Non-neoplastic Thyroid Tissue Research Articles

The expression of c-MYC, Cyclin D1 and Ki-67/MIB-1 in benign and malignant thyroid tissues: is there any diagnostic value?

To investigate the immunohistochemical (IHC) expression and the diagnostic value of c-MYC, Cyclin D1, and Ki-67∕MIB-1 in follicular adenomas (FAs), follicular carcinomas (FCs), and anaplastic carcinomas (ACs) of the thyroid gland, as well as in their corresponding adjacent, non-neoplastic thyroid tissue (NNTT). We conducted a retrospective study of patients who were pathologically diagnosed with FA, FC, or AC after total thyroidectomy. Tissue microarrays with cores taken from neoplastic and adjacent NNTT were constructed. Immunohistochemistry for anti-c-MYC, anti-Cyclin D1, and anti-Ki-67∕MIB-1 antibodies was performed, and the positivity was evaluated. Twenty-eight specimens were included. Nuclear c-MYC positivity was observed in 4∕11 FCs, and 3∕4 ACs, whereas cytoplasmic c-MYC positivity was found in 16∕24 NNTTs. Globally, there were statistically significant differences between neoplasms and NNTTs, with higher nuclear c-MYC and Cyclin D1 expression observed in neoplasms (p=0.017 and p=0.001, respectively). In contrast, cytoplasmic positivity was seen solely in NNTTs (p=0.001). Cyclin D1 positivity was noted in 11∕13 FAs, 7∕11 FCs, 2∕4 ATCs, and only in one NNTT. A statistically significant correlation was found between MIB1 and c-MYC nuclear positivity (p=0.040). Our findings exhibit a clear difference in the IHC expression of c-MYC and Cyclin D1 between different types of thyroid tumors, as well as between the neoplastic and NNTT. Nuclear c-MYC positivity excludes the benign nature of a thyroid lesion, in contrast to cytoplasmic positivity, which demonstrates normal or hyperplastic nature.

Transcriptomic and immunophenotypic characterization of two cases of adamantinoma-like Ewing sarcoma of the thyroid gland.

Adamantinoma-like Ewing sarcoma (ALES) is a rare aggressive malignancy occasionally diagnosed in the thyroid gland. ALES shows basaloid cytomorphology, expresses keratins, p63, p40, frequently CD99, and harbours the t(11;22) EWSR1::FLI1 translocation. There is debate on whether ALES resembles more sarcoma or carcinoma. We performed RNA sequencing from two ALES cases and compared findings with skeletal Ewing's sarcomas and nonneoplastic thyroid tissue. ALES was investigated by in situ hybridization (ISH) for high-risk human papillomavirus (HPV) DNA and immunohistochemistry for the following antigens: keratin 7, keratin 20, keratin 5, keratins (AE1/AE3 and CAM5.2), CD45, CD20, CD5, CD99, chromogranin, synaptophysin, calcitonin, thyroglobulin, PAX8, TTF1, S100, p40, p63, p16, NUT, desmin, ER, FLI1, INI1, and myogenin. An uncommon EWSR1::FLI transcript withretained EWSR1 exon 8 was detected in both ALES cases. Regulators of EWSR1::FLI1 splicing (HNRNPH1, SUPT6H, SF3B1) necessary for production of a functional fusion oncoprotein, as well as 53 genes (including TNNT1, NKX2.2) activated downstream to the EWSR1::FLI1 cascade, were overexpressed. Eighty-six genes were uniquely overexpressed in ALES, most of which were related to squamous differentiation. Immunohistochemically, ALES strongly expressed keratins 5, AE1/AE3 and CAM5.2, p63, p40, p16, and focally CD99. INI1 was retained. The remaining immunostains and HPV DNA ISH were negative. Comparative transcriptomic profiling reveals overlapping features of ALES with skeletal Ewing's sarcoma and an epithelial carcinoma, as evidenced by immunohistochemical expression of keratin 5, p63, p40, CD99, the transcriptome profile, and detection of EWSR1::FLI1 fusion transcript by RNA sequencing.

DICER1 RNase IIIb domain mutations trigger widespread miRNA dysregulation and MAPK activation in pediatric thyroid cancer.

