TARGET RECETTORIALI DELLA LIQUIRIZIA NELLA TIROIDE SANA E PATOLOGICA: EVIDENZE PER UN SUO POSSIBILE RUOLO ANTIPROLIFERATIVO NEL CARCINOMA TIROIDEO DIFFERENZIATO

Presupposti dello studio: Sono vari i composti antitumorali isolati dalla liquurizia (Li), i cui possibili meccanismi anticancerogeni sono stati studiati in vitro e in vivo in modelli animali: tra questi l’acido glicirretinico (GA). Il GA è un noto agonista del recettore dei mineralocorticoidi (MR). Non vi è invece alcun dato in letteratura sulla possibile presenza ed attività del MR sulle cellule di tiroide sana e di carcinoma papillare della tiroide (PTC). Scopo dello studio: Questo progetto si poneva l’obbiettivo di: 1) dimostrare per la prima volta in letteratura l’espressione genica (NR3C2) e proteica dell’MR nelle cellule tiroidee sane e patologiche; 2) studiare l’effetto in vitro dell’estratto di radice di Li (fito-Li) e del suo principio attivo GA su colture cellulari di PTC. Materiali e metodi: Sono state utilizzate due linee cellulari stabilizzate di carcinoma papillare tiroideo: BCPAP e K1. È stato studiato l’effetto della Li, sia sotto forma di fito-Li che di principio attivo GA, e del canrenoato di potassio (CA) sulla vitalità cellulare (test MTT) e sulla migrazione cellulare (test wound healing) su entrambe le linee cellulari. Dai campioni di tessuto tiroideo fresco congelato provenienti da 49 pazienti risultati affetti da PTC di cui 29 presentanti la controparte di tessuto sano è stato estratto l’RNA sia dal tessuto patologico (PTC) che dal parenchima tiroideo sano adiacente. Mediante RT-PCR è stata valutata l’espressione genica di NR3C2. Per lo studio dell’espressione proteica del MR è stata utilizzata la tecnica dell’immunofluorescenza (IF) associata alla microscopia confocale sia sui campioni di tessuto tiroideo fresco congelato che sulle due colture cellulari di PTC. Risultati: I risultati del MTT test mostrano complessivamente un significativo effetto di riduzione della vitalità cellulare dose-dipendente e tempo-dipendente per entrambi i composti e in entrambe le linee cellulari. Soprattutto nella linea cellulare BCPAP è evidente un’attenuazione dell’effetto di riduzione della vitalità cellulare a parità di concentrazione di fito-Li e GA dopo l’aggiunta di CA. Si evidenzia un chiaro effetto di riduzione della migrazione cellulare sia per il fito-Li che per il GA rispetto al controllo su entrambe le linee cellulari. Inoltre, si evidenzia un’aumentata migrazione cellulare dopo incubazione con CA. Tramite RT-PCR si dimostra un elevato livello di espressione dell’mRNA per MR sia nella tiroide sana che nel PTC. Inoltre, si evidenzia una riduzione statisticamente significativa dell’espressione del mRNA per MR nel PTC rispetto al tessuto sano tiroideo. Inoltre, l’espressione del MR è ridotta nel PTC variante Hobnail e nel carcinoma anaplastico rispetto al PTC variante classica e al tessuto tiroideo sano. Successivamente abbiamo dimostrato per la prima volta l’espressione proteica del MR sia nelle cellule tiroidee sane che nel PTC e attraverso la quantificazione dell’intensità del segnale di MR in IF abbiamo osservato una minor espressione proteica del MR nel PTC rispetto alla controparte sana. Dopo incubazione delle linee cellulari BCPAP e K1 con aldosterone e come conferma della sua attivazione abbiamo evidenziato la migrazione del MR dal citoplasma al nucleo dopo 24 h. Conclusione: questo studio ha evidenziato per la prima volta in letteratura l’espressione genica e proteica del MR sulle cellule tiroidee sane e tumorali, riconoscendo la tiroide come nuovo organo bersaglio dei mineralocorticoidi. Inoltre, ha generato le prime evidenze di un effetto antitumorale in vitro della Li e del suo principio attivo GA nel PTC, almeno in parte legate all’attività agonista sul MR.Backgrounds: There are various anticancer compounds isolated from liquorice (Li), whose possible anticarcinogenic mechanisms have been studied in vitro and in vivo in animal models: among them glycyrrhetinic acid (GA). GA is a known mineralocorticoid receptor (MR) agonist. On the other hand, there is no data in the literature on the possible presence and activity of MR on normal thyroid cells and papillary thyroid carcinoma (PTC). Aim of the study: This project aimed to: 1) demonstrate for the first time in literature the gene (NR3C2) and protein expression of MR in healthy and pathological thyroid cells; 2) to study the in vitro effect of Li root extract (phyto-Li) and its active compound GA on PTC cell cultures. Materials and Methods: Two stabilized papillary thyroid carcinoma cell lines were used: BCPAP and K1. The effect of Li, both in the form of phyto-Li and of the active compound GA, and of potassium canrenoate (CA) on cell viability (MTT test) and on cell migration (wound healing test) on both cell lines was investigated. RNA was extracted from both the pathological tissue (PTC) and from the adjacent healthy thyroid parenchyma from fresh frozen thyroid tissue samples from 49 patients found to be affected by PTC, 29 of which had the healthy tissue counterpart. The gene expression of NR3C2 was evaluated by RT-PCR. To study the protein expression of the MR, the immunofluorescence (IF) technique was used associated with confocal microscopy both on fresh frozen thyroid tissue samples and on the two PTC cell cultures. Results: The MTT test show an overall significant dose-dependent and time-dependent cell viability reduction effect for both compounds and in both cell lines. Especially in the BCPAP cell line, an attenuation of the effect of reducing cell viability is evident at the same concentration of phyto-Li and GA after the addition of CA. There is a clear effect of reducing cell migration for both phyto-Li and GA compared to the control on both cell lines. In addition, there is an increased cell migration after incubation with CA. RT-PCR demonstrates a high level of mRNA expression for MR in both healthy thyroid and PTC. In addition, there is a statistically significant reduction in mRNA expression for MR in PTC compared to healthy thyroid tissue. In addition, MR expression is reduced in Hobnail variant PTC and anaplastic carcinoma compared to classic variant PTC and healthy thyroid tissue. We subsequently demonstrated for the first time the protein expression of MR in both healthy thyroid cells and in the PTC and through the quantification of the intensity of the MR signal in IF we observed a lower protein expression of the MR in the PTC compared to the healthy counterpart. After incubation of the BCPAP and K1 cell lines with aldosterone, and as confirmation of its activation, we highlighted the migration of the MR from the cytoplasm to the nucleus after 24 h. Conclusion: this study highlighted for the first time in the literature the gene and protein expression of MR on healthy and cancer thyroid cells, recognizing the thyroid as a new target organ for mineralocorticoids. In addition, it generated the first evidence of an in vitro antitumor effect of Li and its active ingredient GA in PTC, at least in part related to the agonist activity on MR

