Thyroid Tissue Samples Research Articles

Receptor-Binding Cancer Antigen Expression in Thyroid Neoplasms: A Retrospective Study.

Background: Receptor-binding cancer antigen (RCAS1) is a membrane protein, regarded as a tumor-associated antigen. Cancer cells evade immune response with RCAS1 up-regulation, inducing apoptosis to tumor infiltrating lymphocytes. Thyroid cancer incidence is rising and its accurate diagnosis in early stage is targeted. The aim of this study is to access RCAS1 expression in benign and malignant thyroid pathology. Methods: This is a retrospective study of 110 patients, who had thyroidectomy in a single tertiary referral centre between January 2008 until December 2014. Immunohistochemistry study for RCAS1 expression was carried out and correlation with clinical and histopathological data is attempted. Results: RCAS1 immunostaining was found positive in 81 out of 110 cases. Notably it was deemed positive in all malignant thyroid tissue samples (p 0.001). In thyroid malignancy, tumor size, thyroid capsule invasion and positive lymph nodes status were positively correlated with moderate and strong expression of RCAS1. For papillary thyroid carcinoma, the vast majority (35/37 cases, 94.6%) were also classified as having moderate or strong RCAS1 expression. Conclusions: RCAS1 expression can aid in differential diagnosis between benign and malignant thyroid pathology, while its strong expression correlates with worse oncological features.

BCL2 and hsa-miR-181a-5p are potential biomarkers associated with papillary thyroid cancer based on bioinformatics analysis

BackgroundThe morbidity of thyroid carcinoma has been rising worldwide and increasing faster than any other cancer type. The most common subtype with the best prognosis is papillary thyroid cancer (PTC); however, the exact molecular pathogenesis of PTC is still not completely understood.MethodsIn the current study, 3 gene expression datasets (GSE3678, GSE3467, and GSE33630) and 2 miRNA expression datasets (GSE113629 and GSE73182) of PTC were selected from the Gene Expression Omnibus (GEO) database and were further used to identify differentially expressed genes (DEGs) and deregulated miRNAs between normal thyroid tissue samples and PTC samples. Then, Gene Ontology (GO) and pathway enrichment analyses were conducted, and a protein-protein interaction (PPI) network was constructed to explore the potential mechanism of PTC carcinogenesis. The hub gene detection was performed using the CentiScaPe v2.0 plugin, and significant modules were discovered using the MCODE plugin for Cytoscape. In addition, a miRNA-gene regulatory network in PTC was constructed using common deregulated miRNAs and DEGs.ResultsA total of 263 common DEGs and 12 common deregulated miRNAs were identified. Then, 6 significant KEGG pathways (P < 0.05) and 82 significant GO terms were found to be enriched, indicating that PTC was closely related to amino acid metabolism, development, immune system, and endocrine system. In addition, by constructing a PPI network and miRNA-gene regulatory network, we found that hsa-miR-181a-5p regulated the most DEGs, while BCL2 was targeted by the most miRNAs.ConclusionsThe results of this study suggested that hsa-miR-181a-5p and BCL2 and their regulatory networks may play important roles in the pathogenesis of PTC.

Targeted Mutational Profiling and a Powerful Risk Score as Additional Tools for the Diagnosis of Papillary Thyroid Cancer

Nowadays, the complementary diagnostics based on the suspicious thyroid lesion specific mutational state analysis is indispensable in the clinical practice. We aimed to test and validate our novel 568-mutational hotspot panel (23 cancer-related genes) on papillary thyroid cancers (PTCs) and their tumor-free pairs to find the most powerful mutation pattern related to PTC. The sequencing method was carried on with Ion Torrent PGM on 67 thyroid tissue samples. The most commonly detected mutation was the BRAF c.1799 T > A in all non-classical PTC cases. We utilized a multivariate statistical method (CVA) to determine a discrimination score based on mutational data array and to assess malignancy risk. Based on variants, the BRAF gene has by far the highest indicative power, followed by TSHR and APC. We highlighted novel aspects of the mutational profile and genetic markers of PTC. CVA has correctly assigned most of the samples based on the mutation frequencies and different variables of the selected genes, with high analytical probabilities. The final goal is to set up a new comprehensive rule-in and rule-out test to support the clinical decision making mainly in inconclusive fine-needle aspiration biopsy cases.

