Focal adhesion kinase splicing and protein activation in papillary thyroid carcinoma progression.

Papillary thyroid carcinoma (PTC), a common endocrine malignancy, presents a challenge from a prognostic standpoint. Molecular alterations underlying PTC progression include deregulation of focal adhesion kinase (FAK) at post-transcriptional and post-translational levels. Searching for candidate markers of PTC progression, we investigated the prognostic significance of FAK alterations on mRNA/protein level. The expression levels and subcellular localisation of auto-phosphorylated FAK (pY397-FAK) were determined by western blot (WB) and immunohistochemistry. The quantity of total FAK mRNA, alternatively spliced FAK-Del26 and FAK-Del33 variants were analysed by RT-qPCR and related to pY397-FAK expression and subcellular distribution. The results were correlated with clinicopathological parameters of the patients. The expression of pY397-FAK was significantly elevated in malignant samples. Active FAK showed predominant cytoplasmic distribution with co-occurrence along the membrane, while nuclear staining was found less frequently. Expression of pY397-FAK in separate cellular compartments correlated with adverse clinicopathological parameters, but the strongest association was found when their mean scores were calculated. Alternatively spliced FAK-Del33 and total FAK transcripts positively correlated to pY397-FAK protein levels as well as to characteristics of PTC advancement. Over-expression of FAK on mRNA (total and Del-33) and activated protein (pY397-FAK) levels is a feature of PTC advanced stages. Of the analysed alterations, the mean pY397-FAK IHC score showed the best predictive performance. Correlation between mRNA FAK-Del33 and pY397-FAK expression implies a regulatory role of alternative splicing in PTC patients.

Detecting thyrotropin receptor mRNA from peripheral blood of patients with differentiated thyroid cancer rules out non-aggressive cases.

BACKGROUND: Early diagnosis of thyroid cancer is hampered by the inability of fine-needle aspiration biopsy (FNAB) to accurately classify ∼30% of cases while preoperative cancer staging detects lymph nodal involvement in only half of cases. Liquid biopsy may present an accurate, non-invasive alternative for preoperative thyroid nodule assessment. Thyrotropin receptor (TSHR) mRNA, a surrogate marker for circulating cancer cells (CTC), may be an option for early detection of malignancy from peripheral blood, but requires methodological improvements. We aimed to investigate if TSHR mRNA can be detected in low sample volumes by employing an ultrasensitive method - droplet digital PCR (ddPCR). METHODS: Less than 5 mL of blood was collected from 47 patients with thyroid nodules (25 benign and 22 malignant). RNA was isolated from the fraction of mononuclear cells where CTCs segregate. Samples were analysed for the presence of TSHR mRNA by ddPCR. RESULTS: Thyrotropin receptor mRNA was detectable in 4 mL sample volumes, with the test having good specificity (80%) but modest diagnostic accuracy (68.1%). Combining TSHR mRNA with ultrasound features and FNAB diagnosis, the test reaches high rule-out performances (sensitivity = 90% and NPV = 88.2%). Strikingly, TSHR mRNA correctly classified all samples with thyroid capsule invasion, lymph node metastasis and extrathyroidal extension. If aggressiveness is defined using these parameters, TSHR mRNA test reaches 100% sensitivity and 100% NPV for detecting high-risk cases. CONCLUSIONS: Employing ddPCR for TSHR mRNA improves its measurement by enabling detection in sample volumes common for laboratory testing. The test displays high prognostic performance, showing potential in preoperative risk assessment.

MiR-203a-3p, miR-204-3p, miR-222-3p as useful diagnostic and prognostic tool for thyroid neoplasia spectrum.