Safety and efficacy of prophylactic treatment for hyperthyroidism induced by iodinated contrast media in a high-risk population

IntroductionThe use of iodinated contrast media (ICM) can lead to thyrotoxicosis, especially in patients with risk factors, such as Graves' disease, multinodular goiter, older age, and iodine deficiency. Although hyperthyroidism may have clinically relevant effects, whether high-risk patients should receive prophylactic treatment before they are administered ICM is still debated. Aim of the studyWe aimed to demonstrate the safety and efficacy of prophylactic treatment with sodium perchlorate and/or methimazole to prevent ICM-induced hyperthyroidism (ICMIH) in a population of high-risk cardiac patients. We ran a cost analysis to ascertain the most cost-effective prophylactic treatment protocol. We also aimed to identify possible risk factors for the onset of ICMIH. Materials and methodsWe performed a longitudinal retrospective study on 61 patients admitted to a tertiary-level cardiology unit for diagnostic and/or therapeutic ICM-procedures. We included patients with available records of thyroid function tests performed before and after ICM were administered, who were at high risk of developing ICMIH. Patients were given one of two different prophylactic treatments (methimazole alone or both methimazole and sodium perchlorate) or no prophylactic treatment. The difference between their thyroid function at the baseline and 11-30 days after the ICM-related procedure was considered the principal endpoint. ResultsTwenty-three (38%) of the 61 patients were given a prophylactic treatment. Thyroid function deteriorated after the administration of ICM in 9/61 patients (15%). These cases were associated with higher plasma creatinine levels at admission, higher baseline TSH levels, lower baseline FT4 levels, and no use of prophylactic treatment. The type of prophylaxis provided did not influence any onset of ICMIH. A cost-benefit analysis showed that prophylactic treatment with methimazole alone was less costly per person than the combination protocol. On multivariate analysis, only the use of a prophylactic treatment was independently associated with a reduction in the risk of ICMIH. Patients not given any prophylactic treatment had a nearly five-fold higher relative risk of developing ICMIH. ConclusionProphylactic treatment can prevent the onset of ICMIH in high-risk populations administered ICM. Prophylaxis is safe and effective in this setting, especially in cardiopathic patients. Prophylaxis with methimazole alone seems to be the most cost-effective option

