Noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) is an encapsulated or clearly delimited, noninvasive neoplasm with a follicular growth pattern and nuclear features of papillary thyroid carcinoma (PTC). It is considered a ‘pre-malignant’ lesion of the RAS-like group. Ultrasonography (US), cytology and molecular tests are useful to suspect thyroid nodules that correspond to NIFTP but there is wide overlap of the results with the encapsulated follicular variant of PTC (E-FVPTC). In these nodules that possibly or likely correspond to NIFTP, if surgery is indicated, lobectomy is favored over total thyroidectomy. The diagnosis of NIFTP is made after complete resection of the lesion by observing well-defined criteria. In the case of patients who received the diagnosis of FVPTC and whose pathology report does not show findings of malignancy (lymph node metastasis, extrathyroidal invasion, vascular/capsular invasion), if the tumor was encapsulated or well delimited, the slides can be revised by an experienced pathologist to determine whether the diagnostic criteria of NIFTP are met, but special attention must be paid to the adequate representativeness of the capsule and tumor. Since NIFTP is not ‘malignant’, tumor staging is not necessary and patients are not submitted to thyroid cancer protocols or guidelines. We believe that patients with NIFTP without associated malignancy and without nodules detected by US of the remnant lobe (if submitted to lobectomy) can be managed like those with follicular adenoma.
Pedro Weslley Rosario and Gabriela Franco Mourão
Johan O Paulsson, Na Wang, Jiwei Gao, Adam Stenman, Jan Zedenius, Ninni Mu, Weng-Onn Lui, Catharina Larsson, and C Christofer Juhlin
Mutations in the miRNA enzyme gene DICER1 have been reported in several endocrine malignancies and is associated with the rare tumour-predisposing DICER1 syndrome. DICER1 mutations have been reported in subsets of follicular thyroid carcinoma (FTC), but the role of DICER1 in follicular thyroid tumorigenesis has not been extensively studied. In this study, we investigate the role of DICER1 in 168 follicular thyroid tumours and in an FTC cell line. We found rare DICER1 mutations in paediatric FTC cases and a general DICER1 down-regulation in FTCs visualized both on mRNA and protein level, especially pronounced in Hürthle cell carcinoma (HuCC). The down-regulation was also evident in follicular thyroid adenomas (FTAs), suggesting a potential early step in tumorigenesis. The expression of DICER1 was lower in FTCs of older patients in which TERT promoter mutations are more frequent. In FTCs, DICER1 down-regulation was not caused by gene copy number loss but significantly correlated to expression of the transcription factor GABPA in clinical cases. GABPA was found to bind to the DICER1 promoter and regulate DICER1 expression in vitro, as GABPA depletion in FTC cell lines reduced DICER1 expression. This in turn stimulated cell proliferation and affected the miRNA machinery, evident by altered miRNA expression. To conclude, we show that GABPA directly regulates DICER1 in FTC, acting as a tumour suppressor and displaying down-regulation in clinical samples. We also show reduced expression of DICER1 in benign and malignant follicular thyroid tumours, suggesting a potentially early tumorigenic role of this gene aberrancy.
María Jesús Larriba, Noelia Valle, Héctor G Pálmer, Paloma Ordóñez-Morán, Silvia Álvarez-Díaz, Karl-Friedrich Becker, Carlos Gamallo, Antonio García de Herreros, José Manuel González-Sancho, and Alberto Muñoz
The Wnt/β-catenin signalling pathway is activated in 90% of human colon cancers by nuclear accumulation of β-catenin protein due to its own mutation or to that of adenomatous polyposis coli. In the nucleus, β-catenin regulates gene expression promoting cell proliferation, migration and invasiveness. 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) inhibits β-catenin signalling by inducing its binding to vitamin D receptor (VDR) and by promoting β-catenin nuclear export. The transcription factor Snail1 represses VDR expression and we demonstrate here that Snail1 also abolishes the nuclear export of β-catenin induced by 1,25(OH)2D3 in SW480-ADH cells. Accordingly, Snail1 relieves the inhibition exerted by 1,25(OH)2D3 on genes whose expression is driven by β-catenin, such as c-MYC, ectodermal-neural cortex-1 (ENC-1) or ephrin receptor B2 (EPHB2). In addition, Snail1 abrogates the inhibitory effect of 1,25(OH)2D3 on cell proliferation and migration. In xenografted mice, Snail1 impedes the nuclear export of β-catenin and the inhibition of ENC-1 expression induced by EB1089, a 1,25(OH)2D3 analogue. The elevation of endogenous SNAIL1 protein levels reproduces the effect of an ectopic Snail1 gene. Remarkably, the expression of exogenous VDR in cells with high levels of Snail1 normalizes the transcriptional responses to 1,25(OH)2D3. However, this exogenous VDR failed to fully restore the blockage of the Wnt/β-catenin pathway by 1,25(OH)2D3. This suggests that the effects of Snail1 on this pathway are not merely due to the repression of VDR gene. We conclude that Snail1 is a positive regulator of the Wnt/β-catenin signalling pathway in part through the abrogation of the inhibitory action of 1,25(OH)2D3.