EZH2 upregulation by ER\u03b1 induces proliferation and migration of papillary thyroid carcinoma

BackgroundThe incidence of papillary thyroid carcinoma (PTC) has been increasing worldwide in recent years. Therefore, novel potential therapeutic targets for PTC are urgently needed. Enhancer of zeste homolog 2 (EZH2), a methyltransferase belonging to PRC2, plays important roles in epigenetic silencing and cell cycle regulation. EZH2 overexpression has been found in several malignant tumor tissues, while its expression and function in PTC are largely unknown.MethodsSixty-five cases of PTC tissue confirmed by pathology and 30 cases of normal thyroid tissue adjacent to PTC tissue were collected from patients undergoing surgical treatment, between February 2003 and February 2006. We investigated the clinic pathologic significance of EZH2 expression using Realtime-PCR and IHC in 65 human PTC tissues and 30 normal thyroid tissue samples. The EZH2 expression in human PTC cell lines (K1 and W3) and the normal thyroid follicular epithelial cell line Nthy-ori 3–1 was analyzed by Western blotting and Realtime PCR. The expressions of ERα and ERβ in cell lines were analyzed by Realtime PCR.The tumor cell biological behavior was evaluated by CCK8 assay, colony formation assay, transwell migration assay and xenograft tumors model.ResultsHigher rate of EZH2 expression was found in PTC tissues than in normal thyroid tissues, EZH2 expression is associated with lymph node metastasis and recurrent. Inhibition of EZH2 in PTC cell lines downregulates cellular proliferation and migration. PTC is a disease with high incidence of female and E2-ERα upregulates EZH2 expression.ConclusionsThese results suggest a potential role of EZH2 for the PTC growth and metastasis. As a novel therapy, a pharmacological therapy targeting EZH2 has full potential in treatment of PTC.

Expression of thyroglobulin by regulatory T cells in thyroid tissue.

The follicles are the minimal functional unit of the thyroid; the morphology and the function of each follicle can vary significantly. However, the reasons for the apparent follicular heterogeneity are poorly understood. Some tissue-resident regulatory T cells (Tregs) have a special phenotype that expresses unique molecules related to local tissue and regulates the tissue functions. The aim of this study was to identify the phenotype of thyroid Tregs and the roles of thyroid Tregs in thyroid physiological regulation. Thyroid tissue and peripheral blood samples were obtained from patients with benign thyroid nodules. Microarray-based gene expression, flow cytometry, immunofluorescence microscopy, and functional analysis of thyroid Tregs were performed. Here, we demonstrated that human thyroid Tregs expressed high level of thyroglobulin (Tg), both gene and protein. The immunofluorescence microscopy of thyroid section showed that the FOXP3+Tg+ cells concentrated in some of the thyroid follicles, at the side of the thyroid follicle. The peripheral blood Tregs expressed minimal levels of Tg, and low levels of Tg could effectively induce peripheral blood Tregs to express Tg, which was independent of thyrotropin simulation. Furthermore, the Tg secreted freely from thyroid Tregs that negatively regulated some thyroid-related genes expression. Our results revealed that the thyroid Tregs was a distinct population of Tregs, which expressed high level of Tg. The thyroid Tregs regulate thyroid function by Tg that is paracrine from the cells.

Localization and characterization of thyroid microcalcifications: A histopathological study.

Thyroid calcification is frequent in thyroid nodules. The aim of our study was to evaluate the prevalence of calcifications in thyroid tissue samples of patients with various thyroid diseases, and to identify their composition according to their localization. Among 50 thyroid samples included, 56% were malignant (papillary carcinoma) and 44% were benign (adenoma, multinodular goiter, Graves’ disease, sarcoidosis). Calcifications were found in 95% of samples using polarised light microscopy, whereas only 12% were described in initial pathological reports. Three types were individualised and analyzed by infrared spectrometry (μFTIR): colloid calcifications composed of calcium oxalate, capsular calcifications and psammoma bodies, both composed of calcium phosphate. Of notice, psammoma bodies characterized by FE-SEM were composed of concentric structure suggesting a slow process for crystal deposition. Calcium phosphates were found only in malignant samples whereas calcium oxalate was not associated with a define pathology. Proliferation assessed by KI67 staining was high (33% of positive follicles), and RUNX2, OPN, and CD44 positive staining were detected in thyrocytes with a broad variation between samples. However, thyrocyte proliferation and differentiation markers were not associated with the number of crystals. TRPV5 and CaSR expression was also detected in thyrocytes. mRNA transcripts expression was confirmed in a subgroup of 10 patients, altogether with other calcium transporters such as PMCA1 or Cav1.3. Interestingly, TRPV5 mRNA expression was significantly associated with number of colloid calcifications (rho = -0.72; p = 0.02). The high prevalence of calcium oxalate crystals within colloid gel raises intriguing issues upon follicle physiology for calcium and oxalate transport.