PROGNOSTIC SIGNIFICANCE OF TERT PROMOTER AND BRAF MUTATIONS IN TIR-4 AND TIR-5 THYROID CYTOLOGY

Objective: Follicular-derived thyroid cancers generally have a good prognosis, but in a minority of cases, they have an aggressive behavior and develop distant metastases, with an increase in the associated mortality. None of the prognostic markers currently available prior to surgery can identify such cases. Methods: TERT promoter and BRAF gene mutations were examined in a series of 436 consecutive TIR-4 and TIR-5 nodes referred for surgery. Follow-up (median: 59 months, range: 7-293 months) was available for 384/423 patients with malignant nodes. Results: TERT promoter and BRAF mutations were detected in 20/436 (4.6%) and 257/434 thyroid nodules (59.2%), respectively. At the end of the follow-up, 318/384 patients (82.8%) had an excellent outcome, 48/384 (12.5%) had indeterminate response or biochemical persistence, 18/384 (4.7%) had a structural persistence or died from thyroid cancer. TERT promoter mutations correlated with older age (P < 0.0001), larger tumor size (P = 0.0002), oxyntic and aggressive PTC variants (P = 0.01), higher tumor stages (P < 0.0001), distant metastases (<0.0001) and disease outcome (P < 0.0001). At multivariate analysis, TERT promoter mutation was not an independent predictor of disease outcome. TERT promoter mutation- (OR: 40.58; 95% CI: 3.06-539.04), and N1b lymph node metastases (OR: 40.16, 95% CI: 3.48-463.04) were independent predictors of distant metastases. BRAF mutation did not predict the outcome, and it correlated with a lower incidence of distant metastases (P = 0.0201). Conclusions: TERT promoter mutation proved an independent predictor of distant metastases, giving clinicians the chance to identify many of the patients who warranted more aggressive initial treatment and closer follow-up

The role of procalcitonin in the follow-up of medullary thyroid cancer

Objective: Calcitonin (Ct) represents the most important biochemical marker of medullary thyroid cancer (MTC), but has certain limits. We analyzed the performance of procalcitonin (ProCt) in follow-up MTC patients. Methods: In this monocentric and retrospective study, we consecutively obtained ProCt and Ct values from all MTC patients that we visited during the period from April 2021 to May 2022. Patients were defined as having structural evidence of disease (29/90, 32.2%) irrespective of Ct values or, in its absence, as not evident disease (NED) if Ct was ≤10 ng/L (47/90, 52.2%), or minimal residual disease if Ct was >10 ng/L (14/90, 15.6%). Results: Ct and ProCt values were highly correlated (r = 0.883, P 0.12 ng/mL (P < 0.01, area under the curve: 0.963), with the following sensitivity, specificity, positive predictive value, and negative predictive value (NPV): 100%, 83.61%, 74.4%, and 100.0%. Conclusions: ProCt and Ct have a high correlation in MTC follow-up. ProCt may be useful as an adjunct to Ct, especially for its NPV concerning the structural disease

THE NATURAL HISTORY OF AUTOIMMUNE ADDISON'S DISEASE FROM THE DETECTION OF AUTOANTIBODIES TO DEVELOPMENT OF THE DISEASE: A LONG FOLLOW-UP STUDY ON 143 PATIENTS

