The rs2910164 Genetic Variant of miR-146a-3p Is Associated with Increased Overall Mortality in Patients with Follicular Variant Papillary Thyroid Carcinoma.

Aberrant expression of the sodium-iodide symporter (NIS) and the resistance to post-operative radioactive iodide treatment is a crucial cause of higher mortality of some thyroid cancer patients. In this study, we analyzed the impact of miR-146a on the expression and function of NIS and on the overall survival of thyroid cancer patients. The study included 2441 patients (2163 women; 278 men); including 359 cases with follicular variant of papillary thyroid carcinoma (fvPTC). miR:NIS interactions were analyzed in cell lines using in vivo binding and inhibition assays and radioactive iodine uptake assays. Tumor/blood DNA was used for rs2910164 genotyping. Overall survival was assessed retrospectively. In the results, we showed that miR-146a-3p directly binds to and inhibits NIS. Inhibition of miR-146a-3p restores the expression and function of NIS, increasing radioactive iodine uptake. Rs2910164 functional variant within miR-146a-3p is associated with increased overall mortality among fvPTC female patients. The deaths per 1000 person-years were 29.7 in CC carriers vs. 5.08 in GG/GC-carriers (HR = 6.21, p = 0.006). Higher mortality of CC vs. GG/GC carriers was also observed in patients with lower clinical stage (HR = 22.72, p < 0.001), smaller tumor size (pT1/pT2) (HR = 25.05, p < 0.001), lack of extrathyroidal invasion (HR = 9.03, p = 0.02), lack of nodular invasion (HR = 7.84, p = 0.002), lack of metastases (HR = 6.5, p = 0.005) and older (age at diagnosis >50 years) (HR = 7.8, p = 0.002). MiR-146a-3p underwent somatic mutations in 16.1% of analyzed specimens, mainly towards the deleterious C allele. In this report we propose a novel molecular marker of the clinical outcome of fvPTC patients. Rs2910164 increases the overall mortality with inhibition of NIS and disruption of radioiodine uptake as a possible mechanism.

Modifying impact of RET gene haplotypes on medullary thyroid carcinoma clinical course.

The clinical course of medullary thyroid carcinoma (MTC) associated with the MEN2A syndrome as well as of sporadic MTC shows considerable heterogeneity. The disease picture varies not only between the same RET proto-oncogene mutation carriers but also among sporadic MTC patients with no RET germinal mutations, which suggests the involvement of additional modulators of the disease. However, genetic factors responsible for this heterogeneity of the MTC clinical course still remain unknown. The aim of this study was to determine if polymorphic variants or specific haplotypes of the RET gene may modify the MTC clinical course. We genotyped the following loci: c.73+9277T>C, c.135G>A, c.1296A>G, c.2071G>A, c.2307T>C, c.2508C>T and c.2712C>G in 142 MTC patients and controls. We demonstrated considerable differences in the genotypes distribution within c.73+9277T>C, c.135G>A and c.2307T>C loci Our results show that the c.73+9277T variant associated with a decreased activity of the MCS+9.7 RET enhancer is rare in hereditary MTC patients with primary hyperparathyroidism, and thus, may influence the MTC clinical picture. The decreased activity of the RET promoter enhancer reduces RET expression level and may counterbalance the activating mutation in this gene. Frequent co-occurrence of the c.73+9277T allele with p.E768D, p.Y791F, p.V804M or p.R844Q RET mutations may be associated with their attenuation and milder clinical picture of the disease. Haplotypes analysis showed that C-G-A-G-T-(C)-C (c.73+9277T>C - c.135G>A - c.1296A>G - c.2071G>A - c.2307T>G - (c.2508C>T) - c.2712C>G) alleles combination predisposes to pheochromocytomas and primary hyperparathyroidism. We consider that RET haplotypes defining may become an auxiliary diagnostic tool in MTC patients.

Analysis of Newly Identified and Rare Synonymous Genetic Variants in the RET Gene in Patients with Medullary Thyroid Carcinoma in Polish Population.

Gain-of-function germline mutations of the RET proto-oncogene are responsible for initiation of carcinogenesis within the thyroid gland and development of hereditary form of medullary thyroid carcinoma and MEN2 syndrome. Genotype-phenotype correlations are established for most RET mutations, but the importance of the synonymous changes in this gene remains debatable. We aimed to analyze RET gene variants in Polish population. Genetic testing for the RET gene variants was performed with standard methods in 585 people aged 1-85, including 448 patients with medullary thyroid carcinoma and 131 of their first- and second-degree relatives, as well as six patients suspected of MTC/MEN2. Besides the most frequent synonymous changes, p.Leu769Leu, p.Ser836Ser, and p.Ser904Ser, four rare changes-c.1827C>T (p.Cys609Cys), c.2364C>T (p.Ile788Ile), c.2418C>T (p.Tyr806Tyr), and c.2673G>A (p.Ser891Ser)-were found in the RET gene, in the Polish population. Two of the rare changes, p.Cys609Cys and p.Ile788Ile, had not been previously described. The frequency of molecular synonymous variants in the general population was evaluated by testing 400 anonymous blood samples of neonates. Our findings may contribute to a better understanding of the genetic diversity of the RET gene and the involvement of synonymous variants in this diversity.

