RET fusion genes in pediatric and adult thyroid carcinomas: cohort characteristics and prognosis.

Thyroid cancer is associated with a broad range of different mutations, including RET (rearranged during transfection) fusion genes. The importance of characterizing RET fusion-positive tumors has recently increased due to the possibility of targeted treatment. The aim of this study was to identify RET fusion-positive thyroid tumors, correlate them with clinicopathological features, compare them with other mutated carcinomas, and evaluate long-term follow-up of patients. The cohort consisted of 1564 different thyroid tissue samples (including 1164 thyroid carcinoma samples) from pediatric and adult patients. Samples were analyzed for known driver mutations occurring in thyroid cancer. Negative samples were subjected to extensive RET fusion gene analyses using next-generation sequencing and real-time PCR. RET fusion genes were not detected in any low-risk neoplasm or benign thyroid tissue and were detected only in papillary thyroid carcinomas (PTCs), in 113/993 (11.4%) patients, three times more frequently in pediatric and adolescent patients (29.8%) than in adult patients (8.7%). A total of 20 types of RET fusions were identified. RET fusion-positive carcinomas were associated with aggressive tumor behavior, including high rates of lymph node (75.2%) and distant metastases (18.6%), significantly higher than in NTRK fusion, BRAF V600E and RAS-positive carcinomas. Local and distant metastases were also frequently found in patients with microcarcinomas positive for the RET fusions. 'True recurrences' occurred rarely (2.4%) and only in adult patients. The 2-, 5-, 10-year disease-specific survival rates were 99%, 96%, and 95%, respectively. RET fusion-positive carcinomas were associated with high invasiveness and metastatic activity, but probably due to intensive treatment with low patient mortality.

NTRK Fusion Genes in Thyroid Carcinomas: Clinicopathological Characteristics and Their Impacts on Prognosis.

Chromosomal rearrangements of NTRK genes are oncogenic driver mutations in thyroid cancer (TC). This study aimed to identify NTRK fusion-positive thyroid tumors and to correlate them with clinical and pathological data and determine their prognostic significance. The cohort consisted of 989 different TC samples. Based on the detected mutation, samples were triaged, and those that were positive for a BRAF, HRAS, KRAS, NRAS, RET, RET/PTC or PAX8/PPARγ mutation were excluded from further analyses. NTRK fusion gene testing was performed in 259 cases, including 126 cases using next-generation sequencing. NTRK fusion genes were detected in 57 of 846 (6.7%) papillary thyroid carcinomas and in 2 of 10 (20.0%) poorly differentiated thyroid carcinomas. A total of eight types of NTRK fusions were found, including ETV6/NTRK3, EML4/NTRK3, RBPMS/NTRK3, SQSTM1/NTRK3, TPM3/NTRK1, IRF2BP2/NTRK1, SQSTM1/NTRK1 and TPR/NTRK1.NTRK fusion-positive carcinomas were associated with the follicular growth pattern, chronic lymphocytic thyroiditis and lymph node metastases. NTRK1-rearranged carcinomas showed a higher frequency of multifocality and aggressivity than NTRK3-rearranged carcinomas. Tumor size, presence of metastases, positivity for the NTRK3 or NTRK1 fusion gene and a late mutation event (TERT or TP53 mutation) were determined as factors affecting patient prognosis. NTRK fusion genes are valuable diagnostic and prognostic markers.

RET, NTRK, ALK, BRAF, and MET Fusions in a Large Cohort of Pediatric Papillary Thyroid Carcinomas.

