Frequency and significance of Ras, Tert promoter, and Braf mutations in cytologically indeterminate thyroid nodules: A monocentric case series at a tertiary-level Endocrinology unit

PurposeThe management of thyroid nodules of indeterminate cytology is controversial. Our study aimed to establish the frequency and significance of H-,K-,N-RAS, TERT promoter, and BRAF gene mutations in thyroid nodes of indeterminate cytology and to assess their potential usefulness in clinical practice.MethodsH-,K-,N-RAS, TERT promoter and BRAF gene mutations were examined in a series of 199 consecutive nodes of indeterminate cytology referred for surgical excision.Results69/199 (35%) were malignant on histopathological review. RAS mutations were detected in 36/199 (18%), and 19/36 cases (53%) were malignant on histological diagnosis. TERT promoter mutations were detected in 7/199 (4%) nodules, which were all malignant lesions. BRAF mutations were detected in 15/199 (8%), and a BRAF K601E mutation was identified in 2 follicular adenomas and 1 noninvasive follicular thyroid neoplasm with papillary-like nuclear features. Altogether, this panel was able to identify 48% of the malignant lesions, achieving a specificity, positive predictive value, and negative predictive value for malignancy of 85, 62, and 75%, respectively.ConclusionThe residual malignancy risk in mutation-negative nodes is 25%. These nodes still need to be resected, but mutation analysis could help to orient the appropriate surgical strategy

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

Neck emergency due to parathyroid adenoma bleeding: a case report

<p>Abstract</p> <p>Introduction</p> <p>The spontaneous rupture of a parathyroid adenoma accompanied by extracapsular hemorrhage is a rare, potentially fatal, condition and is a cervicomediastinal surgical emergency.</p> <p>Case presentation</p> <p>This report describes an atypical two-step spontaneous rupture of an asymptomatic parathyroid adenoma in a 56-year-old Caucasian woman who presented with a painful mass in the right side of her neck.</p> <p>Conclusion</p> <p>Based on this case report and similar cases reported in the medical literature, a diagnosis of extracapsular parathyroid hemorrhage should be considered when a non-traumatic sudden neck swelling coexists with hypercalcemia and regional ecchymosis.</p

