Thyroid carcinoma is the most common endocrine malignancy, and papillary thyroid carcinoma represents the most common thyroid cancer. Papillary thyroid carcinomas that invade locally or metastasize are associated with a poor prognosis. We found that, during epithelial-mesenchymal transition (EMT) induced by transforming growth factor-β1 (TGF-β1), papillary thyroid carcinoma cells acquired increased cancer stem cell-like features and the transcription factor paired-related homeobox protein 1 (PRRX1; alias PRX-1), a newly identified EMT inducer, was markedly up-regulated. miR-146b-5p was also transiently up-regulated during EMT, and in siRNA experiments miR-146b-5p had an inhibitory role on cell proliferation and invasion during TGF-β1-induced EMT. We conclude that papillary thyroid carcinoma tumor cells exhibit increased cancer stem cell-like features during TGF-β1-induced EMT, that miR-146b-5p has a role in cell proliferation and invasion, and that PRRX1 plays an important role in papillary thyroid carcinoma EMT and disease progression.

Epithelial–mesenchymal transition is an important mechanism of epithelial tumor progression, local invasion and metastasis. The E-cadherin (CDH1) repressor SLUG (SNAI2) and the basic helix–loop–helix transcription factor TWIST1 inhibit CDH1 expression in poorly differentiated malignancies as inducers of epithelial– mesenchymal transition. Epithelial–mesenchymal transition has been implicated in progression from well to poorly differentiated/anaplastic thyroid carcinoma but the expression of SNAI2 and TWIST1 proteins and their phenotypic association in human thyroid cancers has not been extensively studied. We examined the expression of SNAI2, TWIST1 and CDH1 by immunohistochemistry in a panel of well-differentiated and anaplastic thyroid cancers and by qRT-PCR in thyroid cell lines. Ten normal thyroids, 33 follicular adenomas, 56 papillary thyroid carcinomas including 28 follicular variants, 27 follicular carcinomas and 10 anaplastic thyroid carcinomas were assembled on a tissue microarray and immunostained for SNAI2, TWIST1 and CDH1. Most (8/10) anaplastic thyroid carcinomas demonstrated strong nuclear immunoreactivity for SNAI2 with associated absence of CDH1 in 6/8 cases (75%). TWIST1 was expressed in 5/10 anaplastic thyroid carcinomas with absence of CDH1 in 3/5 (60%) cases. These findings were confirmed in whole sections of all anaplastic thyroid carcinomas and in a separate validation set of 10 additional anaplastic thyroid carcinomas. All normal thyroids, follicular adenomas, papillary and follicular thyroid carcinomas were negative for SNAI2 and TWIST1 (P<0.0001) and all showed strong diffuse immunoreactivity for CDH1 (P=0.026). Expression of SNAI2, TWIST1 and CDH1 mRNA varied in a normal thyroid, papillary carcinoma and two anaplastic thyroid carcinoma cell lines tested, but the highest levels of CDH1 mRNA were detected in the normal thyroid cell line while the anaplastic thyroid carcinoma cell line demonstrated the highest levels of SNAI2 and TWIST1 mRNA. Our findings support the role of epithelial–mesenchymal transition in the development of anaplastic thyroid carcinoma.

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Understanding the molecular mechanisms involved in thyroid cancer progression may provide targets for more effective treatment of aggressive thyroid cancers. Epithelial-mesenchymal transition (EMT) is a major pathologic mechanism in tumor progression and is linked to the acquisition of stem-like properties of cancer cells. We examined expression of ZEB1 which activates EMT by binding to the E-box elements in the E-cadherin promoter, and expression of E-cadherin in normal and neoplastic thyroid tissues in a tissue microarray (TMA) which included 127 neoplasms and 10 normal thyroid specimens. Thyroid follicular adenomas (FA, n=32), follicular thyroid carcinomas (FTC, n=28), and papillary thyroid carcinomas (PTC, n=57) all expressed E-cadherin and were mostly negative for ZEB1 while most anaplastic thyroid carcinomas (ATC, n=10) were negative for E-cadherin, but positive for ZEB1. A validation set of 10 whole sections of ATCs showed 90% of cases positive for ZEB1 and all cases were negative for E-cadherin. Analysis of three cell lines (normal thyroid, NTHY-OR13-1; PTC, TPC-1 and ATC, THJ-21T) showed that the ATC cell line expressed the highest levels of ZEB1 while the normal thyroid cell line expressed the highest levels of E-Cadherin. Quantitative RT-PCR analyses showed that Smad7 mRNA was significantly higher in ATC than in any other group (p<0.05). These results indicate that ATCs show evidence of EMT including decreased expression of E-cadherin and increased expression of ZEB1 compared to well differentiated thyroid carcinomas and that increased expression of Smad7 may be associated with thyroid tumor progression.