Background: Pediatric papillary thyroid carcinoma (PTC) is a rare malignancy, but with increasing incidence. Pediatric PTCs have distinct clinical and pathological features and even the molecular profile differs from adult PTCs. Somatic point mutations in pediatric PTCs have been previously described and studied, but complex information about fusion genes is lacking. The aim of this study was to identify different fusion genes in a large cohort of pediatric PTCs and to correlate them with clinical and pathological data of patients. Methods: The cohort consisted of 93 pediatric PTC patients (6-20 years old). DNA and RNA were extracted from fresh frozen tissue samples, followed by DNA and RNA-targeted next-generation sequencing analyses. Fusion gene-positive samples were verified by real-time polymerase chain reaction. Results: A genetic alteration was found in 72/93 (77.4%) pediatric PTC cases. In 52/93 (55.9%) pediatric PTC patients, a fusion gene was detected. Twenty different types of RET, NTRK3, ALK, NTRK1, BRAF, and MET fusions were found, of which five novel, TPR/RET, IKBKG/RET, BBIP1/RET, OPTN/BRAF, and EML4/MET, rearrangements were identified and a CUL1/BRAF rearrangement that has not been previously described in thyroid cancer. Fusion gene-positive PTCs were significantly associated with the mixture of classical and follicular variants of PTC, extrathyroidal extension, higher T classification, lymph node and distant metastases, chronic lymphocytic thyroiditis, and frequent occurrence of psammoma bodies compared with fusion gene-negative PTCs. Fusion-positive patients also received more doses of radioiodine therapy. The most common fusion genes were the RET fusions, followed by NTRK3 fusions. RET fusions were associated with more frequent lymph node and distant metastases and psammoma bodies, and NTRK3 fusions were associated with the follicular variant of PTC. Conclusions: Fusion genes were the most common genetic alterations in pediatric PTCs. Fusion gene-positive PTCs were associated with more aggressive disease than fusion gene-negative PTCs.

Somatic genetic alterations in a large cohort of pediatric thyroid nodules.

There is a rise in the incidence of thyroid nodules in pediatric patients. Most of them are benign tissues, but part of them can cause papillary thyroid cancer (PTC). The aim of this study was to detect the mutations in commonly investigated genes as well as in novel PTC-causing genes in thyroid nodules and to correlate the found mutations with clinical and pathological data. The cohort of 113 pediatric samples consisted of 30 benign lesions and 83 PTCs. DNA from samples was used for next-generation sequencing to identify mutations in the following genes: HRAS, KRAS, NRAS, BRAF, IDH1, CHEK2, PPM1D, EIF1AX, EZH1 and for capillary sequencing in case of the TERT promoter. RNA was used for real-time PCR to detect RET/PTC1 and RET/PTC3 rearrangements. Total detection rate of mutations was 5/30 in benign tissues and 35/83 in PTCs. Mutations in RAS genes (HRAS G13R, KRAS G12D, KRAS Q61R, NRAS Q61R) were detected in benign lesions and HRAS Q61R and NRAS Q61K mutations in PTCs. The RET/PTC rearrangement was identified in 18/83 of PTCs and was significantly associated with higher frequency of local and distant metastases. The BRAF V600E mutation was identified in 15/83 of PTCs and significantly correlated with higher age of patients and classical variant of PTC. Germline variants in the genes IDH1, CHEK2 and PPM1D were found. In conclusion, RET/PTC rearrangements and BRAF mutations were associated with different clinical and histopathological features of pediatric PTC. RAS mutations were detected with high frequency in patients with benign nodules; thus, our results suggest that these patients should be followed up intensively.

[Thyroid carcinomas: the present view on diagnostics and therapy]. / Karcinomy stítné zlázy: soucasný pohled na diagnostiku a lécbu.

Thyroid carcinoma (TC) represents 1-2 % of all human tumors, and is the seventh most common tumor. Women are in large majority among new patients. For women, this is the fifth most common tumor. In the Czech Republic, 1 143 new cases of TC were diagnosed in 2015. It is the tumor with the highest increase in incidence. Among newly diagnosed tumors, most of those are differentiated thyroid gland carcinomas (DTCs) originating from follicular thyroid cells. These tumors are follicular and papillary carcinomas and Hurthle carcinoma, accounting for 95 % of new cases. Due to the great progress in treatment, the prognosis is most commonly good for these tumors. Treatment is more difficult for other types of tumors. Anaplastic thyroid cancer (representing less than 1 % of thyroid tumors) is a rare form of thyroid cancer that is very malignant. Also found in the thyroid gland is Euro-C-cell tumor, which originates in C cells. This is the so-called medullary thyroid carcinoma, which is less common (5 % of all thyroid carcinomas). It emerges from the parapolyclic neuroendocrine cells of the thyroid gland. This tumor often metastasizes to the cervical lymph nodes, and frequently occurs in distant bone, liver and lung metastases. In 2015, in this publication we published an article: Thyroid gland carcinomas, current therapeutic procedures. This article was devoted to the diagnosis of thyroid carcinoma and individual treatment procedures. In this article, we look at differentiated thyroid carcinomas (DTCs), especially current opinions on the treatment of low-risk carcinomas.Key words: differentiated thyroid cancer - radioidine - targeted therapy.