Adrenal cortex autoantibodies (ACA) and/or 21-hydroxylase (21OHAb) are markers of autoimmune Addison's disease (AAD) and progression to overt AAD. The reported cumulative risk of developing AAD varies from 0-90% in different studies. Aim To assess the predictive value of different parameters for progression towards AAD in ACA and/or 21OHAb-positive patients with autoimmune polyendocrine syndromes (APS). Materials and Methods 29 patients with APS-1 and 114 patients with APS-2 or APS-4, were followed-up for a median of 10 years (range 6 months-33 years) and assessed by ACTH test. The risk of AAD was estimated according to age, gender, stage of adrenal dysfunction, associated diseases and antibody titer. Univariate and multivariate Cox proportional hazard models were used for statistical analysis. Results The cumulative risk (CR) of developing AAD was higher in APS-1 patients (94.2%) compared to patients with APS-2/APS-4 (38.7%). The CR was high in both males and females with APS-1 patients, while in patients with APS-2/APS-4 it was high only in males. Stage 1 (increased plasma renin) for patients with APS-1 and Stage 2 (no response of cortisol to ACTH-test) for patients with APS-2/APS-4 were established as the points of no return in the progression to AAD. Adjusted hazard ratio analyses by multivariate Cox model for AAD showed that gender, diseases, adrenal function were independent risk factors for developing clinical AAD. The risk of developing clinical AAD appears to subside after 19 years of follow up. Conclusions A model for estimating the probability to survive free of AAD has been developed and should be a useful tool in designing appropriate follow-up intervals and future therapeutic strategies

Validation of miRNAs as diagnostic and prognostic biomarkers, and possible therapeutic targets in medullary thyroid cancers

IntroductionMedullary thyroid cancer (MTC) is a rare type of neuroendocrine tumor that produces a hormone called calcitonin (CT). Thyroidectomy is the preferred treatment for MTC, as chemotherapy has been shown to have limited effectiveness. Targeted therapy approaches are currently being used for patients with advanced, metastatic MTC. Several studies have identified microRNAs, including miR-21, as playing a role in the development of MTC. Programmed cell death 4 (PDCD4) is a tumor suppressor gene that is an important target of miR-21. Our previous research has shown that high levels of miR-21 are associated with low PDCD4 nuclear scores and high CT levels. The aim of this study was to investigate the potential of this pathway as a novel therapeutic target for MTC.MethodsWe used a specific process to silence miR-21 in two human MTC cell lines. We studied the effect of this anti-miRNA process alone and in combination with cabozantinib and vandetanib, two drugs used in targeted therapy for MTC. We analyzed the effect of miR-21 silencing on cell viability, PDCD4 and CT expression, phosphorylation pathways, cell migration, cell cycle, and apoptosis.ResultsSilencing miR-21 alone resulted in a reduction of cell viability and an increase in PDCD4 levels at both mRNA and protein levels. It also led to a reduction in CT expression at both mRNA and secretion levels. When combined with cabozantinib and vandetanib, miR-21 silencing did not affect cell cycle or migration but was able to enhance apoptosis.ConclusionSilencing miR-21, although not showing synergistic activity with TKIs (tyrosine kinase inhibitors), represents a potential alternative worth exploring as a therapeutic target for MTC

Circulating miR-146a predicts glucocorticoid response in thyroid eye disease

Objective: Thyroid eye disease (TED) is an immune-mediated disorder of the eye. Intravenous glucocorticoid (GC) is the first-line treatment for patients with active moderate-to-severe TED. However, the response rate is between 5 0% and 80%. There are still no simple and reliable markers of responsiveness to G C therapy. We aimed to explore the possible role of miR-146a and miR-21 as predictors of responsiveness to GC treatment in TED. Methods: We carried out a prospective longitudinal study on 30 consecutive adult patients with active moderate-to-severe TED and eligible for GC therapy. All patients received the standard GC treatment with methylprednisolone iv. In cases of progressive worsening of Gorman Score for diplopia or with duction restrict ion <30° in at least two consecutive controls, patients also underwent orbital radiotherapy. Response to GC treatment was defined as a decrease of two or more points in the clinical activity score (CAS) or CAS <4/10 at 24 weeks. Circulating miRNAs were extract ed from patients’ serum and quantified by real-time PCR. Results: Twenty-three (77%) patients responded to GC. Thyroid surgery, higher CAS, greater proptosis and higher pre-treatment circulating levels of miR-146a emerged as predictive factors of responsiveness to GC. A ROC analysis revealed that miR-146a could predict responsiveness to GC with a positive predictive value of 100%. Conclusion: This is the first study investigating the role of pre-treatment circulating miR-21 and miR-146a to predict responsiveness to GC in TED. miR-146a emerged as a simple, objective, new marker of GC sensitivity that could be used to avoid ineffective administration of GC therapy to TED patients

