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.

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.

The aim of our study was to address the potential for improvements in thyroid cancer detection in routine clinical settings using a clinical examination, the American College of Radiology Thyroid Imaging Reporting and Database System (ACR TI-RADS), and fine-needle aspiration cytology (FNAC) concurrently with molecular diagnostics. A prospective cohort study was performed on 178 patients. DNA from FNA samples was used for next-generation sequencing to identify mutations in the genes BRAF, HRAS, KRAS, NRAS, and TERT. RNA was used for real-time PCR to detect fusion genes. The strongest relevant positive predictors for malignancy were the presence of genetic mutations (p < 0.01), followed by FNAC (p < 0.01) and ACR TI-RADS (p < 0.01). Overall, FNAC, ACR TI-RADS, and genetic testing reached a sensitivity of up to 96.1% and a specificity of 88.3%, with a diagnostic odds ratio (DOR) of 183.6. Sensitivity, specificity, and DOR decreased to 75.0%, 88.9%, and 24.0, respectively, for indeterminate (Bethesda III, IV) FNAC results. FNA molecular testing has substantial potential for thyroid malignancy detection and could lead to improvements in our approaches to patients. However, clinical examination, ACR TI-RADS, and FNAC remained relevant factors.

Introduction: Medullary thyroid carcinoma (MTC) is a calcitonin-producing tumor that predominantly occurs in a sporadic form (75%) and less commonly in an inherited form. Besides activating germline mutations of the RET proto-oncogene in hereditary syndromes of MEN2, somatic RET mutations are detectable in about 50% of sporadic MTC. Further, also RAS mutations have been discovered in 30% of RET-negative tumor tissues. Other genetic alterations, chromosomal rearrangements or point mutations in minor genes, are very rare. Case Report: We report a case of a 38-year-old woman with a nodule in the left lobe, cytologically suspected of MTC. Serum calcitonin was elevated at 217 pmol/l. The patient underwent total thyroidectomy and histopathological examination revealed a 1.3 cm MTC with positive immunohistochemical staining for calcitonin and chromogranin. Molecular genetic analysis detected neither germline RET mutation, nor RET/RAS somatic mutations in examined fresh frozen tumor tissue. A comprehensive NGS panel targeted especially fusion genes, but also other genetic changes, was used for subsequent analysis. Surprisingly, a common V600E BRAF mutation, typical for papillary thyroid carcinoma (PTC), was found. The mutation was confirmed by allele-specific real-time PCR performed from material isolated from both fresh frozen and FFPE tumor tissue. The histological examination demonstrated morphologic features of MTC, no signs of mixed tumor and no evidence of PTC. Interestingly, beside positivity for calcitonin and chromogranin, it showed strong and diffuse CK19 expression, an immunohistochemical marker, typical for diagnosis of PTC. Conclusions: Only two other cases of BRAF V600E in MTC patients have been reported. In all these studies, the results of molecular genetic analysis were verified by an alternate method, and the tumors were histologically confirmed as pure medullary thyroid carcinomas, without concurrent PTC. The study was supported by projects of the MHCR AZV NU21-01-00448 and MHCR–RVO (00023761).

Introduction: Thyroid carcinomas are the most often endocrine malignancy and their incidence is still growing. Thus, the finding of genetic predispositions to the thyroid cancer is desired. One of the genetic causes can be risk variants of tumor suppressor genes in patients. Our goal was to determine the influence of polymorphisms Val109Gly (T/G) in gene CDKN1B encoding protein p27/Kip1 and Arg72Pro (C/G) in gene TP53 encoding protein p53 on the development of thyroid cancer. Both these genes are crucial in regulation of cell cycle. Methods: Three cohorts were studied – 345 patients with sporadic medullary thyroid carcinoma (MTC), 269 patients with papillary thyroid carcinoma (PTC), and 374 healthy controls. DNA was isolated from peripheral leukocytes or from thyroid cancer tissues using QIAamp DNA Blood Kit or Trizol. Variants of polymorphisms were analyzed using TaqMan specific sonds on LightCycler 480. Statistical evaluation was performed by NCSS 2004 programme. Results: Although total distributions of alleles of each gene were not different in patients compared to controls, the remarkable risk is in combination of specific alleles of these genes. G allele in TP53 in combination with T allele in CDKN1B is more often in patients with PTC than in controls (84.4 vs 74.7%, OR=1.82; 95%CI (1.23–2.70); P=0.003) and specifically the TT genotype in CDKN1B is the most risk in comparison with controls (70.8 vs 56.6%, OR=1.86; 95%CI (1.18–2.94); P=0.01). In MTC patients, C allele in TP53 with GG genotype in CDKN1B is more frequent compared to controls (6.2 vs 2.9%, OR=2.2; 95%CI (1.19–4.08); P=0.016). Conclusion: It seems that genetic variants of tumor suppressor genes and mainly their cumulative risk effect play the role in the development of PTC and MTC. This work was supported by IGA MH CZ NT/13901-4 and MH CZ 00023761.