato estratto dalle componenti PTC, MTC e parenchima sano per essere analizzato tramite PCR e sequenziamento diretto con primer specifici per mutazioni nei codoni 12, 13 e 61 dei geni H-, K- ed N-RAS, 600 del gene BRAF e 634 e 918 dell’oncogene RET. Due/24 pazienti (8.3%) erano affetti da MEN2A con mutazione germinale del gene RET (S981A, V804M), presente quindi in tutte e tre le componenti. Nessuno degli altri pazienti presentava mutazioni nel tessuto sano ed in 8/24 pazienti (33.4%) non e’ stata trovata alcuna mutazione nemmeno nelle porzioni PTC ed MTC. Negli altri pazienti, 8/24 (33.3%) MTC presentavano la mutazione somatica di RET M918T; 3/24 (12.5%) mutazioni del gene H-RAS (G13R, Q61R, D69N) ed 1/24 (4.2%) mutazioni H-RAS (Q61K) e RET (M918T) in contemporanea. Per quanto riguarda la componente PTC: 1/24 (4.2%) presentava la mutazione BRAF (V600E), 1/24 (4.2%) la mutazione H-RAS (T58A) e 1/24 (4.2%) la mutazione K-RAS (M1T). Uno dei pazienti mostrava contemporaneamente la mutazione RET (M918T) nella componente MTC e BRAF (V600E) nella componente PTC. Nessuna delle mutazioni trovate era presente su entrambe le. Da questi dati si evidenzia che le due componenti tumorali presentano mutazioni tipiche per il tipo istologico (PTC e il MTC) ed in nessun caso sono condivise, suggerendo cause indipendenti nell’insorgenza di questi tumori. A supporto di questa osservazione, uno dei pazienti mostrava una mutazione di RET nella componente MTC e la mutazione di BRAF nella componente PTC. Nel pannello di mutazioni trovate, le mutazioni D69N, T58A di H-RAS e M1T di K-RAS risultano nuove e mai descritte, suggerendo che anche mutazioni alternative agli hot spot classici dei geni RAS potrebbero avere un ruolo nella patogenesi di questi tumori

WHOLE EXOME SEQUENCING OF MEDULLARY THYROID CARCINOMA CASES IDENTIFIES 86 VARIATIONS IN GENES POSSIBLY INVOLVED IN TUMORAL TRANSFORMATION

Objectives: About 40% of sporadic Medullary Thyroid Carcinomas (MTC) is still orphan of an oncogenic driver. Purpose of this study was to disclose novel genetic alterations leading to the pathogenesis of MTC using Whole Exome Sequencing (WES) of RET+ and RET- cases. Methods: WES analysis was performed on 6 sporadic MTC cases (2 RET+, 4 RET-) using an Illumina platform. After processing and proper filtering, all non-synonymous Single Nucleotide Variations (SNV) shared by the RET- cases were listed. Validation on 135 MTC cases and 189 healthy controls by PCR and enzymatic restriction analysis was performed in one of the genes of interest identified through WES. Results: WES analysis led to the identification of a panel of 86 non-synonymous SNV shared in the 4 RET- cases and possibly involved in tumoral transformation process. Among the 86 SNV identified, the A133S polymorphism of the RASSF1A oncosuppressor appeared to be of interest. We found A133S in 21/135 (15.6%) MTC cases and in 19/189 (10%) healthy controls (P=0.137). The incidence of A133S appeared to be slightly lower in RET mutated MTC [9/64 (14%)] than in not-mutated [12/71 (17%)], although not statistically significant. Conclusions: Through WES analysis we were able to identify 86 non-synonymous SNV shared in RET- MTC cases. This panel represents the first list of variations containing hypothetically novel genetic drivers involved in MTC oncogenesis. The prevalence of the A133S SNP was found to be higher in MTC cases compared to healthy controls (15.6% vs 10%) although not statistically significant. Further validation of other candidate genes is on-going