W Religa, M Krzeminska-Pakula, J Kasprzak, and W Zieleniewski
Alyson Murray, Stephen F Madden, Naoise C Synnott, Rut Klinger, Darran O'Connor, Norma O'Donovan, William Gallagher, John Crown, and Michael J Duffy
Considerable epidemiological evidence suggests that high levels of circulating vitamin D (VD) are associated with a decreased incidence and increased survival from cancer, i.e., VD may possess anti-cancer properties. The aim of this investigation was therefore to investigate the anti-cancer potential of a low calcaemic vitamin D analogue, i.e., inecalcitol and compare it with the active form of vitamin D, i.e., calcitriol, in a panel of breast cancer cell lines (n = 15). Using the MTT assay, IC50 concentrations for response to calcitriol varied from 0.12 µM to >20 µM, whereas those for inecalcitol were significantly lower, ranging from 2.5 nM to 63 nM (P = 0.001). Sensitivity to calcitriol and inecalcitol was higher in VD receptor (VDR)-positive compared to VDR-negative cell lines (P = 0.0007 and 0.0080, respectively) and in ER-positive compared to ER-negative cell lines (P = 0.043 and 0.005, respectively). Using RNA-seq analysis, substantial but not complete overlap was found between genes differentially regulated by calcitriol and inecalcitol. In particular, significantly enriched gene ontology terms such as cell surface signalling and cell communication were found after treatment with inecalcitol but not with calcitriol. In contrast, ossification and bone morphogenesis were found significantly enriched after treatment with calcitriol but not with inecalcitol. Our preclinical results suggest that calcitriol and inecalcitol can inhibit breast cancer cell line growth, especially in cells expressing ER and VDR. As inecalcitol is significantly more potent than calcitriol and has low calcaemic potential, it should be further investigated for the treatment of breast cancer.
Marc Sinotte, François Rousseau, Pierre Ayotte, Eric Dewailly, Caroline Diorio, Yves Giguère, Sylvie Bérubé, and Jacques Brisson
Vitamin D has been associated with reduced breast cancer risk. We studied the association of two vitamin D receptor (VDR) gene single nucleotide polymorphisms restriction enzyme detecting SNP of VDR (FokI and BsmI) with breast cancer risk in two independent case–control studies carried out in the same population. The modifying effect of family history of breast cancer on this relationship was also evaluated. The first and second studies included respectively 718 (255 cases/463 controls) and 1596 (622 cases/974 controls) women recruited in Quebec City, Canada. FokI and BsmI genotypes were assessed. Relative risks of breast cancer were estimated by multivariate logistic regression. Compared with homozygotes for the common F allele (FF genotype), FokI ff homozygotes had a higher breast cancer risk (study 1: odds ratio (OR)=1.22, 95% confidence interval (CI)=0.76–1.95; study 2: OR=1.44, 95% CI=1.05–1.99; and combined studies: OR=1.33, 95% CI=1.03–1.73). Significant interactions were observed between FokI and family history of breast cancer in the two studies as well as in the combined analysis (P interaction=0.031, 0.050 and 0.0059 respectively). Among women without family history, odds ratios were 1.00, 1.27 (95% CI=1.02–1.58) and 1.57 (95% CI=1.18–2.10) respectively for FF, Ff and ff carriers (P trend=0.0013). BsmI Bb+bb genotypes were associated with a weak non-significant increased risk in the two studies (combined OR=1.22, 95% CI=0.95–1.57) without interaction with family history. Results support the idea that vitamin D, through its signalling pathway, can affect breast cancer risk. They also suggest that variability in observed associations between VDR FokI and breast cancer from different studies may partly be explained by the proportion of study subjects with a family history of breast cancer.