Validation of Reference Genes for Normalization of Relative qRT-PCR Studies in Papillary Thyroid Carcinoma

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) in thyroid tumors require accurate data normalization, however, there are no sufficient studies addressing the suitable reference genes for gene expression analysis in malignant and normal thyroid tissue specimens. The purpose of this study was to identify valid internal control genes for normalization of relative qRT-PCR studies in human papillary thyroid carcinoma tissue samples. The expression characteristics of 12 candidate reference genes (GAPDH, ACTB, HPRT1, TBP, B2M, PPIA, 18SrRNA, HMBS, GUSB, PGK1, RPLP0, and PGM1) were assessed by qRT-PCR in 45 thyroid tissue samples (15 papillary thyroid carcinoma, 15 paired normal tissues and 15 multinodular goiters). These twelve candidate reference genes were selected by a systematic literature search. GeNorm, NormFinder, and BestKeeper statistical algorithms were applied to determine the most stable reference genes. The three algorithms were in agreement in identifying GUSB and HPRT1 as the most stably expressed genes in all thyroid tumors investigated. According to the NormFinder software, the pair of genes including ‘GUSB and HPRT1’ or ‘GUSB and HMBS’ or ‘GUSB and PGM1’ were the best combinations for selection of pair reference genes. The optimal number of genes required for reliable normalization of qPCR data in thyroid tissues would be three according to calculations made by GeNorm algorithm. These results suggest that GUSB and HPRT1 are promising reference genes for normalization of relative qRT-PCR studies in papillary thyroid carcinoma.

&lt;p&gt;Upregulation Of Protein Tyrosine Phosphatase Receptor Type C Associates To The Combination Of Hashimoto’s Thyroiditis And Papillary Thyroid Carcinoma And Is Predictive Of A Poor Prognosis&lt;/p&gt;

IntroductionPTC is not generally considered a lethal disease, but prone to recurrence as the prognosis. Hashimoto’s thyroiditis (HT) is an important factor that affects the prognosis of papillary thyroid carcinoma (PTC). It is crucial to find biomarkers to identify the combination of HT with PTC and to predict the prognosis.MethodsRNASeq data from the Cancer Genome Atlas (TCGA) database was used to screen differentially expressed genes (DEGs) of PTC with HT via the edgeR package of R software version 3.3.0. Also, the DEGs were applied to the DAVID web-based tool to determine the enrichment of gene functions via Gene Ontology (GO) analysis and to identify associated pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. By constructing protein interaction networks within Cytoscape software, we screened candidate genes and explored possible relationships with the clinical phenotype of PTC. Finally, additional thyroid tissue samples were collected to verify the results above.ResultsAfter analyzing the RNA-Seq data of PTC patients from the Cancer Genomic Atlas, 497 differentially expressed PTC genes were found to be associated with HT, of which protein tyrosine phosphatase receptor type C (PTPRC), KIT, and COL1A1 were associated with tumor size and lymph node metastasis (p < 0.05). Verification of these results with another 30 thyroid tissues of clinical PTC patients revealed that the expression level of PTPRC in the PTC with HT group was higher than that in the PTC without HT group (p < 0.05) and the ROC curve showed a good discrimination (area under the curve = 0.846). However, the correlation with the clinical phenotype was not statistically significant (p > 0.05).DiscussionThese data suggest that upregulation of PTPRC enhances the incidence of HT associated with PTC and is also predictive of a poor prognosis.

Ku80 gene knockdown by the CRISPR/Cas9 technique affects the biological functions of human thyroid carcinoma cells.