[Thyroid cancer in children and adolescents and its molecular genetic background]. / Nádory stítné zlázy u detí a dospívajících a jejich molekulárne genetická podstata.

Thyroid cancer is the main endocrine malignancy. Its incidence is steadily growing and what is alarming is its increase in children and adolescent population. Pediatric thyroid carcinomas differ from the adult ones in phenotype as well as in genetics. These carcinomas tend to be clinically more aggressive, with more frequent local and distant metastases. However, their long-term prognosis is better in comparison with the adult thyroid cancers. Due to the rarity of the disease, there is lack of data on genetic changes in this age group. Knowledge on the genetic background of thyroid cancer in children will help to precise diagnosis and prognosis of the disease and to personalized treatment.Key words: adolescents - carcinoma - gene - genetics - children - mutation - next generation sequencing - thyroid.

Polymorphisms in selected DNA repair genes and cell cycle regulating genes involved in the risk of papillary thyroid carcinoma.

BACKGROUND: Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer. In addition to causal somatic mutations in the BRAF gene and RET/PTC rearrangements, the contribution of single nucleotide polymorphisms (SNPs) in low-penetrance genes in the development of PTC has been proposed. METHODS: Four SNPs in the XRCC1 (Arg399Gln, Arg280His, Arg194Trp and T-77C) and one SNP from each of three other genes participating in DNA repair pathways and/or cell cycle regulation (ATM Asp1853Asn, TP53 Arg72Pro, CDKN1B Val109Gly) were selected. The allelic and genotypic distributions of these variants as well as haplotypes of the XRCC1 were examined in 583 individuals comprising well-characterized cohorts of 209 PTC patients and 374 healthy volunteers. Correlations of polymorphism with clinical-pathological data and mutation status were performed. RESULTS: XRCC1 T-77C polymorphism affects the genetic susceptibility for PTC development in men, the specific combination of XRCC1 haplotypes correlates with RET/PTC incidence, CDKN1B Val109Gly significantly influences the risk of developing PTC regardless of gender and in PTC cases, selected genotypes of TP53 Arg72Pro and ATM Asp1853Asn were significantly associated with monitored tumour characteristics. CONCLUSION: It seems that SNPs in studied regulating genes contribute to the development of PTC and modify the tumour behaviour or characteristics.

A novel RET/PTC variant detected in a pediatric patient with papillary thyroid cancer without ionization history.

Papillary thyroid carcinoma (PTC) is the most frequent type of thyroid cancer. Its development is often caused by the formation of RET/PTC fused genes. RET/PTC1 is the most prevalent form, where exon 1 of CCDC6 gene is fused with the intracellular portion of RET protooncogene starting with exon 12. We have discovered a novel RET/PTC1 variant which we have named RET/PTC1ex9 in metastatic PTC of 8-year-old boy. RET/PTC1ex9 detection was performed by real-time polymerase chain reaction with melting curve analysis and subsequent Sanger and next-generation sequencing. A fusion of exon 1 of CCDC6 with exon 9 of extracellular domain of RET followed by exon 12 of RET was revealed. This is the first RET/PTC variant among PTC cases that contain the extracellular part of RET. This observation could be probably explained by incorrect splicing of RET due to the somatic 32-bp deletion in exon-intron 11 boundary of RET.

Search for new genetic biomarkers in poorly differentiated and anaplastic thyroid carcinomas using next generation sequencing.