Association between GWAS-Derived rs966423 Genetic Variant and Overall Mortality in Patients with Differentiated Thyroid Cancer.

PURPOSE: Five germline genetic variants (rs116909374, rs965513, rs944289, rs966423, and rs2439302) have been associated in genome-wide association studies (GWAS) with increased risk of differentiated thyroid cancer (DTC), but their role in mortality of patients has not been established. Also, no preoperative marker of the clinical outcome of thyroid cancer had yet been identified. The aim of the study was to investigate the relationship between the variants and overall mortality in patients with DTC. EXPERIMENTAL DESIGN: Retrospective study of 1,836 patients (1,643 women, 193 men) with median age at diagnosis of 49 years and overall median follow-up time of 8.7 years after initial treatment at a single comprehensive cancer center between 1990 and 2013. RESULTS: Among 5 variants, rs966423 was associated with increased mortality, which was 6.4% (33 of 518) versus 3.7% (47 of 1,259) in TT carriers versus CC/CT carriers (P = 0.017). The HR of TT versus TC/CC carriers was 1.6 [95% confidence interval (CI), 1.02-2.49; P = 0.038] after adjustment for age at diagnosis and sex. Importantly, the association of rs966423 with mortality remained valid when clinicopathologic risk factors were included in the model (HR, 1.89; 95% CI, 1.14-3.13; P = 0.014). Higher rs966423-associated patient mortality of TT versus CC/CT carriers was also observed in interaction with angioinvasion (adjusted HR, 3.48; 95% CI, 1.67-7.22; P < 0.001), lymph node metastasis (adjusted HR, 3.47; 95% CI, 1.16-10.4; P = 0.018), extrathyroidal invasion (adjusted HR, 2.07; 95% CI, 1.15-3.73; P = 0.013). CONCLUSIONS: The presence of the rs966423-TT genotype was associated with a significant increase in overall mortality of patients with DTC. Contrary to BRAF mutation and other somatic changes, the status of germline rs966423 is known before the treatment and might be used in the management of mortality risk by means of modification of therapy.

Outcomes of adrenal-sparing surgery or total adrenalectomy in phaeochromocytoma associated with multiple endocrine neoplasia type 2: an international retrospective population-based study.

BACKGROUND: The prevention of medullary thyroid cancer in patients with multiple endocrine neoplasia type 2 syndrome has demonstrated the ability of molecular diagnosis and prophylactic surgery to improve patient outcomes. However, the other major neoplasia associated with multiple endocrine neoplasia type 2, phaeochromocytoma, is not as well characterised in terms of occurrence and treatment outcomes. In this study, we aimed to systematically characterise the outcomes of management of phaeochromocytoma associated with multiple endocrine neoplasia type 2. METHODS: This multinational observational retrospective population-based study compiled data on patients with multiple endocrine neoplasia type 2 from 30 academic medical centres across Europe, the Americas, and Asia. Patients were included if they were carriers of germline pathogenic mutations of the RET gene, or were first-degree relatives with histologically proven medullary thyroid cancer and phaeochromocytoma. We gathered clinical information about patients'RET genotype, type of treatment for phaeochromocytoma (ie, unilateral or bilateral operations as adrenalectomy or adrenal-sparing surgery, and as open or endoscopic operations), and postoperative outcomes (adrenal function, malignancy, and death). The type of surgery was decided by each investigator and the timing of surgery was patient driven. The primary aim of our analysis was to compare disease-free survival after either adrenal-sparing surgery or adrenalectomy. FINDINGS: 1210 patients with multiple endocrine neoplasia type 2 were included in our database, 563 of whom had phaeochromocytoma. Treatment was adrenalectomy in 438 (79%) of 552 operated patients, and adrenal-sparing surgery in 114 (21%). Phaeochromocytoma recurrence occurred in four (3%) of 153 of the operated glands after adrenal-sparing surgery after 6-13 years, compared with 11 (2%) of 717 glands operated by adrenalectomy (p=0.57). Postoperative adrenal insufficiency or steroid dependency developed in 292 (86%) of 339 patients with bilateral phaeochromocytoma who underwent surgery. However, 47 (57%) of 82 patients with bilateral phaeochromocytoma who underwent adrenal-sparing surgery did not become steroid dependent. INTERPRETATION: The treatment of multiple endocrine neoplasia type 2-related phaeochromocytoma continues to rely on adrenalectomies with their associated Addisonian-like complications and consequent lifelong dependency on steroids. Adrenal-sparing surgery, a highly successful treatment option in experienced centres, should be the surgical approach of choice to reduce these complications.