LE CARATTERISTICHE MOLECOLARI DEL CARCINOMA PAPILLARE DELLA TIROIDE NON IODOFISSANTI

ABSTRACT Background: Differentiated thyroid cancers (DTC) represent the most frequent endocrine cancer. They typically show an indolent behaviour and a good prognosis, mainly due to their capability to trap radioiodine (131-I), the most powerful therapeutic tool in these malignancies. Nevertheless, approximately one third of thyroid cancer metastases do not concentrate radioiodine. The most common genetic event in papillary thyroid carcinoma is a transversion in exon 15 of the BRAF gene which leads to a V600E aminoacid substitution (glutamic acid instead of valine) ranging from 36% to 48%. It has been demonstrated that cancers with the BRAF mutation have a more aggressive behaviour: the mutation is associated with more advanced initial tumor stage at diagnosis and with conversion into less differentiated or anaplastic cancers during the follow-up. BRAF could represent a new independent prognostic factor helpful to identify cancers that both tend to recur more frequently and lose the capability to trap radioiodine. The BRAF mutation is also associated with an increased expression of the glucose transporter (GLUT-1), suggesting the possibility to use 18-FDG-PET-CT examination during the follow-up. The objective of the study: 1) to estimate the frequency of the BRAF mutation in primary papillary cancers, in lymphonodal metastases at diagnosis and in recurrences; to value whether the BRAF mutation frequency is higher in relapsing cancers, particularly in those with no radioiodine uptake; 2) to determine whether the BRAF mutation is associated with increased glucose metabolism phenotypes. Patients and methods: 42 consecutive recurrences, belonging to 42 PTC affected patients who underwent surgery, were analysed. At the time of diagnosis they were subjected to thyroidectomy and to 131-I radioablation; then they were submitted to a total body scintigraphic examination 3 days after a 131-I therapeutic dose (2,7- 5,4GBq). 23/42 patients were submitted to a FDG-PET-CT examination as well. All the samples were paraffin embedded: DNA extraction was performed according to the manufacturer’s recommendations. Genomic DNA was evaluated for the existence of mutations of the BRAF gene by PCR amplification of the target DNA sequences followed by both direct sequencing and MASA. Results: recurrences were analysed under the TNM classification: 10/42 (24%) were T1, 8/42 (19%) were T2, 4/42 (10%) were T3 and 18/42 (43%) were T4. (TNM classification was not available for 2/42 patients). The average age at diagnosis was 40, the median was 35-yr-old (min 16, max 79), the average age at the time of recurrence was 45, the median was 43-yr-old (min 16, max 82). The average serum Tg concentration during suppressive therapy with l-T4 was 181, the median was 7 ng/ml (min <0,5- max 3344). 11/42 patients had anti-Tg autoantibodies. 19/37 (51%) recurrences with no 131-I uptake showed the BRAF mutation, while 1/5 (20%) recurrence with 131-I uptake showed the mutation. In 15 recurrences (14 with no 131-I uptake and 1 with 131-I uptake) we correlated the presence of the BRAF mutation with both the capability to trap 131-I and 18-FDG: among the recurrences with no 131-I uptake 73% could concentrate 18-FDG and 20% could not, the one that trapped 131-I trapped also 18-FDG. The recurrences with BRAF mutation showed also an increased SUV. In 10 patients a simultaneous analysis of BRAF status was conducted in primary cancers (K0), in lymphonodal metastases at diagnosis (N0) and in surgically removed first (R1) and second recurrences (R2): the BRAF mutation was detected in 2/10 (20%) primary cancers, in 3/7 (42%) lymphonodal metastases at diagnosis, in 7/10 (70%) recurrences with no 131-I uptake and in 2/2 (100%) second recurrences. By DNA sequencing we discovered a new BRAF mutation in exon 15 codon 602, which determine an aminoacidic substitution (phenylalanine instead of serine, S602F). Conclusions: 1) BRAF mutations are more frequent in recurrences with no 131-I uptake than in those with 131-I uptake (51% versus 20%); 2) de novo BRAF mutations were shown in thyroidal recurrences during the follow-up (20% versus 42% and 70% respectively in primary cancers, in lymphonodal metastases and in recurrences); 3) the presence of BRAF mutations has an inverse correlation with the capability to trap 131-I, and a direct one with the ability to concentrate 18-FDG and it is related to a higher level of 18-FDG uptake; 4) a new mutation occurs in exon 15, codon 602 of the BRAF gene and it causes the substitution of a polar aminoacid (serine) with a non polar one (phenylalanine). Our study confirms that BRAF is a possible molecular indicator of poor prognosis because its mutation is associated with more aggressive, less differentiated and unable to trap radioiodine recurrences.Presupposti dello studio: i carcinomi papillari tiroidei (PTCs) costituiscono la neoplasia endocrina più frequente e si caratterizzano per un decorso indolente e una bassa mortalità, dovuta principalmente alla capacità di questi tumori di concentrare lo iodio e alla possibilità di essere efficacemente trattati con iodio radioattivo (131-I). Tuttavia in circa un terzo dei pazienti, in particolare in corso di malattia persistente o ricorrente, viene persa la capacità di captare lo 131-I. Una mutazione somatica interessante l’esone 15 della serina-treonina-chinasi BRAF, realizzante la sostituzione di una valina con glutammato (V600E), rappresenta l’evento genico più frequente alla base dello sviluppo del carcinoma papillare tiroideo, documentabile nel 36-48% dei casi. Diversi studi hanno messo in evidenza che i carcinomi papillari con mutazione di BRAF hanno un atteggiamento più aggressivo, presentandosi in stadio più avanzato alla diagnosi e potendo evolvere verso istotipi meno differenziati o anaplastici nel follow-up. BRAF potrebbe pertanto rappresentare un nuovo fattore prognostico indipendente, in grado di identificare i carcinomi papillari a maggior rischio di recidiva e di perdita della capacità di concentrare lo 131-I. La mutazione di BRAF è inoltre associata ad un’aumentata espressione del trasportatore del glucosio GLUT1, suggerendo l’utilità di tecniche di imaging, quali la 18-FDG-PET-TC, nel follow-up dei pazienti con recidiva di tumore tiroideo differenziato. Scopo dello studio: 1) valutare la frequenza di mutazioni BRAF nei carcinomi papillari primitivi, nelle metastasi linfonodali al momento della diagnosi e nelle recidive e definire se tale frequenza sia maggiore nei tumori che recidivano e in particolare in quelli con recidive non iodofissanti; 2) valutare se la presenza di mutazioni BRAF si correli ai fenotipi con accelerato metabolismo del glucosio FDG-PET positivi. Pazienti e metodi: abbiamo selezionato 42 pazienti consecutivi operati per recidiva di carcinoma tiroideo differenziato; alla diagnosi tutti i pazienti erano stati sottoposti a tiroidectomia totale e a terapia radioablativa con 131-I. In questi pazienti la scintigrafia totale corporea è stata eseguita 3 giorni dopo una dose terapeutica di 131-I (2,7-5,4 GBq) dopo aver sospeso la terapia con ormone tiroideo. 23/42 pazienti sono stati sottoposti a 18FDG PET-TC. La presenza di mutazioni a carico dell’esone 15 di BRAF è stata analizzata tramite sequenziaggio e PCR allele-specifica (MASA). Risultati: tra le recidive di carcinoma papillare, in base allo staging tumorale, 10/42 (24%) erano T1, 8/42 (19%) T2, 4/42 (10%) T3 e 18/42 (43%) T4. (Di 2/42 pazienti non era disponibile la classificazione TNM). L'età media alla diagnosi era di 40, la mediana 35 anni (min 16, max 79). L'età media al momento della recidiva era di 45, la mediana 43 anni (min 16, max 82). La media dei valori di Tg durante terapia soppressiva era 181, mentre la mediana era 7 ng/ml (min.<0.5 max 3344). 11/42 pazienti presentavano positività/persistenza degli anticorpi anti-Tg durante il follow-up. BRAF risultava mutato in 19/37 (51%) recidive non iodofissanti e in 1/5 (20%) delle iodofissanti. Abbiamo quindi correlato la presenza di mutazioni BRAF con la capacità di captare il radioiodio e il 18-FDG (15 casi): di tali 15 casi, 14 erano recidive non iodiofissanti, mentre 1 era iodofissante. Nell’insieme delle recidive BRAF-mutate, tra le non iodofissanti 73% captavano il 18-FDG e 20% non lo captavano, mentre l’unica recidiva iodofissante BRAF-mutata era PET-positiva. Inoltre la presenza della mutazione correlava con gli indici di uptake più elevati. In 10 pazienti l’analisi delle mutazioni di BRAF è stata condotta simultaneamente sul tumore primitivo (K0), sulla metastasi linfonodale al momento della diagnosi (N0) e sulla prima recidiva non iodofissante (R1) asportata chirurgicamente e sulle eventuali successive (R2): BRAF risultava mutato in 2/10 (20%) tumori primitivi, in 3/7 (42%) metastasi linfonodali al momento della diagnosi e in 7/10 (70%) prime recidive non iodofissanti e 2/2 (100%) seconde recidive. Tramite sequenziaggio abbiamo inoltre dimostrato una nuova mutazione che interessa il codone 602 dell’esone 15 di BRAF e porta alla sostituzione dell’amminoacido serina con la fenilalanina (S602F). Conclusioni: nel nostro studio abbiamo dimostrato che 1) le mutazioni BRAF sono più frequenti nelle recidive non iodofissanti rispetto a quelle iodofissanti (51 % versus 20 %); 2) esse possono comparire de novo in corso di recidiva di tumore tiroideo primitivo (20% versus 42% e 70% rispettivamente nel tumore primitivo, nella metastasi linfonodale alla diagnosi e nella recidiva non iodofissante); 3) esiste una correlazione inversa tra la presenza della mutazione e la capacità di fissare lo iodio, ed un’associazione diretta tra la presenza della mutazione e la capacità di captare il 18-FDG e le recidive che mostrano la mutazione V600E hanno un indice di captazione elevato, suggerendo quindi l’esistenza di una correlazione tra il grado di captazione di 18-FDG e la presenza della mutazione; 4) una nuova mutazione di BRAF non riportata in Letteratura è presente a livello del codone 602 dell’esone 15 di BRAF e determina la sostituzione di un amminoacido polare - serina - con uno apolare - fenilalanina. In base ai dati del nostro studio sembrerebbe che BRAF si confermi come probabile marcatore molecolare di prognosi povera, in grado di promuovere lo sviluppo di recidive più aggressive, meno differenziate e meno iodocaptanti durante il follow-up