In the present study, to evaluate the role of Ku80 in thyroid carcinoma (TC), 86 thyroid tissue samples from patients with a spectrum of thyroid disorders were examined for protein levels of Ku80, nuclear factor-κB (NF-κB) and RET/TC by immunohistochemistry. Furthermore, in TC cells, Ku80 mRNA was detected by reverse transcription-quantitative PCR analysis and silenced using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technique to assess its role. An antibody array was used to identify Ku80-related regulatory genes. The protein levels of Ku80 in the TC tissues were significantly higher than those in non-neoplastic adjacent tissue samples (P<0.01). The activation of NF-kB and expression of RET/TC in the TC group were significantly increased (P<0.05) and were correlated with the protein expression of Ku80 (P<0.05). In papillary TC cells, the mRNA levels of Ku80 were high; Ku80 knockdown resulted in reductions in proliferation, invasion and colony formation, increased apoptosis, and reduced levels of proteins involved in MAPK signaling, cell proliferation and apoptosis. The high expression of Ku80 in TC was found to be associated with the expression of RET/TC and activation of NF-κB, and Ku80 knockdown decreased the malignancy of TC cells.

DNA Methylation-Based Method to Differentiate Malignant from Benign Thyroid Lesions.

Background: The differential diagnosis of thyroid nodules using fine-needle aspiration biopsy (FNAB) is challenging due to the inherent limitation of the cytology tests. The use of molecular markers has potential to complement the FNAB-based diagnosis and avoid unnecessary surgeries. In this study, we aimed to identify DNA methylation biomarkers and to develop a diagnostic tool useful for thyroid lesions. Methods: Genome-wide DNA methylation profiles (Illumina 450K) of papillary thyroid carcinoma (PTC = 60) and follicular thyroid carcinoma (FTC = 10) were compared with non-neoplastic thyroid tissue samples (NT = 50) and benign thyroid lesions (BTL = 17). The results were confirmed in publicly available databases from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) using the same DNA methylation platform. Two classifiers were trained to discriminate FTC and PTC from BTL. To increase the applicability of the method, six differentially methylated CpGs were selected and evaluated in 161 thyroid tumors and 69 BTL postsurgical specimens and 55 prospectively collected FNAB using bisulfite-pyrosequencing. Results: DNA methylation analysis revealed 2130 and 19 differentially methylated CpGs in PTC and FTC, respectively. The CpGs confirmed by GEO and TCGA databases showing high areas under the receiver operating characteristic curve in all sample sets were used to train our diagnostic classifier. The model based on six CpGs was able to differentiate benign from malignant thyroid lesions with 94.3% sensitivity and 82.4% specificity. A similar performance was found applying the algorithm to TCGA and GEO external data sets (91.3-97.4% sensitivity and 87.5% specificity). We successfully evaluated the classifiers using a bisulfite-pyrosequencing technique, achieving 90.7% sensitivity and 75.4% specificity in surgical specimens (five of six CpGs). The study comprising FNAB cytology materials corroborated the applicability and performance of the methodology, demonstrating 86.7% sensitivity and 89.5% specificity in confirmed malignant tumors, and 100% sensitivity and 89% specificity in cases with indeterminate cytology. Conclusions: A novel diagnostic tool with potential application in preoperative screening of thyroid nodules is reported here. The proposed protocol has the potential to avoid unnecessary thyroidectomies.

Evaluating pre- and post-operation plasma miRNAs of papillary thyroid carcinoma (PTC) patients in comparison to benign nodules

BackgroundThyroid cancer is the most common endocrinology cancer that its incidence has increased in recent decades. miRNAs are new biomarkers in recent studies in the diagnosis and follow-up of these patients.MethodsBlood and thyroid tissue samples were obtained from two groups of included patients (PTC and benign nodules), pre- and post-operation. miRNAs were extracted from these plasma samples and were measured quantitatively. After cDNA synthesis, qPCR was carried out. Then tissue samples were investigated, and their relation to miR expression was studied. These results were analyzed by paired- and independent samples t-test, and non-parametric tests.ResultsmiR-222 and miR-181a declined in PTC patients before and after surgery, significantly (P < 0.001 for both groups), with no significant difference in control group before and after surgery (P = 0.61 for miR-222 and P = 0.06 for miR-181a). The difference between the two groups, pre-and post-operation, was statistically significant (P = 0.01 for miR-222 and P < 0.001 for miR-181a). Comparing case and control groups, pre- and post-operatively, yielded no significant difference, in miR-155-5p levels (P = 0.61 and P = 0.53, respectively). Comparing PTC and control groups before surgery showed a significant difference (P = 0.01), while no significant difference was observed comparing them after surgery, in miR146-a (P = 0.27). Our results depicted a higher miR-155-5p and miR-146a expression before surgery than after it (P < 0.001 in both groups, for both miRs). We found a significant relationship between miR-222 and BRAFV600E mutation and significantly higher levels of miR-181a with increasing tumor size in PTC patients.ConclusionmiR-222 showed overexpression in all PTC cases, which is indicative of a relation between miRNA and PTC. Also, comparing miR-181 and miR-146a showed a significant difference between cancerous and benign cases. miR-155-5p as an inflammatory factor, showed no significant changes, comparing two groups.

