Vladimir Saenko

BRAFV600E mutation is highly prevalent in thyroid carcinomas in the young population in Fukushima: a different oncogenic profile from Chernobyl

After the accident at the Fukushima Daiichi Nuclear Power Plant, the thyroid ultrasound screening program for children aged 0–18 at the time of the accident was started from October 2011. The prevalence of thyroid carcinomas in that population has appeared to be very high (84 cases per 296,253). To clarify the pathogenesis, we investigated the presence of driver mutations in these tumours. 61 classic papillary thyroid carcinomas (PTCs), two follicular variant PTCs, four cribriform-morular variant PTCs and one poorly-differentiated thyroid carcinoma were analysed. We detected BRAFV600E in 43 cases (63.2%), RET/PTC1 in six (8.8%), RET/PTC3 in one (1.5%) and ETV6/NTRK3 in four (5.9%). Among classic and follicular variant PTCs, BRAFV600E was significantly associated with the smaller size. The genetic pattern was completely different from post-Chernobyl PTCs, suggesting non-radiogenic etiology of these cancers. This is the first study demonstrating the oncogene profile in the thyroid cancers discovered by large mass screening, which probably reflects genetic status of all sporadic and latent tumours in the young Japanese population. It is assumed that BRAFV600E may not confer growth advantage on paediatric PTCs, and many of these cases grow slowly, suggesting that additional factors may be important for tumour progression in paediatric PTCs.

Patterns of FOXE1 Expression in Papillary Thyroid Carcinoma by Immunohistochemistry

BACKGROUNDnFOXE1, a thyroid-specific transcription factor also known as TTF-2, was recently identified as a major genetic risk factor for papillary thyroid carcinoma (PTC). Its role in thyroid carcinogenesis, however, remains unknown. The purpose of the present study was to assess the relationship between the FOXE1 immunohistochemical features and the clinical and genetic characteristics of PTC.nnnMETHODSnImmunohistochemical staining of FOXE1 was performed in 48 PTC cases. Two single nucleotide polymorphisms immediately inside (rs1867277) or in the vicinity (rs965513) of the FOXE1 gene were genotyped by direct sequencing. Histopathological, clinical, and genetic data were included in statistical analyses.nnnRESULTSnFOXE1 exhibited cytoplasmic overexpression in tumor tissue compared to the normal counterpart (p<0.001). Both cancer and normal thyroid cells demonstrated the highest FOXE1 scores in the areas closest to the tumor border (<300 μm) compared with more distant areas (p<0.001). No differences in FOXE1 staining distributions were found between microcarcinomas and PTC of larger size, between different histopathological variants of PTC, and encapsulated and nonencapsulated tumors. Multivariate regression analysis revealed that nuclear FOXE1 expression in neoplastic cells in the vicinity of the tumor border independently associated with the genotype at rs1867277 (the dominant model of inheritance, p=0.037) and tumor multifocality (p=0.032), and with marginal significance with capsular invasion (p=0.051).nnnCONCLUSIONSnFOXE1 overexpression and translocation to the cytoplasm are phenotypic hallmarks of tumor cells suggesting that FOXE1 is involved in the pathogenesis of PTC. Nuclear FOXE1 expression in tumor cells in the vicinity of the PTC border is associated with the presence of a risk allele of rs1867277 (c.-238G>A) in the 5 untranslated region of the FOXE1 gene, as well as with pathological characteristics of PTC, suggesting possible FOXE1 involvement in the facilitation of tumor development beginning at an early stage.

The Common Genetic Variant rs944289 on Chromosome 14q13.3 Associates with Risk of Both Malignant and Benign Thyroid Tumors in the Japanese Population

BACKGROUNDnSeveral single nucleotide polymorphisms (SNP) have been identified to be associated with the risk for differentiated thyroid cancer in populations of distinct ethnic background. The relationship of these genetic markers to a benign tumor of the thyroid, follicular adenoma (FA), is not well established.nnnMETHODSnIn a multicenter retrospective case-control study, five thyroid cancer-related SNPs-rs966513 (9q22.33, FOXE1), rs944289 (14q13.3, PTCSC3), rs2439302 (8p12, NRG1), rs1867277 (9q22.23, FOXE1), and rs6983267 (8q24, POU5F1B)-were genotyped in 959 cases of histologically verified FA, 535 papillary thyroid carcinomas (PTC), and 2766 population controls.nnnRESULTSnA significant association was found between FA and rs944289 (p=0.002; OR 1.176 [CI 1.064-1.316]), and suggestively with rs2439302 (p=0.033; OR 1.149 [CI 1.010-1.315]). In PTC, significant associations were confirmed for rs965513 (p=4.21E-04; OR 1.587 [CI 1.235-2.000]) and rs944289 (p=0.003; OR 1.234 [CI 1.075-1.408]), newly found for rs2439302 (p=0.003; OR 1.266 [CI 1.087-1.493]) and rs1867277 (p=1.17E-04; OR 1.492 [CI 1.235-1.818]), and was not replicated for rs6983267 (p=0.082; OR 1.136 [CI 0.980-1.316]) in this series. A significant correlation between rs2439302 genotype and relative expression of NRG1 was detected in normal and tumor counterparts of PTC (about 10% decrease per each risk allele). NRG1 expression also significantly correlated with that of PTCSC3.nnnCONCLUSIONSnAssociation of rs944289, which was previously known to confer risk for thyroid cancer, with FA, and the correlation between PTCSC3 and NRG1 expression demonstrates that predisposing genetic factors are partly common for benign and malignant thyroid tumors, and imply broader roles of the pathways they underlie in thyroid tumorigenesis, not limited to carcinogenesis.