EF24 (a curcumin analog) and ZSTK474 emphasizes the effect of cabozantinib in medullary thyroid cancer

XL184 is a small-molecule kinase inhibitor recently included in first-line systemic therapy for patients with advanced, progressive medullary thyroid cancer (MTC). EF24 is a curcumin analog with a high bio-availability, and ZSTK474 is an inhibitor of the PI3K signaling pathway. We investigated the effect of these compounds, alone and in combination, in two RET-mutated TT and MZ-CRC-1 MTC cell lines, and in six mostly RET wild-type human MTC primary cultures. Low IC50 values demonstrated the efficacy of the drugs, while the Combination Index revealed a significant synergistic effect of combinations of XL184+ZSTK474 and XL184+EF24. Cell cycle changes and the induction of apoptosis or necrosis were modulated by single compounds or combinations thereof. Both XL184 and EF24, alone or combined, were effective in reducing calcitonin secretion. Western blot and in-cell Western analysis showed that the compounds prompted a decrease in general reactivity to phosphorylated antibodies. Our data confirm XL184 alone as the reference drug for RET-mutated MTC, but we also demonstrated for the first time that EF24 alone is effective in inhibiting MTC cell viability. We tested the new combinations XL184+ZSTK474 and XL184+EF24 for the first time too, finding that they act synergistically irrespective of RET mutation status