DICER1 is a highly conserved RNase III endoribonuclease essential for the biogenesis of single-stranded mature microRNAs (miRNAs) from stem-loop precursor miRNAs. Somatic mutations in the RNase IIIb domain of DICER1 impair its ability to generate mature 5p miRNAs and are believed to drive tumorigenesis in DICER1 syndrome-associated and sporadic thyroid tumors. However, the DICER1-driven specific changes in miRNAs and resulting changes in gene expression are poorly understood in thyroid tissue. In this study, we profiled the miRNA (n=2,083) and mRNA (n=2,559) transcriptomes of 20 non-neoplastic, 8 adenomatous and 60 pediatric thyroid cancers (13 follicular thyroid cancers [FTC] and 47 papillary thyroid cancers [PTC]) of which 8 had DICER1 RNase IIIb mutations. All DICER1-mutant differentiated thyroid cancers (DTC) were follicular patterned (six follicular variant PTC and two FTC), none had lymph node metastasis. We demonstrate that DICER1 pathogenic somatic mutations were associated with a global reduction of 5p-derived miRNAs, including those particularly abundant in the non-neoplastic thyroid tissue such as let-7 and mir-30 families, known for their tumor suppressor function. There was also an unexpected increase of 3p miRNAs, possibly associated with DICER1 mRNA expression increase in tumors harboring RNase IIIb mutations. These abnormally expressed 3p miRNAs, which are otherwise low or absent in DICER1-wt DTC and non-neoplastic thyroid tissues, make up exceptional markers for malignant thyroid tumors harboring DICER1 RNase IIIb mutations. The extensive disarray in the miRNA transcriptome results in gene expression changes, which were indicative of positive regulation of cell-cycle. Moreover, differentially expressed genes point to increased MAPK signaling output and loss of thyroid differentiation comparable to the RAS-like subgroup of PTC (as coined by The Cancer Genome Atlas), which is reflective of the more indolent clinical behavior of these tumors.

Overexpression of miR-100-5p inhibits papillary thyroid cancer progression via targeting FZD8.

Papillary thyroid cancer (PTC) is the most prevalent type of TC worldwide; however, its pathological process remains unclear at the molecular level. In the current study, we analyzed the microarray data of PTC tissues and non-neoplastic thyroid tissues, and confirmed miR-100-5p as a downregulated miRNA in PTC. Via bioinformatic approach, western blotting, and TOP/FOP-flash assay, miR-100-5p was observed to be involved in the inactivation of Wnt/β-catenin signaling in TPC-1 and KTC-1. Frizzled Class Receptor 8 (FZD8), the coupled receptor for canonical Wnt/β-catenin signaling, was verified to be targeted and inhibited by miR-100-5p in TPC-1 and KTC-1. In the function assay, miR-100-5p mimic repressed PTC cell proliferation and induced cell apoptosis of TPC-1 and KTC-1; meanwhile, transfection of full-length FZD8 attenuated the effect of miR-100-5p mimic. Moreover, in the collected samples, miR-100-5p was lowly expressed in PTC tissues compared with normal tissues, especially in those of advanced stage (Stage III/IV vs Stage I/II), while FZD8 was highly expressed in PTC tissues, which in PTC tissues was inversely correlated to miR-100-5p. Thus, we suggest that overexpression of miR-100-5p inhibits the development of PTC by targeting FZD8.

Epstein-Barr Virus-Associated Langerhans Cell Histiocytosis of the Thyroid Gland

Thyroid gland involvement of Langerhans cell histiocytosis (LCH) is extremely rare in both systemic and isolated disease. The role of viral infection in LCH development is not yet fully understood. Although several viruses are proposed as etiologic factors, such as Epstein-Barr virus (EBV) and human herpesvirus 6 (HHV-6), they seem to play a bystander role in LCH. A 29-year old female patient with a prior history of multisystemic LCH (pituitary gland and skull bone), presented with a thyroid nodule. The patient underwent a total thyroidectomy and the histological examination revealed nodular lesions composed of sheets and clusters of histiocytes in the inflammatory background. The histiocytes stained positive for S-100 and CD1a and were negative for HHV-8, cytomegalovirus, and VE1 (anti-BRAFV600E) on immunohistochemistry. The EBER in situ hybridization for EBV showed frequent positive-stained cells. The conventional PCR analysis for EBV was positive and qPCR analysis confirmed a significant DNA copy number difference (p = 0.02) between the tumor and adjacent non-neoplastic thyroid tissue. PCR analysis for HHV-6, HPV, HSV was negative in both tumor and benign samples. In conclusion, the presented case showed a rare thyroid involvement by LCH associated with EBV infection, which has not been reported before. Further studies are required to investigate a possible etiologic link between EBV infection and LCH.

Roles of Non-Coding RNAs on Anaplastic Thyroid Carcinomas.