Arivarasan Karunamurthy, Federica Panebianco, Susan J Hsiao, Jennie Vorhauer, Marina N Nikiforova, Simion Chiosea, and Yuri E Nikiforov
The EIF1AX gene mutations have been recently found in papillary thyroid carcinoma (PTC) and anaplastic thyroid carcinoma (ATC). The prevalence of these mutations in other types of thyroid cancers and benign nodules is unknown. In this study, we analyzed the occurrence of EIF1AX mutations in exons 2, 5, and 6 of the gene in a series of 266 thyroid tumors and hyperplastic nodules by either Sanger or next-generation sequencing (ThyroSeq v.2). In addition, 647 thyroid fine-needle aspiration (FNA) samples with indeterminate cytology were analyzed. Using surgically removed samples, EIF1AX mutations were detected in 3/86 (2.3%) PTC, 1/4 (25%) ATC, 0/53 follicular carcinomas, 0/12 medullary carcinomas, 2/27 (7.4%) follicular adenomas, and 1/80 (1.3%) hyperplastic nodules. Among five mutation-positive FNA samples with surgical follow-up, one nodule was PTC and others were benign follicular adenomas or hyperplastic nodules. Overall, among 33 mutations identified, A113_splice mutation at the intron 5/exon 6 splice site of EIF1AX was the most common. All four carcinomas harbored A113_splice mutation and three of them had one or more coexisting mutations, typically RAS. All PTC carrying EIF1AX mutations were encapsulated follicular variants. In summary, this study shows that EIF1AX mutations occur not only in thyroid carcinomas, but also in benign nodules. The most common mutation hotspot is the A113_splice, followed by a cluster of mutations in exon 2. When found in thyroid FNA samples, EIF1AX mutations confer ~20% risk of cancer; the risk is likely to be higher in nodules carrying a A113_splice mutation and when EIF1AX coexists with RAS mutations.
Pei-Pei Xu, Su Zeng, Xiao-Tian Xia, Zi-Heng Ye, Mei-Fang Li, Ming-Yun Chen, Tian Xia, Jing-Jing Xu, Qiong Jiao, Liang Liu, Lian-Xi Li, and Ming-Gao Guo
Our aims were to uncover the role of FAM172A (Family with sequence similarity 172 member A) in the pathogenesis of follicular thyroid carcinoma (FTC) and to evaluate its value in the differential diagnosis between malignant and benign thyroid follicular lesions. FAM172A expression was evaluated by q-PCR, immunoblotting and immunohistochemistry (IHC). The ability of proliferation, migration and invasion of cells were assessed by Cell Counting Kit-8 assay (CCK8), clone-formation and Transwell assays. Nude mouse tumorigenicity assays were used to investigate the role of FAM172A in the pathogenesis of FTC in vivo. The value of FAM172A in the differential diagnosis for FTC was assessed using 120 formalin-fixed paraffin-embedded (FFPE) tissues after the operation and 81 fine-needle aspiration biopsy (FNAB) samples before the operation. FAM172A was highly expressed in FTC tissues and FTC cell lines. Downregulation of FAM172A inhibited the proliferation, invasion and migration of FTC cells through Erk1/2 and JNK pathways. Subcutaneous tumorigenesis in nude mice showed that knockdown of FAM172A inhibited tumor growth and progression in vivo. The FAM172A IHC scores of 3.5 had 92% sensitivity and 63% specificity to separate FTC from benign/borderline thyroid follicular lesions, and 92% sensitivity and 80% specificity to discriminate FTC from benign thyroid follicular lesions in postoperative FFPE samples. The corresponding values were 75 and 78%, and 75 and 89% in preoperative FNA samples, respectively. FAM172A plays an important role in the pathogenesis of FTC through Erk1/2 and JNK pathways. FAM172A may be a potential marker for the preoperative diagnosis of FTC based on the IHC results of thyroid FNAB samples.
Arja Jukkola, Risto Bloigu, Tapani Ebeling, Pasi Salmela, and Guillermo Blanco
Differentiated thyroid carcinomas (DTC) (papillary, follicular and follicular type of papillary) have a favourable prognosis, but a proportion of patients develop recurrences and eventually die of the disease. Various prognostic factors have been identified and been used to create the current staging classifications (AGES, AMES, MACIS, EORTC, UICC-TNM). We examined 499 DTC patients retrospectively to validate known prognostic factors that enable them to be recognised as having either a low or a high risk of death related to a recurrence of DTC, by reference to the current staging classifications. Sixty-nine of them (14%) had local or distant recurrences, the mean time to recurrence being 7.7 years. The 10-year disease-free survival rate was 80%, and the ten-year overall survival rate for the entire group was 91%, with a mean survival time of 8.7 years. Male gender, a follicular type of tumour, larger tumour size, extrathyroidal invasion outside the capsule and nodal metastases were all related to a higher incidence of tumour recurrence, and the follicular type of histology, age >45 years, larger tumour size and local invasion entailed poorer survival. The AMES and to some extent the EORTC classification were not reproducible in this material, mainly because some prognostic variants were no longer encountered or were insufficient in number to allow reliable conclusions to be drawn. The MACIS staging classification leaves the definition of the intermediate and high risk groups too wide and is therefore not very reliable. Pooling of stages I and II improved the relevance of the TNM classification. All the current staging classifications are able to discern a low risk DTC group well. We achieved a highly accurate definition of risk in the present material using only two parameters, age (cut-off value 50 years) and extracapsular invasion of the thyroid gland.