Differential expression of RET isoforms in normal thyroid tissues, papillary and medullary thyroid carcinomas.

We investigated the expression of RET9 and RET51 isoforms in medullary (MTC), papillary (PTC) thyroid carcinoma, normal thyroid tissues, and pheochromocytoma (PHEO) to verify if these isoforms are present also in follicular thyroid cell-derived tissues, and if there is a differential expression of RET9 and RET51 in MTC. Nineteen patients with MTC, 18 patients with PTC, 18 samples of contralateral normal thyroid tissues, and 5 cases of PHEO were included in this study. RET isoform expression was studied by real-time RT-PCR. All MTCs and PHEOs were positive for RET9 and RET51. Fourteen/eighteen (77.7%) PTC cases were positive for RET9 and/or RET51, and four were positive for only one of the genes. In normal thyroid tissues, 3/18 (16.7%) cases were negative for both isoforms, 4/18 (22.2%) were positive for both, and 11/18 (61.1%) were positive for only one. RET isoforms were expressed at different levels in MTC, PHEO, PTC, and normal thyroid tissues: RET9 expression was higher in PHEO than in MTC, PTC, and normal thyroid tissues. RET9 expression was also higher in MTC than in PTC and normal thyroid tissues. No difference was observed between PTC and normal thyroid tissues. A similar pattern of expression was observed for RET51. In addition, RET51 was significantly more expressed than RET9 in MTC, while RET9 was the predominant isoform in PHEO. Our study documented the expression of the RET9 and RET51 isoforms in normal thyroid and PTC tissues. RET9 and RET51 isoforms were also present in MTC and PHEO. RET51 expression was higher than RET9 expression in MTC, while there was no difference in the expression of these two isoforms in PTC and normal thyroid tissues. RET9 was more highly expressed than RET51 in PHEOs.

Prevalence of BRAFV600E Mutation and Human Parvovirus B19 Infection in Thyroid Cancer

Background: Thyroid carcinoma, the most prevalent endocrine malignancy, has increased rapidly in recent decades. A single-base substitution in the BRAF gene is identified as the most common genetic event in thyroid carcinoma. Infections with both DNA and RNA viruses can induce chronic inflammatory diseases and cancer. Human parvovirus B19 (B19V) infection has been implicated in thyroid cancer as the most common inflammation of the endocrine system. Objectives: The study aimed to determine the prevalence of the BRAFV600E gene mutation and the presence of B19V infection in malignant and non-malignant thyroid tissue samples. Methods: A cross-sectional study was performed from January 2012 to December 2017 on 108 paraffin-embedded thyroid tissues from patients with thyroid cancer tumor and nodular goiter. After DNA extraction, PCR restriction fragment length polymorphism (PCR-RFLP) and nested PCR assays were used to detect the BRAFV600E gene mutation and B19V DNA in tissue specimens, respectively. To confirm PCR-RFLP reliability, the amplified products were subjected to DNA sequencing. Statistical analysis was performed to determine a possible correlation between the occurrence of BRAFV600E mutations and clinicopathological characteristics, such as tumor subtypes, gender, age, and B19V presence. Results: Overall, BRAFV600E mutation was detected in 77 out of 108 patients (71.3%) using PCR-RFLP, confirmed by DNA sequencing analysis. Using nested PCR, human parvovirus B19 DNA was detected in 14 out of 108 (13%) of the formalin-fixed, paraffin-embedded (FFPE) tissue specimens. Statistical analysis showed a significant difference in the prevalence of the BRAFV600E mutation in thyroid cancer patients when compared with the control group (P ≤ 0.001). Conclusions: Since cytological examinations depend on fine needle aspiration of the thyroid (FNAB) cannot be conclusive; hence it might be suggested that detection of the BRAFV600E gene mutation can be considered as a feasible assay. However, a low detection rate of the B19V DNA in FFPE tissue samples suggests that B19V infection is not associated with thyroid cancer.

MON-563 Quantitative Assessment Of BRAF Mutations In Thyroid Cancer Using Droplet Digital PCR.