PURPOSE: The challenge in the diagnosis and treatment of thyroid carcinoma is to correctly classify neoplasias with overlapping features and to identify the high-risk patients among those with a less aggressive form, in order to personalize the treatment of thyroid carcinoma patients accordingly. METHODS: MiR-203a-3p, miR-204-3p, and miR-222-3p levels were determined in 99 cases of thyroid neoplasias (77 papillary thyroid carcinomas (PTC) of diverse variants, 12 follicular thyroid adenomas (FTA) and 10 nodular goiters (NG)) along with 99 adjacent non-malignant thyroid tissues using quantitative RT-PCR. The results were evaluated in comparison with the clinicopathological features of the patients and available TCGA data. RESULTS: Down-regulated miR-203a-3p indicates the presence of thyroid tumor (PTC or FTA) with high sensitivity (75%) and specificity (73%), while its up-regulation indicates NG. If miR-203a-3p is down-regulated, up-regulated miR-204-3p with high sensitivity (83.3%) and specificity (74.4%) indicates FTA presence, while up-regulated miR-222-3p, with high sensitivity (76.6%) and specificity (75.0%), points to PTC. The expression of miR-204-3p and miR-222-3p depends on the PTC subtype (P < 0.05). While the deregulated expression of tested miRs is associated with a long-range of unfavorable clinicopathological parameters of PTC, only abundant expression of miR-222-3p may be used as an independent predictive factor for the presence of extrathyroid invasion and advanced pTNM stage of PTC (P < 0.05). CONCLUSION: Successive evaluation of miR-203a-3p, miR-204-3p, and miR-222-3p expression can help in the differential diagnosis of thyroid neoplasias. A high relative value of miR-222-3p expression is an independent predictive factor for the presence of extrathyroid invasion and advanced pTNM stage of PTC. The panel consisting of miR-203a-3p, miR-204-3p, and miR-222-3p could be used as a diagnostic and prognostic tool for personalizing the treatment of thyroid cancer patients.

BRAFV600E, BANCR, miR-203a-3p and miR-204-3p in Risk Stratification of PTC Patients.

In order to enhance the risk stratification of papillary thyroid carcinoma (PTC) patients, we assessed the presence of the most common mutation in PTC (BRAFV600E) with the expression profiles of long non-coding RNA activated by BRAFV600E (BANCR) and microRNAs, which share complementarity with BANCR (miR-203a-3p and miR-204-3p), and thereafter correlated it with several clinicopathological features of PTC. BRAFV600E was detected by mutant allele-specific PCR amplification. BANCR and miRs levels were determined by quantitative RT-PCR. Bioinformatic analysis was applied to determine the miRs' targets. The expression profile of miR-203a-3p/204-3p in PTC was not affected by BRAFV600E. In the BRAFV600E-positive PTC, high expression of miR-203a-3p correlated with extrathyroidal invasion (Ei), but the patients with both high miR-203a-3p and upregulated BANCR were not at risk of Ei. In the BRAFV600E-negative PTC, low expression of miR-204-3p correlated with Ei, intraglandular dissemination and pT status (p < 0.05), and the mutual presence of low miR-204-3p and upregulated BANCR increased the occurrence of Ei. Bioinformatic analysis predicted complementary binding between miR-203a-3p/204-3p and BANCR. The co-occurrence of tested factors might influence the spreading of PTC. These findings partially describe the complicated network of interactions that may occur during the development of PTC aggressiveness, potentially providing a new approach for high-risk PTC patient selection.

Expression of pY397-FAK and Its miR Regulators Drive Dedifferentiation in the Thyroid Neoplasia Spectrum.

Thyroid carcinomas are growing malignancies worldwide. They encompass several diagnostic categories with varying degrees of dedifferentiation. Focal adhesion kinase is involved in cellular communication and locomotion. It is regulated on a posttranscriptional level by miR-7, miR-135a, and miR-138 and on a posttranslational level by autophosphorylation at Y397 (pY397-FAK). We related regulators of FAK with histologic dedifferentiation, clinicopathological factors, and differential diagnosis in the thyroid neoplasia spectrum. We classified 82 cases into 5 groups with increasing aggressiveness: healthy tissue, follicular and classical variants of papillary thyroid carcinoma (PTC), dedifferentiated PTC, and anaplastic carcinoma. MiRs were analyzed by RT-qPCR. Protein expression of pY397-FAK was analyzed by immunohistochemistry (separately in the membrane, cytoplasm, and nuclear compartment) and Western blot. All three miRs were upregulated in healthy tissue compared to malignant, while pY397-FAK was downregulated. MiRs and pY397-FAK were not mutually correlated. MiR-135a-5p was decreasing while membranous and cytoplasmic pY397-FAK increased with dedifferentiation. Neither miR correlated with clinicopathological factors. MiR-135a-5p, miR-138-5p, and membranous and cytoplasmic pY397-FAK discriminated the follicular from the classical variant of PTC. Disturbances of FAK regulation on different levels contribute to neoplastic dedifferentiation. pY397-FAK exerts its oncogenic role in the membrane and cytoplasm. Diagnostically, miRs-135a-5p, miR-138-5p, and membranous and cytoplasmic pY397-FAK differentiated between classical and follicular PTC.

Predictive Significance of Two MMP-9 Promoter Polymorphisms and Acetylated c-Jun Transcription Factor for Papillary Thyroid Carcinoma Advancement.