BACKGROUND: Poorly differentiated thyroid carcinoma (PDTC) and anaplastic thyroid carcinoma (ATC) are very rare tumors with extremely aggressive behavior. Their comprehensive genetic background is still unclear. Some of the main genetic changes of differentiated thyroid carcinomas, such as mutations in BRAF and RAS genes, as well as changes in CTNNB1, PIK3CA, TP53, AXIN1, PTEN or APC genes leading to the dedifferentiation of the tumors, are described. MATERIALS AND METHODS: DNAs from fresh frozen thyroid tissues of 3 PDTCs and 5 ATCs were extracted. The next-generation sequencing (NGS) approach was used to target 94 genes involved in cancer. The samples were prepared using a TruSight Cancer panel and sequenced with a MiSeq sequencer. Analysis of variants was performed by the MiSeq Reporter and NextGENe software and stringent criteria for prioritization of the variants were used in the Illumina VariantStudio software. RESULTS: Using NGS, we identified 26 genetic changes in 18 genes, novel variants included. CONCLUSION: NGS is a useful tool for searching for new variants and genes involved in PDTC and ATC. It seems that each of these rare tumor types has its own specific genetic background. These data could be helpful for recognizing new genetic markers and targets for future personalized therapy.

Polyclonal, newly derived T cells with low expression of inhibitory molecule PD-1 in tonsils define the phenotype of lymphocytes in children with Periodic Fever, Aphtous Stomatitis, Pharyngitis and Adenitis (PFAPA) syndrome.

PURPOSE: PFAPA syndrome is a benign, recurrent inflammatory disease of childhood. Tonsillectomy is one of the therapeutic options with a yet unexplained biological mechanism. We tested whether specific lymphocyte subsets recruited from blood to human tonsils participate in PFAPA pathogenesis. METHODS: Paired tonsils/peripheral blood (PB) samples were investigated (a) from children with PFAPA that successfully resolved after tonsillectomy (n=10) (b) from children with obstructive sleep apnoea syndrome as controls (n=10). The lymphocyte profiles were analysed using 8-colour flow cytometry, immunoglobulin (IGH) and T-cell receptor (TCR) gene rearrangements via PCR and next generation sequencing; a TREC/KREC analysis was performed using qPCR. RESULTS: The PFAPA tonsils in the asymptomatic phase had a lower percentage of B-lymphocytes than controls; T-lymphocyte counts were significantly higher in PB. The percentages of cytotoxic CD8pos T-lymphocytes were approximately 2-fold higher in PFAPA tonsils; the transitional B cells and naïve stages of both the CD4pos and CD8pos T-lymphocytes with a low expression of PD-1 molecule and high numbers of TREC were also increased. With the exception of elevated plasmablasts, no other differences were significant in PB. The expression levels of CXCL10, CXCL9 and CCL19 genes were significantly higher in PFAPA tonsils. The IGH/TCR pattern showed no clonal/oligoclonal expansion. DNA from the Epstein-Barr virus, Human Herpervirus-6 or adenovirus was detected in 7 of 10 PFAPA tonsils but also in 7 of 9 controls. CONCLUSIONS: Our findings suggest that the uninhibited, polyclonal response of newly derived lymphocytes participate in the pathogenesis of PFAPA. Because most of the observed changes were restricted to tonsils and were not present in PB, they partly explain the therapeutic success of tonsillectomy in PFAPA syndrome.

Thyroid carcinoma surgery in children and adolescents - 15 years experience surgery of pediatric thyroid carcinoma.

OBJECTIVES: The purpose of this study is to evaluate the characteristics of thyroid gland surgery focusing on malignancies at the pediatric age with the main concern on treatment results and complications in extensive primary treatment. METHODS: The records of all patients 18 years and younger with surgically treated thyroid diseases in the Prague Hospital, Motol, between 1991 and 2006 were retrospectively reviewed. RESULTS: Thyroid surgery was performed on 148 pediatric patients (including 56 carcinomas). The youngest patient involved in the study was seven years old, the oldest patient 18 years old (mean 13.7 years). Most frequent histological cancer type was PTC (42 cases, 75%). Follicular cancer was diagnosed in five cases (8.9%) and medullar cancer in nine cases (16.1%). A prophylactic thyroidectomy was performed in three cases (5.4%) without clinical signs of thyroid tumor with diagnosed RET gene mutation. CONCLUSIONS: We consider total thyroidectomy with subsequent radioiodine ablation and TSH suppression as the basic approach in the treatment protocol of pediatric WDTC. The observed 100% recurrence-free and overall survival together with a low incidence of postoperative complications strongly supports the idea of a total thyroidectomy with selective neck dissection in the treatment of metastases of WDTC and MTC.