Background:Identification of somatic mutations is crucial for guiding therapeutic decisions in patients with thyroid cancer. Droplet digital PCR (ddPCR) is a sensitive and particularly useful method of nucleic acid detection for quantification of gene mutations. This study aimed to determine the analytic and clinical validity of BRAF ddPCR mutational testing in thyroid cancer tissue. Material and Methods: Initially we examined a training cohort of 30 patients with thyroid cancer that had been previously tested for BRAF mutation with Sanger sequencing and included 15 BRAF positive and 15 BRAF negative subjects. A second group included 65 samples from 18 patients with thyroid cancer. Samples were obtained from central and invasive areas of tumors, normal thyroid tissue and lymph node metastasis. The DNA was extracted using Thermo Fisher Scientific King Fisher Duo Prime Purification System, and DNA concentration was measured by NanoDrop. Digital PCR was performed using a QuantStudio 3D Digital PCR platform. QuantStudio Software was used for relative and quantitative data analysis. Results: In the training set, BRAF mutations were detected in 15/15 BRAF-positive thyroid cancers and in 0/15 BRAF- negative tumors. Analysis of tissue samples from the second group of patients demonstrated detection of wild type BRAF alleles in normal thyroid as well as in thyroid cancer tissue. In contrast, BRAFV600 mutant alleles were detected only in thyroid cancer tissue samples (13/18 (72%) examined patients). In cancer samples, the ratios of mutant/total BRAF alleles ranged from 1.47% to 42.8% (average 24.6%). Quantification of BRAF mutant alleles did not reveal differences between male and female patients or patient age. The ratios of mutant/total BRAF did not correlate with tumor size, and were not different in central and invasive areas of the thyroid tumors. Metastases at the time of surgery were detected in 2/5 patients with BRAF-negative tumors, and in 6/13 patients with BRAF-positive tumors. The ratios of mutant/total BRAF alleles were higher in cancers presenting with metastases at the time of surgery (average 26.6%) compared to tumors without metastases (average 19.6%). However, these differences were not statistically significant. The quantifications of mutant BRAF alleles in metastatic lesions showed that the ratios of mutant/total BRAF were similar to corresponding primary tumors in 3 cases, and were decreased in 3 cases. Conclusion: Overall, droplet digital PCR allows specific, sensitive and rapid detection of BRAF mutations in thyroid tissue samples. Quantitative evaluation of BRAF mutations offers an opportunity for additional assessment of prognosis and potential response to treatment with tyrosine kinase inhibitors. Further research on implementation of ddPCR-based assays of thyroid tumors may provide important insights to the management of patients with thyroid cancer.

A novel microfluidic device capable of maintaining functional thyroid carcinoma specimens ex vivo provides a new drug screening platform

BackgroundThough the management of malignancies has improved vastly in recent years, many treatment options lack the desired efficacy and fail to adequately augment patient morbidity and mortality. It is increasingly clear that patient response to therapy is unique to each individual, necessitating personalised, or ‘precision’ medical care. This demand extends to thyroid cancer; ~ 10% patients fail to respond to radioiodine treatment due to loss of phenotypic differentiation, exposing the patient to unnecessary ionising radiation, as well as delaying treatment with alternative therapies.MethodsHuman thyroid tissue (n = 23, malignant and benign) was live-sliced (5 mm diameter × 350-500 μm thickness) then analysed or incorporated into a microfluidic culture device for 96 h (37 °C). Successful maintenance of tissue was verified by histological (H&E), flow cytometric propidium iodide or trypan blue uptake, immunohistochemical (Ki67 detection/ BrdU incorporation) and functional analysis (thyroxine [T4] output) in addition to analysis of culture effluent for the cell death markers lactate dehydrogenase (LDH) and dead-cell protease (DCP). Apoptosis was investigated by Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL). Differentiation was assessed by evaluation of thyroid transcription factor (TTF1) and sodium iodide symporter (NIS) expression (western blotting).ResultsMaintenance of gross tissue architecture was observed. Analysis of dissociated primary thyroid cells using flow cytometry both prior to and post culture demonstrated no significant change in the proportion of viable cells. LDH and DCP release from on-chip thyroid tissue indicated that after an initial raised level of release, signifying cellular damage, detectable levels dropped markedly. A significant increase in apoptosis (p < 0.01) was observed after tissue was perfused with etoposide and JNK inhibitor, but not in control tissue incubated for the same time period. No significant difference in Ki-67 positivity or TTF1/NIS expression was detected between fresh and post-culture thyroid tissue samples, moreover BrdU positive nuclei indicated on-chip cellular proliferation. Cultured thyroid explants were functionally viable as determined by production of T4 throughout the culture period.ConclusionsThe described microfluidic platform can maintain the viability of thyroid tissue slices ex vivo for a minimum of four days, providing a platform for the assessment of thyroid tissue radioiodine sensitivity/adjuvant therapies in real time.

