Thyroid nodule molecular profiling: The clinical utility of Afirma Xpression Atlas for nodules with Afirma Genomic Sequencing Classifier-suspicious results.

BACKGROUND: The Afirma Genomic Sequencing Classifier uses whole transcriptome RNA sequencing to identify thyroid nodules as benign or suspicious. The Afirma Xpression Atlas became available in 2018 and reports findings across 593 genes, including 905 variants and 235 fusions. When an alteration is identified, its risk of malignancy and associated neoplasm type is listed. We report the results of Afirma Xpression Atlas testing at our institution during its first 2 years of clinical use. METHODS: All patient charts with indeterminate thyroid nodules and Afirma Xpression Atlas results at our institution were reviewed. Thyroid nodule characteristics, cytology, Afirma Genomic Sequencing Classifier results, Afirma Xpression Atlas results, and final histopathology were reported. RESULTS: Afirma Xpression Atlas was performed on 136 indeterminate nodules since May 2018, and 103 met inclusion criteria. Forty-three nodules had positive Afirma Xpression Atlas results, and of these, 83.7% were follicular cell-derived thyroid cancer on surgical histopathology. This is similar to the overall 82.5% positive predictive value among Afirma Genomic Sequencing Classifier-suspicious indeterminate nodules during the same time period. Of the 60 nodules with negative Afirma Xpression Atlas, 73.3% were follicular cell-derived thyroid cancer on surgical histopathology. CONCLUSION: Afirma Xpression Atlas positivity is predictive of follicular cell-derived thyroid cancer, but its positive predictive value is similar to that of Genomic Sequencing Classifier-suspicious results alone at our institution, which is higher than previously published. Specific mutations likely predict follicular cell-derived thyroid cancer with higher accuracy, but our current sample size of any given mutation is too small to evaluate this further. Larger studies are needed to determine whether Afirma Xpression Atlas results predictably inform the risk of malignancy and tumor characteristics in thyroid nodules.

The sonic hedgehog signaling pathway stimulates anaplastic thyroid cancer cell motility and invasiveness by activating Akt and c-Met.

The sonic hedgehog (Shh) pathway is highly activated in thyroid neoplasms and promotes thyroid cancer stem-like cell phenotype, but whether the Shh pathway regulates thyroid tumor cell motility and invasiveness remains unknown. Here, we report that the motility and invasiveness of two anaplastic thyroid tumor cell lines, KAT-18 and SW1736, were inhibited by two inhibitors of the Shh pathway (cyclopamine and GANT61). Consistently, the cell motility and invasiveness was decreased by Shh and Gli1 knockdown, and was increased by Gli1 overexpression in KAT-18 cells. Mechanistic studies revealed that Akt and c-Met phosphorylation was decreased by a Gli1 inhibitor and by Shh and Gli1 knockdown, but was increased by Gli1 overexpression. LY294002, a PI-3 kinase inhibitor, and a c-Met inhibitor inhibited the motility and invasiveness of Gli1-transfected KAT-18 cells more effectively than the vector-transfected cells. Knockdown of Snail, a transcription factor regulated by the Shh pathway, led to decreased cell motility and invasiveness in KAT-18 and SW1736 cells. However, key epithelial-to-mesenchymal transition (EMT) markers including E-cadherin and vimentin as well as Slug were not affected by cyclopamine and GANT61 in either SW1736 or WRO82, a well differentiated follicular thyroid carcinoma cell line. Our data suggest that the Shh pathway-stimulated thyroid tumor cell motility and invasiveness is largely mediated by AKT and c-Met activation with little involvement of EMT.

The sonic hedgehog signaling pathway maintains the cancer stem cell self-renewal of anaplastic thyroid cancer by inducing snail expression.

CONTEXT: Cancer stem cells (CSCs) have been recently identified in thyroid neoplasm. Anaplastic thyroid cancer (ATC) contains a higher percentage of CSCs than well-differentiated thyroid cancer. The signaling pathways and the transcription factors that regulate thyroid CSC self-renewal remain poorly understood. OBJECTIVE: The objective of this study is to use two ATC cell lines (KAT-18 and SW1736) as a model to study the role of the sonic hedgehog (Shh) pathway in maintaining thyroid CSC self-renewal and to understand its underlying molecular mechanisms. DESIGN: The expression and activity of aldehyde dehydrogenase (ALDH), a marker for thyroid CSCs, was analyzed by Western blot and ALDEFLUOR assay, respectively. The effect of three Shh pathway inhibitors (cyclopamine, HhAntag, GANT61), Shh, Gli1, Snail knockdown, and Gli1 overexpression on thyroid CSC self-renewal was analyzed by ALDEFLUOR assay and thyrosphere formation. The sensitivity of transfected KAT-18 cells to radiation was evaluated by a colony survival assay. RESULTS: Western blot analysis revealed that ALDH protein levels in five thyroid cancer cell lines (WRO82, a follicular thyroid cancer cell line; BCPAP and TPC1, two papillary thyroid cancer cell lines; KAT-18 and SW1736, two ATC cell lines) correlated with the percentage of the ALDH(High) cells as well as Gli1 and Snail expression. The Shh pathway inhibitors, Shh and Gli1 knockdown, in KAT-18 cells decreased thyroid CSC self-renewal and increased radiation sensitivity. In contrast, Gli1 overexpression led to increased thyrosphere formation, an increased percentage of ALDH(High) cells, and increased radiation resistance in KAT-18 cells. Inhibition of the Shh pathway by three specific inhibitors led to decreased Snail expression and a decreased number of ALDH(High) cells in KAT-18 and SW1736. Snail gene knockdown decreased the number of ALDH(High) cells in KAT-18 and SW1736 cells. CONCLUSIONS: The Shh pathway promotes the CSC self-renewal in ATC cell lines by Gli1-induced Snail expression.