Prevalence of Helicobacter pylori in adenotonsillar hypertrophy in children.

CONCLUSION: Our results encourage the notion that the pharynx could be an extragastric reservoir of Helicobacter pylori (HP). The study confirmed the presence of HP in adenotonsillar tissue in children. It could have importance in the pathophysiology of upper respiratory diseases. However, its precise role in these processes remains unclear and requires further studies. OBJECTIVE: A prospective study was carried out to evaluate the presence of HP in tonsillar and adenoid tissue in children. The study focused on real-time PCR analyzing CagA and VacA genotypes of HP strains. METHODS: A total of 37 consecutive pediatric patients with adenotonsillar hypertrophy indicated for surgery were observed in a prospective study. Adenoidectomy and/or tonsillectomy was performed in each patient; 49 specimens were taken, 32 from adenoids and 17 from tonsils. The presence of HP and its genotype were tested in all samples by real-time PCR analysis. RESULTS: Of 49 samples analyzed, 48 were positive for the presence of HP (98%), so only 1 sample was negative. While the genotype VacAs1bm2 was definitely dominant in adenoid tissue, wider distribution was observed in tonsillar tissue. Cag(+) strains represented one-fifth of all samples (21%).

Detection of Helicobacter pylori in oropharyngeal lymphatic tissue with real-time PCR and assessment of its carcinogenic potential.

Helicobacter pylori (HP) is considered a major gastric pathogen with oncogenic potential. The aim of this study was to determine whether HP is present in oropharyngeal lymphoid tissue and whether oropharyngeal HP strains carry virulence factor genes known to be involved in gastric carcinogenesis. The study included 104 subjects (41 patients with tonsillar carcinoma, 38 with chronic tonsillitis and 25 with obstructive sleep apnoea syndrome--OSAS). Detection of specific serum anti-HP antibodies was performed with an ELISA. The presence of HP in tissue was determined by culture and real-time PCR. Detection of virulence factors genes was also performed. Specific antibodies were found in 78.05% of tumour cases, 34.21% of chronic tonsillitis cases, and 72.0% of OSAS cases. The presence of HP in the tissue was detected in 73.91% of tonsillar tumours, 70.0% of tonsillitis cases, and 69.23% of OSAS specimens. The results of the virulence factor gene analysis showed the majority of the s1b (52.4%) and m2 (59.5%) alleles of vacA gene and limited abundance of cagA gene (12.5%). Results confirm that HP may colonise oropharyngeal lymphoid tissue. Oropharyngeal HP colonisation was frequently found in the oropharyngeal cancer group and in patients with benign oropharyngeal diseases. A virulence factor gene analysis showed differences from the predominant strains most commonly found in the stomach. The strains obtained from the oropharynx differed primarily by the lower abundance of the cagA gene and carried the less virulent vacA gene allele combination.

Presence of different genotypes of Helicobacter pylori in patients with chronic tonsillitis and sleep apnoea syndrome.