Immune Profiling of Thyroid Carcinomas Suggests the Existence of Two Major Phenotypes: An ATC-Like and a PDTC-Like.

The understanding of the mechanisms underlying thyroid cancer immune escape can lead to the identification of new molecular targets and/or efficacy biomarkers. For this purpose, we performed immune expression profiling in thyroid cancers to obtain a comprehensive view on immune mechanisms activated during cancer progression. The study was conducted retrospectively in 25 papillary thyroid carcinomas (PTCs), 14 poorly differentiated thyroid carcinomas (PDTC), 13 anaplastic thyroid carcinomas (ATCs), and 7 normal thyroid (NT) tissue samples. Gene expression profiling was obtained on RNA samples using the Nanostring platform and its nCounter PanCancer Immune Profiling Panel. Gene expression comparison of ATC, PTC, and PDTC vs NT showed high number of regulated genes in cancer samples. In detail, immune-related gene sets were significantly upregulated (ATC > PTC > > PDTC). Most ATC and approximately half of PTC showed a microenvironment infiltrated by macrophages and T-cells with CD8+ effector phenotype, part of which appeared to be functionally exhausted. Conversely, most PDTC, as NT samples, as the remaining part of PTC, displayed a poor or absent infiltration by immune cells. Interestingly, an upregulation of inhibitory immune checkpoint mediators, including PDL1, PDL2, PD1, LAG-3, TIM-3, PVR, and TIGIT, could be detected in ATC and PTC. These data indicated the existence of two major immune phenotypes in thyroid carcinoma: an ATC-like one, including hot and altered-immunosuppressed tumors and a PDTC-like one, including altered-excluded and cold tumors. Confirmation of the findings in locally advanced or metastatic cancer tissues is expected to have important immunotherapeutic implications.

Characterization of human telomerase reverse transcriptase promoter methylation and transcription factor binding in differentiated thyroid cancer cell lines.

Telomerase reverse transcriptase (TERT) activation plays an important role in cancer development by enabling the immortalization of cells. TERT regulation is multifaceted, and its promoter methylation has been implicated in controlling expression through alteration in transcription factor binding. We have characterized TERT promoter methylation, transcription factor binding, and TERT expression levels in five differentiated thyroid cancer (DTC) cell lines and six normal thyroid tissue samples by targeted bisulfite sequencing, ChIP-qPCR, and qRT-PCR. DTC cell lines express varying levels of TERT and exhibit TERT promoter methylation patterns similar to patterns seen in other telomerase positive cancer cell lines. The minimal promoter immediately surrounding the transcription start site is hypomethylated, while further upstream portions show dense methylation. In contrast, the TERT promoter in normal thyroid tissue is largely unmethylated throughout and expresses TERT minimally. Transcription factor binding is also affected by TERT mutation status. The E-twenty-six (ETS) factor GABPA exhibits TERT binding in the TERT mutant DTC cells only, and allele-specific methylation patterns at the minimal promoter were observed as well, which may indicate allele-specific factor recruitment at the minimal promoter. Furthermore, we identified binding sites for activators MYC and GSC in the hypermethylated upstream region, pointing to its possible importance in TERT regulation. Overall, TERT expression and telomerase activity depend on the interplay of multiple regulatory mechanisms including TERT promoter methylation, mutation status, and recruitment of transcription factors. This work explores of the interplay between these regulatory mechanisms and offers insight into cellular control of active telomerase in human cancer.

Monitoring photodynamic oxygen consumption by endogenous oxygen contrast MRI.