Circulating cell-free DNA, SLC5A8 and SLC26A4 hypermethylation, BRAFV600E: A non-invasive tool panel for early detection of thyroid cancer

Purpose: In the latest years, high levels of circulating cell-free DNA (cf-DNA) have been found to be associated with cancer diagnosis and progression, and cf-DNA has become a potential candidate as biomarker for tumor detection. cf-DNA has been investigated in plasma or serum of many tumor patients affected by different malignancies, but not yet in thyroid cancer (TC). Furthermore, in TC cells the capability to metabolize iodine is frequently lost. SLC5A8 and SLC26A4 genes are both involved in the iodine metabolism, and SLC5A8 hypermethylation status is associated with the BRAF(V600E) mutation, which is the most frequent genetic event underlying the development of papillary TC. The aim of our study is the development of a new non-invasive tool for the diagnosis and prognosis of TC based on cf-DNA, SLC5A8 and SLC26A4 hypermethylation, and BRAF(V600E) analysis. Methods: cf-DNA was measured by quantitative real-time PCR in nine cases of anaplastic thyroid cancer (ATC), 58 medullary thyroid cancers (MTC), five of synchronous medullary and follicular thyroid cancers (SMFC), 23 follicular adenomas (FA), 86 papillary thyroid cancers (PTC). A control group of 19 healthy subjects was taken. Moreover, in the PTC group we analyze the state of hypermethylation of SLC5A8 and SLC26A4, BRAF(V600E) mutation, and their involvement in the loss of function of the thyroid. Results: cf-DNA showed a high ability to discriminate healthy individuals from cancer patients. cf-DNA(ALU83) and cf-DNA(ALU244) values were significantly correlated with the histological type of TC (P-value < 0.0001). A significant increase in the amount of cf-DNA(ALU83) and cf-DNA(ALU244) when methylation occurs was observed (P-value = 0.02). A correlation between BRAF(V600E) and cf-DNA(ALU244/ALU83) was also found (P-value = 0.02). Conclusions: According to our experimental results, the panel including cf-DNA, SLC5A8 and SLC26A4 hypermethylation, and BRAF(V600E) analysis appears easy, reproducible, and non-invasive for the diagnosis on TC. Its possible implication in clinical setting remains to be elucidated. (C) 2013 Elsevier Masson SAS. All rights reserved

The Hobnail variant of Papillary Thyroid Carcinoma: clinical/molecular characteristics of a large monocentric series and comparison with conventional histotypes

Background: The hobnail variant of papillary thyroid carcinoma (HPTC) has an aggressive behavior. The aims of this prospective study were to define the clinical/molecular characteristics of HPTC, and to compare them to those of conventional papillary thyroid carcinoma (PTC). Methods: From 2010 to 2016, 25 cases of HPTC, characterized clinically and molecularly (BRAF, RAS, TERT promoter, and TP53 mutations), were compared to a series of 165 consecutive cases of PTC. All patients underwent total thyroidectomy and received radioactive iodine treatment. Follow-up was available for 19 HPTC patients. Results: Among the HPTC patients, 64% had a hobnail component 30%, and 64% had multifocal disease. The mean tumor size was 30 mm; 96% of tumors were angio-invasive; 68% were N1, and 12% were M1; 58% harbored the BRAF(V600E) mutation, 12% had a mutation in the TERT promoter, 17% had a TP53 mutation, and not had a RAS mutation. At a mean follow-up of 39 months, 32% of patients had biochemical and/or structural disease. Tumor size was the only significant difference between patients with persistent disease and those with an excellent response (40 mm and 24 mm, respectively; p = 0.02). Compared to the PTC control group, the HPTC patients had larger tumors (30 mm vs. 16 mm; p < 0.001), more frequent lymph node involvement (68% vs. 38%; p = 0.01), and remote disease (16% vs. 3%; p < 0.0001), a similar prevalence of the BRAF(V600E) mutation (58% vs. 59%), a higher prevalence of TP53 mutations (17% vs. 1%; p < 0.05), and a worse outcome (structural/biochemical disease: 32% vs. 9%; p < 0.0001). Conclusions: HPTC is an aggressive variant, characterized by large tumor size, lymph node involvement, a tendency to metastasize, and a worse outcome