Radiation signatures in childhood thyroid cancers after the Chernobyl accident: Possible roles of radiation in carcinogenesis

After the Tokyo Electric Power Company Fukushima Daiichi nuclear power plant accident, cancer risk from low‐dose radiation exposure has been deeply concerning. The linear no‐threshold model is applied for the purpose of radiation protection, but it is a model based on the concept that ionizing radiation induces stochastic oncogenic alterations in the target cells. As the elucidation of the mechanism of radiation‐induced carcinogenesis is indispensable to justify the concept, studies aimed at the determination of molecular changes associated with thyroid cancers among children who suffered effects from the Chernobyl nuclear accident will be overviewed. We intend to discuss whether any radiation signatures are associated with radiation‐induced childhood thyroid cancers.

The 8q24 rs6983267G variant is associated with increased thyroid cancer risk.

The G allele of the rs6983267 single-nucleotide polymorphism, located on chromosome 8q24, has been associated with increased risk of several cancer types. The association between rs6983267G and thyroid cancer (TC) has been tested in different populations, mostly of European ancestry, and has led to inconclusive results. While significant associations have been reported in the British and Polish populations, no association has been detected in populations from Spain, Italy and the USA. To further investigate the role of rs6983267G in TC susceptibility, we evaluated rs6983267 genotypes in three populations of different continental ancestry (British Isles, Colombia and Japan), providing a total of 3067 cases and 8575 controls. We detected significant associations between rs6983267G and TC in the British Isles (odds ratio (OR)=1.19, 95% CI: 1.11-1.27, P=4.03×10(-7)), Japan (OR=1.20, 95% CI: 1.03-1.41, P=0.022) and a borderline significant association of similar effect direction and size in Colombia (OR=1.19, 95% CI: 0.99-1.44, P=0.069). A meta-analysis of our multi-ethnic study and previously published non-overlapping datasets, which included a total of 5484 cases and 12u200a594 controls, confirmed the association between rs6983267G and TC (P=1.23×10(-7), OR=1.13, 95% CI: 1.08-1.18). Our results therefore support the notion that rs6983267G is a bona fide TC risk variant that increases the risk of disease by ∼13%.

Thyrotropin Signaling Confers More Aggressive Features with Higher Genomic Instability on BRAFV600E-Induced Thyroid Tumors in a Mouse Model

BACKGROUNDnThe BRAF(V600E) mutation is the most common genetic alteration in papillary thyroid carcinomas (PTCs). Transgenic mice overexpressing BRAF(V600E) in their thyroids under control of the thyroglobulin promoter (Tg-BRAF2 mice) developed invasive PTCs with high penetrance. However, these mice showed elevated thyrotropin (TSH) levels, which also stimulate the proliferation of thyrocytes and tumorigenesis. The purpose of the present study was to investigate how TSH signaling cooperates with BRAF(V600E) in the process of thyroid carcinogenesis.nnnMETHODSnWe crossed Tg-BRAF2 mice with TSH receptor knockout (TshR(-/-)) mice. Four genetically distinct mice groups-Braf(wt)/TshR(+/-) (group 1), Braf(wt)/TshR(-/-) (group 2), Tg-BRAF2/TshR(+/-) (group 3), and Tg-BRAF2/TshR(-/-) (group 4)--were sacrificed at 12 and 24 weeks of age. We performed histopathological analysis. Genomic instability was evaluated by immunofluorescence for p53-binding protein 1 (53BP1) and γH2AX. Invasiveness and genomic instability were also evaluated using thyroid PCCL3 cells expressing BRAF(V600E).nnnRESULTSnGroups 3 and 4 developed distinct neoplasias comparable to human PTCs. Group 3 developed typically larger, more aggressive, invasive tumors compared to group 4. The frequency of 53BP1 and γH2AX foci-indicators of genomic instability--in group 3 was higher than that in group 4. TSH also enhanced invasiveness and genomic instability in PCCL3 cells with BRAF(V600E) expression.nnnCONCLUSIONSnThese data demonstrate that the TSH signaling confers more aggressive features in BRAF(V600E)-induced thyroid tumors in mice. This might be due, in part, to accelerated genomic instability.

Significance of p53-binding protein 1 (53BP1) expression in thyroid papillary microcarcinoma: association with BRAFV600E mutation status.