I CARCINOMI PAPILLARI E MIDOLLARI DELLA TIROIDE PRESENTI CONTEMPORANEAMENTE HANNO ORIGINE GENETICA INDIPENDENTE

Il Carcinoma Papillare (PTC) ed il Carcinoma Midollare (MTC) della tiroide si sviluppano entrambi nella tiroide ma hanno un’origine embrionale distinta: il PTC deriva dalle cellule follicolari mentre il MTC dalle cellule C-parafollicolari. In letteratura sono riportati casi in cui questi due tipi di tumore si presentano contemporaneamente ed una possibile causa genetica comune e’ stata ipotizzata anche se i dati a disposizione rimangono tuttora controversi. Scopo di questo studio è stato quello di analizzare la presenza di alterazioni genetiche caratteristiche dei due tipi di tumore (PTC e MTC) che si presentano contemporaneamente. Allo scopo abbiamo studiato 24 pazienti (6 maschi, 18 femmine) che presentavano simultaneamente un PTC ed un MTC. Il DNA genomico è stato estratto dalle componenti tumorali e dal parenchima sano ed e’ stato analizzato con sequenziamento diretto per le mutazioni nei codoni 12, 13 e 61 dei geni H-, K- ed N-RAS, 600 del gene BRAF e 634 e 918 dell’oncogene RET. Due/24 pazienti (8.3%) erano affetti da MEN2A con mutazione germinale del gene RET (S981A, V804M). Nessuno degli altri pazienti presentava mutazioni nel tessuto sano ed in 8/24 pazienti (33.4%) non presentavano mutazioni nemmeno nelle porzioni tumorali. Negli altri pazienti: 8/24 (33.3%) MTC presentavano la mutazione somatica di RET M918T; 3/24 (12.5%) mutazioni del gene H-RAS (G13R, Q61R, D69N) ed 1/24 (4.2%) mutazioni H-RAS (Q61K) e RET (M918T) contemporanea. Nella componente PTC: 1/24 (4.2%) presentava la mutazione BRAF (V600E), 1/24 (4.2%) la mutazione H-RAS (T58A) e 1/24 (4.2%) la mutazione K-RAS (M1T). In un caso, si aveva contemporaneamente la mutazione RET (M918T) nella componente MTC e BRAF (V600E) nella componente PTC. Nessuna delle mutazioni trovate era presente su entrambe le componenti tumorali. Questi dati suggeriscono cause indipendenti nell’insorgenza di PTC ed MTC che si presentano contemporaneamente in cui si osservano mutazioni tipiche per il tipo istologico (PTC e il MTC). A conferma di questa ipotesi, uno dei pazienti mostrava contemporaneamente una mutazione di RET nella componente MTC e la mutazione di BRAF nella componente PTC. Nel pannello di mutazioni trovate, le mutazioni D69N, T58A di H-RAS e M1T di K-RAS risultano nuove e mai descritte, suggerendo che anche mutazioni al di fuori degli hot spot classici dei geni RAS potrebbero avere un ruolo nella patogenesi di questi tumori

SIMULTANEOUS MEDULLARY AND PAPILLARY THYROID CARCINOMAS SHOW INDEPENDENT GENETIC ORIGIN

Papillary (PTC) and Medullary (MTC) thyroid carcinomas have distinct embryonic origins although they share the activation of common oncogenes (RET, RAS). The occurrence of these tumors in the same gland is not a rare event. Aim of this study was to investigate the hypothetical involvement of common genetic alterations in the symultaneous occurrence of the PTC and MTC. We studied 24 patients presenting simultaneous PTC and MTC. DNA was obtained from both tumor components and healthy tissue and sequenced for mutations in codons 12, 13 and 61 of H-, K- and N-RAS, 600 of BRAF and 634 and 918 of RET. Two/24 patients (8.3 %) were affected by MEN2A and carrying germline mutations of the RET gene (S981A, V804M). None of the patients showed mutations in the healthy tissue and 8/24 patients (33.4 %) had no mutations either in the tumor components. In the other cases, considering the MTC component: 8/24 (33.3 %) harboured the RET/M918T mutation; 3/24 (12.5 %) mutations in the H-RAS gene (G13R, Q61R, D69N) and 1/24 (4.2 %) showed simultaneously the H-RAS/Q61K and the RET/M918T mutations. In the PTC component: 1/24 (4.2 %) harboured the BRAF/V600E mutation, 1/24 (4.2 %) the H-RAS/T58A mutation and 1/24 (4.2 %) the K-RAS/M1T mutation. In one case, we found the RET (M918T) mutation in the MTC component and the BRAF/V600E in the PTC component. None of the mutations found were present in both tumors. These data suggest independent genetic causes in the development of simultaneous PTC and MTC. Confirmation of this hypothesis is that one of the patients showed simultaneous mutation of RET in MTC and the BRAF mutation in PTC. In this study we also found novel H-RAS and K-RAS mutations suggesting that alterations outside the classic hot spots may play a role in the pathogenesis of these tumors

Predictive factors of short and long-term vandetanib response in locally advanced or metastatic medullary thyroid cancer: a single center experience