Variants in the ATM-CHEK2-BRCA1 axis determine genetic predisposition and clinical presentation of papillary thyroid carcinoma.

The risk of developing papillary thyroid carcinoma (PTC), the most frequent form of thyroid malignancy, is elevated up to 8.6-fold in first-degree relatives of PTC patients. The familial risk could be explained by high-penetrance mutations in yet unidentified genes, or polygenic action of low-penetrance alleles. Since the DNA-damaging exposure to ionizing radiation is a known risk factor for thyroid cancer, polymorphisms in DNA repair genes are likely to affect this risk. In a search for low-penetrance susceptibility alleles we employed Sequenom technology to genotype deleterious polymorphisms in ATM, CHEK2, and BRCA1 in 1,781 PTC patients and 2,081 healthy controls. As a result of the study, we identified CHEK2 rs17879961 (OR = 2.2, P = 2.37e-10) and BRCA1 rs16941 (odds ratio [OR] = 1.16, P = 0.005) as risk alleles for PTC. The ATM rs1801516 variant modifies the risk associated with the BRCA1 variant by 0.78 (P = 0.02). Both the ATM and BRCA1 variants modify the impact of male gender on clinical variables: T status (P = 0.007), N status (P = 0.05), and stage (P = 0.035). Our findings implicate an important role of variants in the ATM- CHEK2- BRCA1 axis in modification of the genetic predisposition to PTC and its clinical manifestations.

In-depth characterization of the microRNA transcriptome in normal thyroid and papillary thyroid carcinoma.

CONTEXT: A single microRNA gene may give rise to several mature products that differ in length, called isomiRs. IsomiRs are known to be tissue specific and functionally relevant. The microRNA sequence heterogeneity of the thyroid gland has yet to be determined. OBJECTIVE: The objective of the study was to provide a comprehensive view of the microRNA transcriptome in normal thyroid and papillary thyroid carcinoma (PTC). DESIGN: We used next-generation deep sequencing to analyze microRNA length heterogeneity and expression profiles of PTC tumors (n = 14), unaffected tissue adjacent to tumors (n = 14), and control, noncancerous thyroid tissue (n = 14). The results were validated with a microarray on an additional set of 9 PTC tumor/normal tissue pairs. RESULTS: Eighty-nine microRNAs were significantly deregulated in PTC compared with normal thyroid tissue (false discovery rate < 0.05, fold change 0.13-20.7). Top deregulated miRNAs included miR-146b-5p, miR-221-3p, miR-7-3p, miR-551b-3p, miR-486-3p, and miR-144-3p, confirming previous microarray profiling. The expression of miRNAs did not depend on the BRAF mutation status. Interestingly, 85% of the most abundant microRNAs consisted of isoforms that differed from the standard reference sequence deposited in miRBase. Moreover, the reference microRNAs were completely absent in 42.4% and 35.9% of the microRNAs expressed in normal thyroid and PTC tumors, respectively. Numerous isomiRs had altered seed sequences, which led to a different set of target genes. For highly deregulated miR-146b-5p, we detected 6 isoforms (tumor/normal fold change 14.4-28.7, false discovery rate < 0.002) that varied at their 5' ends with a 1-nt difference that created 2 alternative seeds. The target genes for those 2 seeds overlapped in only 13.1% of genes. CONCLUSIONS: Almost all microRNAs exhibit isoforms of variable length and potentially distinct function in thyroid tumorigenesis.

Cumulative risk impact of five genetic variants associated with papillary thyroid carcinoma.