Helicobacter pylori, a well-known gastric pathogen, has been detected in the oral cavity and oropharynx in tonsillar tissue. In our study, the presence of H. pylori in the tonsillar tissue of patients with chronic tonsillitis and sleep apnoea syndrome (SAS) was investigated. The aim was to detect and genotype H. pylori for a collection of data supporting the possible role of H. pylori in the aetiology of chronic tonsillitis and SAS. Helicobacter pylori was detected by real-time polymerase chain reaction (rt-PCR). 89 patients, 60 with a diagnosis of chronic tonsillitis and 29 with SAS, were tested. In the chronic tonsillitis group, Helicobacter was detected in 48 (80 %) specimens, cagA gene was detected in 12 samples (25 %) and 12 samples were negative. In SAS group, Helicobacter was found in 24 samples (82.76 %), cagA gene was detected in 5 (20.83 %) and 5 samples (17.24 %) were negative. Helicobacter pylori-specific immunoglobulins were tested by ELISA in the serum of 57 patients only with 41 (71.93 %) showing positive. Our results on H. pylori DNA detection and H. pylori seropositivity show 26.32 % discrepancy, slightly in favour of rt-PCR (15.79 % compared to 10.53 %). The H. pylori presence in tonsillar tissue does not depend on the type of oropharyngeal disease (p = 0.756). This study shows that oropharynx constitutes an extragastric reservoir of H. pylori infection which could serve as an aetiopathogenetic factor for chronic tonsillitis and tonsillar hyperplasia by SAS. No conclusion has yet been drawn about the mechanism of the process.

PFAPA syndrome: clinical characteristics and treatment outcomes in a large single-centre cohort.

OBJECTIVES: This paper aims to describe clinical and laboratory features and disease outcome in a single-centre cohort of patients with PFAPA syndrome (Periodic Fever, Aphtous stomatitis, Pharyngitis, and Adenitis) and to test performance of diagnostic and therapeutic algorithms. METHODS: Patients fulfilling criteria were selected from the fever clinic population. Prospective follow-up together with recruitment of newly diagnosed patients followed pre-defined guidelines. Diagnostic and therapeutic algorithms and definitions of outcome and therapy response were formulated. Paired blood samples during febrile and afebrile periods were compared. RESULTS: Out of 176 patients referred for suspected periodic fever 125 children fulfilled criteria. Their age at onset was 23 months, median episode duration 3.5 days at 4-week intervals. Fever was associated with pharyngitis (91%), cervical adenitis (78%) and aphtae (41%). Among therapeutic options, episodic prednisone proved to be the most common first-line treatment. Administered to 77 patients, it reduced symptoms in 94%. Tonsillectomy led to the full symptom resolution in all 18 patients. Forty-six patients reached disease remission. CONCLUSIONS: Distribution of typical symptoms, response to therapies and disease outcome in a large patient cohort were documented. We offer diagnostic and therapeutic algorithms that have proven effective during this prospective trial. Our findings support the general belief of benign nature of this aetiologically unclear condition, despite proportion of patients having persistent disease for years. Maintenance of normal findings in afebrile intervals, striking response to a single dose of prednisone and normal growth and development together with spontaneous tendency towards prolongation of afebrile intervals are important confirmatory features of PFAPA syndrome.

Bacterial infection complications in children with cochlear implants in the Czech Republic.

OBJECTIVE: Studies describing wound infections after cochlear implantation are rare. Meticulous operative techniques and sufficient surgical skill can help to avoid severe postoperative complications. Minor complications such as seromas, superficial wound infections, skin emphysema, and swelling can all be successfully treated using conservative methods. Serious problems, however, could be caused by Pseudomonas aeruginosa and Staphylococcus aureus infections. STUDY DESIGN: Retrospective case series. SETTING: Pediatric cochlear implant center in the Czech Republic. RESULTS: In our cohort of 360 children operated on between 1994 and 2009, there were no major surgical complications. However, there were four serious wound infections, two of which resulted in explantation of the device. Two patients with a well-bordered abscess formation were successfully treated with antibiotics and drainage. Explantation was required in another two children with refractory P. aeruginosa infection. Despite favorable sensitivity to a number of antibiotics, intravenous and intensive local treatment failed. After explantation, the operative sites healed immediately. Both children were successfully implanted contralaterally 6 months later. Detailed bacteriological tests from different parts of the device were performed. CONCLUSION: The consequences of explantation were discussed in our cochlear implant center. Confirmed by the microbiological results and reports in the scientific literature, it seems preferable to cut the electrode near the cochlear array and leave it inside the cochlea in order to use the same ear for reimplantation at a later date.