Objectives: Vandetanib (V) is an important drug in the metastatic medullary thyroid cancer (MTC) treatment. The objective of this study was to evaluate the presence of predictors of V response, in short and long period, in locally advanced or metastatic MTC patients (pts). Methods: Seventy-nine locally advanced or metastatic MTC pts with progressive or symptomatic disease, referred to our Center between 2007 and 2018 and already treated surgically and with other systemic therapies, were treated with V. During follow-up it was performed clinical examination, biochemical and morphological evaluation. Twenty-five pts were treated with V for less than 12 months (short responders, Group 1), 54 patients were treated with V for at least 12 months (long responders, Group 2). Results: The genetic screening showed that in the Group 1, 4/25 (16%) pts were inherited forms and 21/25 (84%) pts were sporadic cases. In the Group 2, 8/54 (14.8%) pts were inherited forms and 46/54 (85.2%) pts were sporadic cases. The evaluation of somatic mutations showed that RET mutation was present in 82.3% and in 95.3% of pts in Group 1 and in Group 2, respectively. However, the presence of RET mutations, it wasn’t a predictor of response to treatment. The metastases site wasn’t correlated with the outcome. Otherwise, we observed that in long responders group, 47/54 (87%) pts showed at least one adverse events (AE) during V treatment with a correlation between AE and V response (P=0.02). In this group we also observed a statistically significant correlation between the younger age (<45 yrs) at screening and a greater response to V (P=0.01) and between the absence of progression disease at screening and response to V (P<0.0001). In the long term outcome, considering the last CT scan performed at the data cut-off during the treatment, 29/54 (53.7%) pts showed a persistent response to V after a median follow-up of 41 months. Moreover, we observed that the pts in the Group1 had a more aggressive disease and a more advanced age at screening than pts in Group 2. The estimated median Progression Free-Survival of all patients was 47 months. Conclusions: In our study, it was observed that the appearance of AE during V treatment, the younger age and the absence of progression disease at screening were predictive factors of long-term response to V in MTC pts. Moreover, RET somatic mutations were very frequent in the metastatic MTC patients but it wasn’t a predictor of response to V

V804M RET MUTATION AND VANDETANIB RESPONSE IN METASTATIC MEDULLARY THYROID CANCER

Objective: Vandetanib is the most important treatment for metastatic medullary thyroid cancer (MTC). “In vitro” studies showed that the presence of the V804M RET mutation is associated with a selective resistance to vande- tanib. The objective of this study was to evaluate the response to vandetanib in metastatic V804M positive MTC patients. Methods: Four metastatic MTC patients, treated with vandetanib, were V804M RET positive. In 2 cases this mutation was a germinal mutation, while in other 2 was a somatic mutation. During the follow-up serum calcitonin lev- els and computed tomography scan were performed. Results: All cases had multiple metastatic lesions distributed in several organs. In 1 case (n 1) the enrollment was due to the symptom (diarrhea) while in 3 cases (n 2-3-4) to progression of the disease. The patient-1 had a symp- tomatic familial MTC. During the 55 months of follow up, she showed a stable disease (SD). The patient-2, 3 and 4 had a progressive metastatic sporadic MTC. During follow up, the patient-2 had an initial partial response (PR) and then a SD for 9 months, the patient-3 had an initial PR and then a SD for 14 months, the patient-4 did not respond to treatment. The most common adverse events were: acneiform rash in all patients, asthenia in patients 1 and 3, nau- sea, anorexia and weight loss in patient-2. Conclusions: In our study we observed that patients with „resistant“ mutations, such as V804M RET mutation, can respond (3/4) or not respond (1/4) to vandetanib. Therefore, also in the presence of this mutation it is indi- cated to start vandetanib since it can likely act also through mechanisms of action independently from RET inhibition such as VEGF-R inhibition

Predictors Of Vandetanib Response In The Locally Advanced Or Metastatic Medullary Thyroid Cancer: A Single Center Experience