BACKGROUND: Two recent genome-wide association studies (GWASs) identified five single nucleotide polymorphisms (SNPs; rs965513, rs944289, rs966423, rs2439302, and rs116909374) associated with papillary thyroid carcinoma (PTC). Each variant showed highly significant but moderate to low disease risk. Here we assessed the cumulative risk and predictive value of the five SNPs. METHODS: We genotyped two cohorts of individuals, 747 PTC cases and 1047 controls from Ohio and 1795 PTC cases and 2090 controls from Poland. Cumulative genetic risk scores were calculated using unweighted and weighted approaches. RESULTS: All five SNPs showed significant association with PTC. The average cumulative risk score in cases was significantly higher than in controls (p<2.2×10(-16)). Each additional risk allele increased the risk of having PTC by 1.51 [95% confidence interval (CI) 1.4, 1.64] in Ohio and by 1.35 [95% CI 1.27, 1.44] in Poland. An analysis was performed weighing risk alleles by effect size and assigning individuals to three weighted risk score groups, low (≤2), medium (2-5), and high (>5). Individuals in the high group were significantly more susceptible to PTC compared with individuals in the low group with an odds ratio of 8.7 [95% CI 5.8, 13.3] in Ohio and 4.24 [95% CI 3.10, 5.84] in Poland. Almost identical results were obtained when follicular variant PTCs and microPTCs were omitted. These five SNPs explained 11% of the familial risk of thyroid cancer in the Ohio cohort and 6% in the Polish cohort. CONCLUSION: As the genetic risk score increases, the risk of having PTC increases. However, the predictive power of the cumulative effect of these five variants is only moderately high and clinical use may not be feasible until more variants are detected.

Ultra-rare mutation in long-range enhancer predisposes to thyroid carcinoma with high penetrance.

Thyroid cancer shows high heritability but causative genes remain largely unknown. According to a common hypothesis the genetic predisposition to thyroid cancer is highly heterogeneous; being in part due to many different rare alleles. Here we used linkage analysis and targeted deep sequencing to detect a novel single-nucleotide mutation in chromosome 4q32 (4q32A>C) in a large pedigree displaying non-medullary thyroid carcinoma (NMTC). This mutation is generally ultra-rare; it was not found in 38 NMTC families, in 2676 sporadic NMTC cases or 2470 controls. The mutation is located in a long-range enhancer element whose ability to bind the transcription factors POU2F and YY1 is significantly impaired, with decreased activity in the presence of the C- allele compared with the wild type A-allele. An enhancer RNA (eRNA) is transcribed in thyroid tissue from this region and is greatly downregulated in NMTC tumors. We suggest that this is an example of an ultra-rare mutation predisposing to thyroid cancer with high penetrance.

SRGAP1 is a candidate gene for papillary thyroid carcinoma susceptibility.

BACKGROUND: Papillary thyroid carcinoma (PTC) shows high heritability, yet efforts to find predisposing genes have been largely negative. OBJECTIVES: The objective of this study was to identify susceptibility genes for PTC. METHODS: A genome-wide linkage analysis was performed in 38 families. Targeted association study and screening were performed in 2 large cohorts of PTC patients and controls. Candidate DNA variants were tested in functional studies. RESULTS: Linkage analysis and association studies identified the Slit-Robo Rho GTPase activating protein 1 gene (SRGAP1) in the linkage peak as a candidate gene. Two missense variants, Q149H and A275T, localized in the Fes/CIP4 homology domain segregated with the disease in 1 family each. One missense variant, R617C, located in the RhoGAP domain occurred in 1 family. Biochemical assays demonstrated that the ability to inactivate CDC42, a key function of SRGAP1, was severely impaired by the Q149H and R617C variants. CONCLUSIONS: Our findings suggest that SRGAP1 is a candidate gene in PTC susceptibility. SRGAP1 is likely a low-penetrant gene, possibly of a modifier type.

Risk profiles and penetrance estimations in multiple endocrine neoplasia type 2A caused by germline RET mutations located in exon 10.

Multiple endocrine neoplasia type 2 is characterized by germline mutations in RET. For exon 10, comprehensive molecular and corresponding phenotypic data are scarce. The International RET Exon 10 Consortium, comprising 27 centers from 15 countries, analyzed patients with RET exon 10 mutations for clinical-risk profiles. Presentation, age-dependent penetrance, and stage at presentation of medullary thyroid carcinoma (MTC), pheochromocytoma, and hyperparathyroidism were studied. A total of 340 subjects from 103 families, age 4-86, were registered. There were 21 distinct single nucleotide germline mutations located in codons 609 (45 subjects), 611 (50), 618 (94), and 620 (151). MTC was present in 263 registrants, pheochromocytoma in 54, and hyperparathyroidism in 8 subjects. Of the patients with MTC, 53% were detected when asymptomatic, and among those with pheochromocytoma, 54%. Penetrance for MTC was 4% by age 10, 25% by 25, and 80% by 50. Codon-associated penetrance by age 50 ranged from 60% (codon 611) to 86% (620). More advanced stage and increasing risk of metastases correlated with mutation in codon position (609→620) near the juxtamembrane domain. Our data provide rigorous bases for timing of premorbid diagnosis and personalized treatment/prophylactic procedure decisions depending on specific RET exon 10 codons affected.