Some thyroid nodules such as follicular adenomas (FAs), follicular variant of papillary thyroid carcinomas (FVPTCs), and follicular thyroid carcinomas (FTCs) exhibit similar clinical presentations and gross morphologic appearances. The differential diagnosis of these lesions is sometimes difficult based on morphologic, cytologic, or clinical features alone. miR-146b-5p and miR-21 deregulation has been associated with progression and metastasis of thyroid cancers. However, the utility of in situ hybridization (ISH) to determine the cellular localization, diagnostic, and prognostic significance of miR-146b-5p and miR-21 expression in thyroid tumors has not been extensively analyzed. In order to examine the expression of miR-146b-5p and miR-21 in benign and malignant thyroid tissues and to determine if these microRNAs could be assigned to distinct histomorphological types of thyroid nodules, we analyzed miR-146b-5p and miR-21 expression in thyroid nodules on tissue microarrays (TMAs) with 193 thyroid specimens by ISH. miR-146b-5p and miR-21 expression in thyroid tissues was also analyzed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). miR-146b-5p was highly expressed (89%) in papillary thyroid carcinomas (PTCs) and 41% of FVPTC. The expression of miR-146b-5p was not expressed in most FTCs, anaplastic thyroid carcinomas (ATCs), poorly differentiated thyroid carcinomas (PDTCs), or FAs (7, 8, 0, and 0%, respectively). MiR-21 was overexpressed in 83% of ATCs, 79 % of PTCs, 34% of FVPTCs, and 19% of PDTCs. The expression of miR-21 was not expressed in most FAs (9%) or FTCs (4%). Normal thyroid tissues and most benign goiters were negative for miR-146b-5p and miR-21. qRT-PCR analysis supported the ISH findings. PTC cases with positive expression of miR-146b-5p and miR-21 had significantly poorer disease-free survival rates. Immunohistochemical staining for HBME-1 showed positive staining in PTCs (100 %) and FVPTCs (92 %) with a subset of FTC (40%) staining positive, while all FAs were negative. Since miR-146b-5p was mainly expressed in PTC including FVPTC and was not expressed in most FTC, PDTC, or ATC, it may serve as a useful diagnostic marker for PTC. ISH is a useful method to analyze microRNA expression in formalin-fixed paraffin-embedded thyroid tissues.

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Cancer stem-like cells are a subpopulation of self-renewing cells that are more resistant to chemotherapy and radiation therapy than the other surrounding cancer cells. The cancer stem cell model predicts that only a subset of cancer cells possess the ability to self-renew and produce progenitor cells that can reconstitute and sustain tumor growth. Evidence supporting the existence of cancer stem-like cells in the thyroid, pituitary, and in other endocrine tissues is rapidly accumulating. These cells have been studied using specific biomarkers including: CD133, CD44, Nestin, Nanog, and aldehyde dehydrogenase enzyme. Putative cancer stem-like cells can be studied in vitro using serum-free media supplemented with basic fibroblast growth factor and epidermal growth factor grown in low attachment plates or in extracellular matrix leading to sphere formation in vitro. Cancer stem-like cells can also be separated by fluorescent cell sorting and used for in vitro or in vivo studies. Injection of enriched populations of cancer stem-like cells (also referred to as tumor initiating cells) into immunodeficient mice results in growth of xenografts which express cancer stem-like biomarkers. Human cancer stem-like cells have been identified in thyroid cancer cell lines, in primary thyroid cancers, in normal pituitary, and in pituitary tumors. Other recent studies suggest the existence of stem cells and cancer stem-like cells in endocrine tumors of the gastrointestinal tract, pancreas, lungs, adrenal, parathyroid, and skin. New discoveries in this field may lead to more effective therapies for highly aggressive and lethal endocrine cancers.

BACKGROUND The 1000 Genomes Project provides a database of genomic variants from whole genome sequencing of 2504 individuals across five continental superpopulations. This database can enrich our background knowledge of worldwide blood group variant geographic distribution and identify novel variants of potential clinical significance. STUDY DESIGN AND METHODS The 1000 Genomes database was analyzed to 1) expand knowledge about continental distributions of known blood group variants, 2) identify novel variants with antigenic potential and their geographic association, and 3) establish a baseline scaffold of chromosomal coordinates to translate next‐generation sequencing output files into a predicted red blood cell (RBC) phenotype. RESULTS Forty‐two genes were investigated. A total of 604 known variants were mapped to the GRCh37 assembly; 120 of these were reported by 1000 Genomes in at least one superpopulation. All queried variants, including the ACKR1 promoter silencing mutation, are located within exon pull‐down boundaries. The analysis yielded 41 novel population distributions for 34 known variants, as well as 12 novel blood group variants that warrant further validation and study. Four prediction algorithms collectively flagged 79 of 109 (72%) known antigenic or enzymatically detrimental blood group variants, while 4 of 12 variants that do not result in an altered RBC phenotype were flagged as deleterious. CONCLUSION Next‐generation sequencing has known potential for high‐throughput and extended RBC phenotype prediction; a database of GRCh37 and GRCh38 chromosomal coordinates for 120 worldwide blood group variants is provided as a basis for this clinical application.