Photodynamic oxygen consumption was measured by changes in spin-lattice relaxation time (T1) in aqueous solution in a clinical GE scanner at 1.5 T. Similar measurements were attempted in excised laryngeal and thyroid tissues that were infused with Rose Bengal. First, T1 was measured as a function of dissolved oxygen in argon and in oxygen pre-saturated water samples that were opened to the atmosphere in a series of steps allowing air to diffuse into or out of solution; for both argon and oxygen saturated water solutions, stepwise air re-equilibration resulted in a return to air-saturated water T1. Secondly, T1 was measured as a function of time under type II photooxidative conditions in aqueous solution. Under type II photooxidative conditions, a 492 ± 53 ms increase in T1 was measured following 300 s of visible light illumination of aqueous solutions containing the photosensitizer Rose Bengal (2.5 × 10-6 M) and the singlet oxygen trap methionine (0.0012 M). The 492 ± 53 ms increase in T1 corresponded to consumption of all the measurable dissolved oxygen (˜ 0.1 mg O2 in 15.0 mL of H2O) during photooxidation of methionine in air saturated water. This rapid oxygen consumption, indicated by an increase in T1, is due to irreversible trapping of photogenerated singlet oxygen by methionine. Thirdly, an increase in T1 was observed in Rose Bengal infused normal laryngeal tissue, and in normal and cancerous thyroid tissue samples following 20 min of exposure to visible light. An increase in T1 was not observed after 40 min of illumination which suggests that the increases in T1 observed after 20 min were not due to water uptake, but rather to photoconsumption of interstitial dissolved oxygen.

Analysis of the role of thyroidectomy and thymectomy in the surgical treatment of secondary hyperparathyroidism

PurposeParathyroidectomy can be subtotal or total with an autograft for the treatment of renal hyperparathyroidism. In both cases, it may be extended with bilateral thymectomy and total or partial thyroidectomy. Thymectomy may be recommended in combination with parathyroidectomy in order to prevent mediastinal recurrence. Also, the occurrence of thyroid disease observed in patients with hyperparathyroidism is poorly understood and the incidence of cancer is controversial. The aim of the present study was to report the experience of a single center in the surgical treatment of renal hyperparathyroidism and to analyse the role of thyroid and thymus surgery in association with parathyroidectomy. Materials and methodsWe analysed parathyroid surgery data, considering patient demographics, such as age and gender, and surgical procedure data, such as type of hyperparathyroidism, associated thyroid or thymus surgery, surgical duration and mediastinal recurrence. Histopathological results of thyroid and thymus samples were also analysed. ResultsMedical records of 109 patients who underwent parathyroidectomy for secondary hyperparathyroidism were reviewed. On average, thymectomy did not have impact on time of parathyroidectomy (p = 0.62) even when thyroidectomy was included (p = 0.91). Intrathymic parathyroids were detected in 7.5% of the thymuses removed and papillary carcinoma was detected in 20,8% of thyroid tissue samples. Two patients showed recurrence of supernumerary intrathymic parathyroids and a single case of mediastinitis was observed. ConclusionsParathyroidectomy with thymectomy and/or thyroidectomy has an important role in the treatment of renal hyperparathyroidism since thyroid cancer can frequently occur and require surgery. Thymectomy should be considered to avoid recurrence and a risky re-operation.

The significance of hypoxia as a molecular and cellular event in patients with toxic and non-toxic goitre: A statistical inference based on cross-sectional analytic of Iraqi patients

Background: Disorders of the thyroid gland, including toxic and non-toxic goitre, are more common in adult female patients. Hypoxia-inducible factors, including HIF-1 and HIF-2, represent transcriptional activators that function as regulators of oxygen homeostasis. Rapid progress is being made in clarifying the homeostatic functions of HIFs in several physiological systems. However, there are much to be learned in connection with the thyroid gland and its pathologies.Aims and Objectives: To assess the biochemical and clinical significance of hypoxia-inducible factors in patients with multinodular goitre.Materials and Methods: The study is observational and cross-sectional analytic conducted among a population of individuals with diffuse and nodular thyroid goitre including clinically toxic and non-toxic patients. It will attempt to answer the research question concerning the significance of HIFs, via applying techniques of immunohistochemistry to histological samples of resected thyroid tissue, and in pertinence with the demographic and clinical parameters of patients.Results: The total number of patients was forty-three,and most was in their fifth decades of life. The percentile contribution of males and females was 11.63% and 88.37% respectively. Individuals with toxic goitres accounted for 13.95%, and those had significantly higher levels of both HIF-1 and HIF-2 than non-toxic patients (p=0.019, p=0.072). Clinically-toxic patients also had notably more elevated levels of HIF-1 as they grew older when compared to non-toxic patients.Conclusion: There is an evident inadequacy of published literature on hypoxia-inducible factors in patients with goitre. There are no studies whatsoever in correspondence to the research questions explored in this study. Future attempts should explore experimental designs while using human as well as animal models and from an interdisciplinary perspective.Asian Journal of Medical Sciences Vol.9(5) 2018 44-49