In a previous report, we proposed that analysis of 53BP1 expression by immunofluorescence could be a useful tool in estimating the level of genomic instability (GIN), as well as the malignant potential, of thyroid tumours. In an attempt to clarify the value of 53BP1 expression as a new molecular marker for the aggressiveness of thyroid papillary microcarcinoma (PMC), we assessed the association between the type of 53BP1 expression and clinicopathological features such as tumour size, extrathyroidal invasion, lymph node metastasis and BRAFV600E mutation of PMC.

TERT promoter mutations and Ki-67 labeling index as a prognostic marker of papillary thyroid carcinomas: combination of two independent factors

Although most papillary thyroid carcinomas (PTCs) have a good prognosis, a small but certain fraction shows aggressive behavior. Therefore, a novel and well-performing molecular marker is needed. In the present study, we assessed the impact of the combination of the TERT promoter/BRAF mutations and Ki-67 labeling index (LI) as a prognostic marker in PTC patients. Of 400 PTC samples, 354 were successfully genotyped for both TERT promoter/BRAF and analyzed for Ki-67 LI. Based on the combination of the mutational status and Ki-67 LI, the cases were categorized into three groups: high-, middle-, and low-risk. The recurrence rates of low-, middle-, and high-risk group were 1.9% (6 of 323), 18.2% (4 of 22), and 44.4% (4 of 9), respectively. The Kaplan-Meier curve and log-rank analyses demonstrated that there were statistical differences between any two groups. The hazard ratios for recurrence remained significant after adjustment for age, sex, tumor size, and extrathyroidal extension (low vs. middle: 8.80, 95% CI: 2.35–32.92, pu2009=u20090.001; middle vs. high: 6.255, 95% CI: 1.13–34.51, pu2009=u20090.035). In conclusion, the combination of the TERT promoter/BRAFV600E mutations and Ki-67 LI performed excellent in predicting PTC recurrence and may be clinically useful.

Targeted Foxe1 Overexpression in Mouse Thyroid Causes the Development of Multinodular Goiter But Does Not Promote Carcinogenesis

Recent genome-wide association studies have identified several single nucleotide polymorphisms in the forkhead box E1 gene (FOXE1) locus, which are strongly associated with the risk for thyroid cancer. In addition, our recent work has demonstrated FOXE1 overexpression in papillary thyroid carcinomas. To assess possible contribution of Foxe1 to thyroid carcinogenesis, transgenic mice overexpressing Foxe1 in their thyroids under thyroglobulin promoter (Tg-Foxe1) were generated. Additionally, Tg-Foxe1 mice were exposed to x-rays at the age of 5 weeks or crossed with Pten(+/-) mice to examine the combined effect of Foxe1 overexpression with radiation or activated phosphatidylinositol-3-kinase/Akt pathway, respectively. In 5- to 8-week-old Tg-Foxe1 mice, severe hypothyroidism was observed, and mouse thyroids exhibited hypoplasia of the parenchyma. Adult 48-week-old mice were almost recovered from hypothyroidism, their thyroids were enlarged, and featured colloid microcysts and multiple benign nodules of macrofollicular-papilloid growth pattern, but no malignancy was found. Exposure of transgenic mice to 1 or 8 Gy of x-rays and Pten haploinsufficiency promoted hyperplastic nodule formation also without carcinogenic effect. These results indicate that Foxe1 overexpression is not directly involved in the development of thyroid cancer and that proper Foxe1 dosage is essential for achieving normal structure and function of the thyroid.

Radiation-Associated Small Cell Neuroendocrine Carcinoma of the Thyroid: A Case Report with Molecular Analyses

BACKGROUNDnNeuroendocrine tumor (NET) of the thyroid other than medullary carcinoma is extremely rare. We describe here a case of calcitonin-negative small cell neuroendocrine carcinoma (SCNEC), which occurred in a thyroid gland that had previously been irradiated at high dose (60 Gy) for pharyngeal cancer, with molecular analyses for follicular cell origin.nnnPATIENT FINDINGSnThe tumor cells were small with fine chromatin, inconspicuous nucleoli, and inapparent cytoplasm, and showed neuroendocrine architectures such as palisading, rosettes, and trabeculae. Mitotic figures were numerous exceeding 10 mitoses per 10 high-power fields. The tumor cells invaded into several vessels and metastasized to regional lymph nodes. Immunohistochemically, the tumor cells were strongly positive for neuroendocrine markers and thyroglobulin (Tg), a marker of thyroid follicular cells but negative for calcitonin and carcinoembryonic antigen (CEA). Expression of Tg and thyrotropin receptor (TSHR) were confirmed by quantitative real-time polymerase chain reaction (RT-PCR). Ki-67 labeling index was more than 70% in the tumor cells. Taken together, the tumor was diagnosed as SCNEC of the thyroid. Genetic analyses also revealed microsatellite abnormalities of the phosphatase and tensin homolog (PTEN) gene, suggesting that functional loss of PTEN contributes to carcinogenesis.nnnCONCLUSIONSnThis is the first report describing a SCNEC of the thyroid with molecular analyses that provide evidence for a follicular epithelial origin.