The frequency of selected polymorphic variants of the RET gene in patients with medullary thyroid carcinoma and in the general population of central Poland.

The object of this work was to compare the frequency of three polymorphic changes in the RET proto-oncogene: L769L, S836S, and S904S in patients with medullary thyroid carcinoma (MTC; n = 246) and in the general population (n = 420 for single-nucleotide polymorphism [SNP] L769L and S904S; n = 411 for SNP 836). We tried to investigate how the harbored SNPs affect the age at onset of sporadic medullary thyroid carcinoma (sMTC) and MTC in carriers of known pathogenic mutations at codons 634 and 791 of the RET gene. A statistically significant difference was found in the frequency of the heterozygous change L769L in patients with sMTC (48.3%) and in unaffected individuals (39.5%). The presence of the polymorphic change L769L in the RET gene predisposes to the development of sMTC and also lowers the age of onset of MTC in carriers of the homozygous polymorphic variant L769L. The presence of this polymorphic change in MTC patients carrying, at the same time, the RET codon 634 mutation lowers the age of onset of MTC in this group.

[Diagnosis and treatment of thyroid cancer in children in the multicenter analysis in Poland for PPGGL]. / Diagnostyka i leczenie raka tarczycy u dzieci w Polscew analizie wieloosrodkowej dla PPGGL.

INTRODUCTION: Differentiated thyroid carcinoma (DTC) in children presents different biological behavior in comparison to adults. Authors presents preliminary results of multicenter analysis concerning incidence, diagnostics and treatment of DTC in children. MATERIAL AND METHODS: The study is a retrospective analysis of 107 pediatric patients from 14 academic centers based on the data from 2000 to 2005 obtained by questionnaire in hospitals involved in the treatment of DTC in children. RESULTS: Papillary thyroid cancer was diagnosed in 83 children, follicular thyroid cancer in 10 children and medullary thyroid cancer in 14 children. Incidence of DTC in children was estimated between 18 and 23 cases per year. The biggest group of patients consisted of children between 11 and 15 years of age, with girls to boys ratio 3.3 : 1. Clinically DTC in children presented most often as solitary thyroid nodule. Cervical lymphadenopathy was observed in 42% of patients. Intraoperative verification indicated metastatic nodes in 50% of children. Low stage DTC predominated (T1 in 36% and T2 in 26% of children). One step surgery was performed in 65% of children with DTC, two step surgery in 25% of patients. I131 therapy was undertaken in 80% of children. Lung metastases were indicated in post therapeutic studies in 14% of children with DTC. Prophylactic thyroidectomies were performed in 79% of children in the group of patients with MTC and RET gene mutations. CONCLUSIONS: The necessity of introduction of unified therapeutic standard in children with DTC in Poland is underlined.

[Clinical and genetic profile of patients with medullary thyroid cancer treated in the Cancer Centre--Institute of Oncology in Warsaw]. / Analiza kliniczna i genetyczna chorych na raka rdzeniastego tarczycy w Centrum Onkologii--Instytucie w Warszawie.

INTRODUCTION: The aim of this study was to analyse the distribution and frequency of mutations and their correlations with clinical phenotypes of patients with MTC, to reveal the differences between sporadic and familial type of MTC, and to describe the phenotypes of patients. MATERIALS AND METHODS: 212 patients with medullary thyroid cancer (MTC) were treated in Cancer Centre in Warsaw between 1997 and 2005. In most patients, DNA isolated from peripheral blood leukocytes was tested for RET gene mutations by sequencing and accordingly MTC form was assessed. Genetic testing was performed in the relatives of patients with familial MTC in order to distinguish asymptomatic mutation carriers from noncarriers. RESULTS: RET gene mutations were identified in 46 patients (22%). The others were found noncarriers and sporadic MTC was diagnosed. MEN 2A/FMTC syndrome (multiple endocrine neoplasia type 2A/ familial type of MTC) was diagnosed in 44 patients, MEN 2B syndrome (multiple endocrine neoplasia type 2B) in 2 patients. In patients with sporadic and familial MTC, age at diagnosis and multifocal occurrence was analysed, and the results were found to be in accordance with those of other research centres. However, the distribution and frequency of mutations, as well as some clinical data, such as the frequency of pheochromocytoma occurrence as the first manifestation of MEN syndrome, differed from the published data, and further studies are necessary to reveal the reasons of these differences. CONCLUSIONS: DNA testing for RET gene mutations is reliable as a diagnostic tool and therefore it should be performed for screening of all patients with MTC or other diseases of MEN syndrome.