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Free access

Marco Marino, Maria Laura Monzani, Giulia Brigante, Katia Cioni, Bruno Madeo, Daniele Santi, Antonino Maiorana, Stefania Bettelli, Valeria Moriondo, Elisa Pignatti, Lara Bonacini, Cesare Carani, Vincenzo Rochira, and Manuela Simoni

Objective: The diagnostic accuracy of thyroid fine needle aspiration biopsy (FNAB) can be improved by the combination of cytological and molecular analysis. In this study, washing liquids of FNAB (wFNAB) were tested for the BRAF V600E mutation, using the sensitive and cost-effective technique called high-resolution melting (HRM). The aim was to demonstrate the feasibility of BRAF analysis in wFNAB and its diagnostic utility, combined with cytology. Design: Prospective cohort study. Methods: 481 patients, corresponding to 648 FNAB samples, were subjected to both cytological (on cells smeared onto a glass slide) and molecular analysis (on fluids obtained washing the FNAB needle with 1 ml of saline) of the same aspiration. BRAF V600E analysis was performed by HRM after methodological validation for application to wFNAB (technique sensitivity: 5.4%). Results: The cytological results of the FNAB were: 136 (21%) nondiagnostic (THY1); 415 (64%) benign (THY2); 80 (12.4%) indeterminate (THY3); 9 (1.4%) suspicious for malignancy (THY4); 8 (1.2%) diagnostic of malignancy (THY5). The BRAF V600E mutation was found in 5 THY2, 2 THY3, 6 THY4 and 6 THY5 samples. Papillary carcinoma diagnosis was histologically confirmed in all BRAF+ thyroidectomized patients. BRAF combined with cytology improved the diagnostic value compared to cytology alone in a subgroup of 74 operated patients. Conclusions: HRM was demonstrated to be a feasible method for BRAF analysis in wFNAB. Thanks to its sensitivity and cost-effectiveness, it might be routinely used on a large scale in clinical practice. In perspective, standby wFNAB samples could be analyzed a posteriori in case of indeterminate cytology and/or suspicious findings on ultrasound.

Free access

Giulia Brigante, Giorgia Spaggiari, Daniele Santi, Katia Cioni, Valentina Gnarini, Chiara Diazzi, Elisa Pignatti, Livio Casarini, Marco Marino, Frank Tüttelmann, Cesare Carani, and Manuela Simoni

Background: Thyroidectomized patients need variable doses of levothyroxine (LT<sub>4</sub>) to obtain target thyroid-stimulating hormone (TSH) levels. Individual feedback set-points have been hypothesized and the influence of several genes in the regulation of the pituitary-thyroid axis has been demonstrated. Objectives: We hypothesized that genetic variants of the TRHR gene could be associated with a different hypothalamo-pituitary sensitivity to thyroid hormone feedback. Methods: We retrospectively analyzed 84 thyroidectomized patients with no residual thyroid function and undetectable thyroglobulin levels. Patients were evaluated under LT<sub>4</sub> resulting in TSH levels detectable but <0.5 μIU/ml. The two SNPs rs3134105 and rs3110040 were identified as informative markers of the TRHR gene. Genotyping was performed using high-resolution melting technology. Genotype distribution was compared between the patients and 99 euthyroid controls. Results: The selected SNPs were in linkage disequilibrium and only rs3134105 was further considered. A significant difference between the three possible genotypes for rs3134105 was found for TSH (p = 0.04) and free thyroxine (fT<sub>4</sub>)/TSH ratio (p = 0.02). Moreover, despite similar serum concentrations of free triiodothyronine (fT<sub>3</sub>) and fT<sub>4</sub>, carriers of at least one A allele of rs3134105 had significantly lower serum TSH levels (p = 0.01) as well as higher fT<sub>3</sub>/TSH (p = 0.01) and fT<sub>4</sub>/TSH ratios (p < 0.01). Conclusions: We demonstrated an association between serum TSH levels and discrete alleles of the TRHR gene in totally thyroidectomized patients under LT<sub>4</sub> therapy. Therefore, the TRHR gene seems to be a determinant of hypothalamo-pituitary sensitivity to LT<sub>4</sub>.

Open access

Giulia Brigante, Clara Lazzaretti, Elia Paradiso, Federico Nuzzo, Martina Sitti, Frank Tüttelmann, Gabriele Moretti, Roberto Silvestri, Federica Gemignani, Asta Försti, Kari Hemminki, Rossella Elisei, Cristina Romei, Eric Adriano Zizzi, Marco Agostino Deriu, Manuela Simoni, Stefano Landi, and Livio Casarini

To identify a peculiar genetic combination predisposing to differentiated thyroid carcinoma (DTC), we selected a set of single nucleotide polymorphisms (SNPs) associated with DTC risk, considering polygenic risk score (PRS), Bayesian statistics and a machine learning (ML) classifier to describe cases and controls in three different datasets. Dataset 1 (649 DTC, 431 controls) has been previously genotyped in a genome-wide association study (GWAS) on Italian DTC. Dataset 2 (234 DTC, 101 controls) and dataset 3 (404 DTC, 392 controls) were genotyped. Associations of 171 SNPs reported to predispose to DTC in candidate studies were extracted from the GWAS of dataset 1, followed by replication of SNPs associated with DTC risk (P < 0.05) in dataset 2. The reliability of the identified SNPs was confirmed by PRS and Bayesian statistics after merging the three datasets. SNPs were used to describe the case/control state of individuals by ML classifier. Starting from 171 SNPs associated with DTC, 15 were positive in both datasets 1 and 2. Using these markers, PRS revealed that individuals in the fifth quintile had a seven-fold increased risk of DTC than those in the first. Bayesian inference confirmed that the selected 15 SNPs differentiate cases from controls. Results were corroborated by ML, finding a maximum AUC of about 0.7. A restricted selection of only 15 DTC-associated SNPs is able to describe the inner genetic structure of Italian individuals, and ML allows a fair prediction of case or control status based solely on the individual genetic background.