Objectives: Vandetanib (V) is a new option in the metastatic medullary thyroid cancer (MTC) treatment. In this study it was evaluated the presence of epidemiological, clinical and genetic predictors of V response in locally advanced or metastatic MTC patients (pts) treated for at least 12 months. Methods: Forty-five locally advanced or metastatic MTC pts with pro- gression or symptomatic disease, referred to our Center and already treated surgically and with other systemic therapies, were treated with V. During fol- low-up it was performed clinical examination, biochemical and morphological evaluation. All pts have taken V for at least 12 months. Results: The genetic screening showed that 7/45 (15.6%) pts were inher- ited forms and 38/45 (84.4%) were sporadic cases. The evaluation of somatic mutations were performed in 36/38 (94.7%) pts and it was observed that 34/36 (94.4%) were carriers of a hereditary or somatic genetic mutation. However, the presence of RET mutations, it wasn’t a predictor of response to treatment. All three pts with V804M RET mutation, that was demonstrated to confer resistance to V in ‘in vitro’ studies, responded to treatment. In our study group, the metastases site wasn’t correlated with the outcome. In 36/45 (80%) pts it was observed the presence of adverse events (AE) with a correlation between AE, particularly cutaneous rash, and V response (p = 0.01). A long-term out- come showed a morphologic and biochemical response in 39/45 (86.7%) pts. The Progression Free-Survival was 85% after six months. Conclusion: It was observed that the presence of AE was the only predic- tor of response to V treatment. Moreover, RET somatic mutations were very frequent in the metastatic MTC patients and also patients with ‘resistant muta- tions’ responded to treatment

AFTER 20 YEARS, RET GENETIC SCREENING STILL INDENTIFIES NEW GERMILINE AND SOMATIC MUTATIONS

Objectives: In the last 20 years we performed RET genetic screening in more than 1000 MTC patients either hereditary or sporadic. Methods: RET genetic screening was performed in DNA extracted from blood and/or tissue by direct sequencing. TA cloning was performed to characterize new mutations and deletions. Site-directed mutagenesis, focus formation and soft agar assays were performed to test in vitro the activity of the new mutations. The Align GVGD program was employed for the in silico analysis. Results: in the last year we identified 3 MTC patients with new RET alterations. The first case had a 7bp “somatic” in frame deletion in exon 11 encompassing codon 629-631. The second case showed the simultaneous presence of a “somatic” E616Q mutation in exon 10 and a “somatic” C630G mutation in exon 11 on different alleles. Moreover, in the same patient, we found an alternative splicing causing the in frame skip of exon 10 in the allele carrying the C630G mutation. The third case harboured a new “germline” mutation (E632K in exon 11) although the MTC was apparently sporadic. According to the in vitro and the in silico tests, both E616Q and E632K RET mutations were not transforming while the C630G RET mutation showed a high transforming activity. Conclusions: 1) RET genetic screening should be performed by sequencing analysis in all MTC patients to detect also new RET mutations that would be missed when looking only at the “hot spot” mutations; 2) all new mutations must be evaluated by in silico and/or in vitro analysis to define their transforming ability since in some cases they may be inactive mutations

Differential expression of RET isoforms in normal thyroid tissues, papillary and medullary thyroid carcinomas

POURPOSES: We investigated the expression of RET9 and RET51 isoforms in medullary (MTC), papillary (PTC) thyroid carcinoma, normal thyroid tissues, and pheochromocytoma (PHEO) to verify if these isoforms are present also in follicular thyroid cell-derived tissues, and if there is a differential expression of RET9 and RET51 in MTC. METHODS: Nineteen patients with MTC, 18 patients with PTC, 18 samples of contralateral normal thyroid tissues, and 5 cases of PHEO were included in this study. RET isoform expression was studied by real-time RT-PCR. RESULTS: All MTCs and PHEOs were positive for RET9 and RET51. Fourteen/eighteen (77.7%) PTC cases were positive for RET9 and/or RET51, and four were positive for only one of the genes. In normal thyroid tissues, 3/18 (16.7%) cases were negative for both isoforms, 4/18 (22.2%) were positive for both, and 11/18 (61.1%) were positive for only one. RET isoforms were expressed at different levels in MTC, PHEO, PTC, and normal thyroid tissues: RET9 expression was higher in PHEO than in MTC, PTC, and normal thyroid tissues. RET9 expression was also higher in MTC than in PTC and normal thyroid tissues. No difference was observed between PTC and normal thyroid tissues. A similar pattern of expression was observed for RET51. In addition, RET51 was significantly more expressed than RET9 in MTC, while RET9 was the predominant isoform in PHEO. CONCLUSIONS: Our study documented the expression of the RET9 and RET51 isoforms in normal thyroid and PTC tissues. RET9 and RET51 isoforms were also present in MTC and PHEO. RET51 expression was higher than RET9 expression in MTC, while there was no difference in the expression of these two isoforms in PTC and normal thyroid tissues. RET9 was more highly expressed than RET51 in PHEOs