Goodbye Hartmann trial: a prospective, international, multicenter, observational study on the current use of a surgical procedure developed a century ago

Background: Literature suggests colonic resection and primary anastomosis (RPA) instead of Hartmann's procedure (HP) for the treatment of left-sided colonic emergencies. We aim to evaluate the surgical options globally used to treat patients with acute left-sided colonic emergencies and the factors that leading to the choice of treatment, comparing HP and RPA. Methods: This is a prospective, international, multicenter, observational study registered on ClinicalTrials.gov. A total 1215 patients with left-sided colonic emergencies who required surgery were included from 204 centers during the period of March 1, 2020, to May 31, 2020. with a 1-year follow-up. Results: 564 patients (43.1%) were females. The mean age was 65.9 ± 15.6&nbsp;years. HP was performed in 697 (57.3%) patients and RPA in 384 (31.6%) cases. Complicated acute diverticulitis was the most common cause of left-sided colonic emergencies (40.2%), followed by colorectal malignancy (36.6%). Severe complications (Clavien-Dindo ≥ 3b) were higher in the HP group (P &lt; 0.001). 30-day mortality was higher in HP patients (13.7%), especially in case of bowel perforation and diffused peritonitis. 1-year follow-up showed no differences on ostomy reversal rate between HP and RPA. (P = 0.127). A backward likelihood logistic regression model showed that RPA was preferred in younger patients, having low ASA score (≤ 3), in case of large bowel obstruction, absence of colonic ischemia, longer time from admission to surgery, operating early at the day working hours, by a surgeon who performed more than 50 colorectal resections. Conclusions: After 100&nbsp;years since the first Hartmann's procedure, HP remains the most common treatment for left-sided colorectal emergencies. Treatment's choice depends on patient characteristics, the time of surgery and the experience of the surgeon. RPA should be considered as the gold standard for surgery, with HP being an exception

Azimuthal anisotropy of charged jet production in root s(NN)=2.76 TeV Pb-Pb collisions

We present measurements of the azimuthal dependence of charged jet production in central and semi-central root s(NN) = 2.76 TeV Pb-Pb collisions with respect to the second harmonic event plane, quantified as nu(ch)(2) (jet). Jet finding is performed employing the anti-k(T) algorithm with a resolution parameter R = 0.2 using charged tracks from the ALICE tracking system. The contribution of the azimuthal anisotropy of the underlying event is taken into account event-by-event. The remaining (statistical) region-to-region fluctuations are removed on an ensemble basis by unfolding the jet spectra for different event plane orientations independently. Significant non-zero nu(ch)(2) (jet) is observed in semi-central collisions (30-50% centrality) for 20 <p(T)(ch) (jet) <90 GeV/c. The azimuthal dependence of the charged jet production is similar to the dependence observed for jets comprising both charged and neutral fragments, and compatible with measurements of the nu(2) of single charged particles at high p(T). Good agreement between the data and predictions from JEWEL, an event generator simulating parton shower evolution in the presence of a dense QCD medium, is found in semi-central collisions. (C) 2015 CERN for the benefit of the ALICE Collaboration. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).Peer reviewe

Forward-central two-particle correlations in p-Pb collisions at root s(NN)=5.02 TeV

Two-particle angular correlations between trigger particles in the forward pseudorapidity range (2.5 2GeV/c. (C) 2015 CERN for the benefit of the ALICE Collaboration. Published by Elsevier B. V.Peer reviewe