Papillary thyroid carcinoma represents a challenge from a prognostic standpoint. Molecular alterations responsible for PTC advancement include MMP-9 genetic promoter polymorphisms that bind transcription factors with varying degrees of affinity and, hence, constitute a predisposition for MMP-9 expression. We examined how two promoter polymorphisms (the -1562 C/T transition and -131 (CA)n tandem repeats) as well as levels of the c-Jun transcription factor and its modified form acetylated at Lys271 influence MMP-9 expression and PTC progression. A significant proportion of PTC samples were heterozygous for the (CA)n tandem repeat number, had a transcription-promoting T allele at -1562, and expressed high levels of c-Jun, acetylated c-Jun, and MMP-9 protein. The T allele at the -1562 position accompanied the elevated MMP-9 protein expression, while high acetylated c-Jun levels accompanied the high MMP-9 protein levels on mRNA. The -1562 C/T transition, MMP-9, and acetylated c-Jun were associated with the presence of extra-thyroid invasion and degree of tumor infiltration, while the T allele and acetylated c-Jun also correlated with tumor stage. We conclude that the -1562 MMP-9 polymorphism and levels of acetylated c-Jun affect PTC progression via modulation of MMP-9 levels. Genotyping the MMP-9 at -1562 and estimating the levels of MMP-9 and acetylated c-Jun in PTC may prove beneficial in identifying high-risk patients.

Circulating MicroRNA Profiles as Potential Biomarkers for Differentiated Thyroid Cancer Recurrence.

CONTEXT: Circulating microRNAs (miRNAs) are emerging biomarkers of thyroid cancer. OBJECTIVE: This study sought to identify the profile of circulating miRNAs and its response to human recombinant TSH (rhTSH) in thyroid cancer patients with recurrent/persistent disease. METHODS: We obtained serum samples from 30 patients with differentiated thyroid cancer, 14 with recurrent/persistent disease and 16 with complete remission. We used next-generation sequencing to define the miRnomes along with a comprehensive quantitative PCR (qPCR) validation using 2 different platforms. We made a transversal study by comparing serum miRNA profiles of patients with or without recurrent/persistent disease and a longitudinal study looking at differences before and after rhTSH stimulation. Selected miRNAs were then studied in human thyroid cancer cell lines TPC-1, FTC-133, and OCUT-2 in response to TSH stimulation. RESULTS: We could not demonstrate any consistent differences in serum profiles of known miRNAs between patients with and without recurrent/persistent disease or before and after rhTSH stimulation. However, our sequencing data revealed 2 putative novel miRNAs that rise with rhTSH stimulation in the serums of patients with recurrent/persistent disease. We further confirmed by qPCR the upregulation of these putative miRNAs both in serums and in TSH-stimulated cells. We also show miRNAs that are good candidates for housekeeping genes in the serum of patients independently of the levels of TSH. CONCLUSIONS: The present study does not provide evidence that known miRNAs can be used as circulating markers for recurrence of thyroid cancer. However, we suggest that novel miRNA molecules may be related to thyroid cancer pathogenesis.

Elevated BANCR expression levels have different effects on papillary thyroid carcinoma progression depending on the presence of the BRAFV600E mutation.

INTRODUCTION: The role of BRAF-activated non-protein coding RNA (BANCR) in papillary thyroid carcinoma (PTC) is controversial, its clinical significance is unclear and no study has correlated the presence of the BRAFV600E mutation in PTC with BANCR expression. METHODS: BANCR levels in PTC and matched nonmalignant thyroid epithelial tissues from 85 patients were determined using quantitative RT-PCR. BRAFV600E was detected by mutant allele-specific PCR amplification. The results were correlated with clinicopathological characteristics of the patients. RESULTS: The presence of BRAFV600E associates with lower relative BANCR expression (RBE) in PTC (p = 0.008). RBE is down-regulated in BRAFV600E positive PTC, while it is unchanged or up-regulated in BRAFV600E negative PTC compared to the levels in paired nonmalignant tissue (p = 0.001). At the cut-off of 31.3%, sensitivity of fold change of BANCR for the presence of BRAFV600E is 68.0% and specificity is 67.2%. In BRAFV600E positive PTC up-regulated BANCR predicts lymph node metastasis (p = 0.001), while in BRAFV600E negative PTCs high RBE predicts thyroid capsule invasion (p = 0.028). CONCLUSIONS: Depending on the presence of BRAFV600E, elevated BANCR levels demonstrated different effects on lymphatic spreading and local PTC invasion. Therefore, BANCR could be a useful prognostic biomarker in risk stratification of PTC patients.