Simple SummaryAnaplastic thyroid carcinoma (ATC) is a biological aggressive human carcinoma and causes most of the thyroid cancer related deaths. Non-protein producing RNAs, such as long non-coding RNAs (lncRNAs) and microRNAs (miRNAs), play crucial roles in formation and therapy resistance of ATC by regulating the production of key proteins. These RNAs alter the proteome of cells, which in turn controls cancer cells’ growth, division, invasion, migration, metastasis, and recurrence of ATC. Thus, the exogenous manipulation of these RNAs could interfere cells growth and development of ATC. Considering this, we discuss the roles of various miRNAs and lncRNAs in the pathogenesis of ATC and their potential therapeutic applications.Anaplastic thyroid cancer (ATC) remains as one of the most aggressive human carcinomas with poor survival rates in patients with the cancer despite therapeutic interventions. Novel targeted and personalized therapies could solve the puzzle of poor survival rates of patients with ATC. In this review, we discuss the role of non-coding RNAs in the regulation of gene expression in ATC as well as how the changes in their expression could potentially reshape the characteristics of ATCs. A broad range of miRNA, such as miR-205, miR-19a, miR-17-3p and miR-17-5p, miR-618, miR-20a, miR-155, etc., have abnormal expressions in ATC tissues and cells when compared to those of non-neoplastic thyroid tissues and cells. Moreover, lncRNAs, such as H19, Human leukocyte antigen (HLA) complex P5 (HCP5), Urothelial carcinoma-associated 1 (UCA1), Nuclear paraspeckle assembly transcript 1 (NEAT1), etc., participate in transcription and post-transcriptional regulation of gene expression in ATC cells. Dysregulations of these non-coding RNAs were associated with development and progression of ATC by modulating the functions of oncogenes during tumour progression. Thus, restoration of the abnormal expression of these miRNAs and lncRNAs may serve as promising ways to treat the patients with ATC. In addition, siRNA mediated inhibition of several oncogenes may act as a potential option against ATC. Thus, non-coding RNAs can be useful as prognostic biomarkers and potential therapeutic targets for the better management of patients with ATC.

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.

Interactions of Vascular Endothelial Growth Factor and p53 with miR-195 in Thyroid Carcinoma: Possible Therapeutic Targets in Aggressive Thyroid Cancers.

The clinical pathological features, as well as the cellular mechanisms of miR-195, have not been investigated in thyroid carcinoma. The aim of this study is to identify the interactions of vascular endothelial growth factor (VEGF), p53 and miR-195 in thyroid carcinoma. The clinical and pathological features of miR-195 were also investigated. The expression levels of miR-195 were identified in 123 primary thyroid carcinomas, 40 lymph nodes with metastatic papillary thyroid carcinomas and seven non-neoplastic thyroid tissues (controls) as well as two thyroid carcinoma cell lines, B-CPAP (from metastasizing human papillary thyroid carcinoma) and MB-1 (from anaplastic thyroid carcinoma), by the real-time polymerase chain reaction. Using Western blot and immunofluorescence, the effects of exogenous miR-195 on VEGF-A and p53 protein expression levels were examined. Then, cell cycle and apoptosis assays were performed to evaluate the roles of miR-195 in cell cycle progression and apoptosis. The expression of miR-195 was downregulated in majority of the papillary thyroid carcinoma tissue as well as in cells. Introduction of exogenous miR-195 resulted in downregulation of VEGF-A and upregulation of p53 protein expressions. Upregulation of miR-195 in thyroid carcinoma cells resulted in cell cycle arrest. Moreover, we demonstrated that miR-195 inhibits cell cycle progression by induction of apoptosis in the thyroid carcinoma cells. Our findings showed for the first time that miR-195 acts as a tumour suppressor and regulates cell cycle progression and apoptosis by targeting VEGF-A and p53 in thyroid carcinoma. The current study exhibited that miR-195 might represent a potential therapeutic target for patients with thyroid carcinomas having aggressive clinical behaviour.

Analysis of lncRNA and mRNA Transcriptomes Expression in Thyroid Cancer Tissues Among Patients With Exposure of Medical Occupational Radiation.

Background:Occupational exposure of radiation among medical radiation workers contributes to the subsequent increased risk of thyroid cancer. Long noncoding RNAs (lncRNAs) are emerging as important regulators of cancer biology. However, little is known about lncRNA expression in thyroid cancer tissues from patients who are exposed to medical occupational radiation. The purpose of this study is to reveal the transcriptomes difference between thyroid cancer tissues and adjacent nonneoplastic thyroid tissues.Methods:Microarray technology was used in this study. Quantitative reverse transcription polymerase chain reaction was adopted to verify 6 differentially expressed lncRNAs. Gene ontology and pathway analyses were performed using standard enrichment computational methods. Potential target genes of the differentially expressed lncRNAs were predicted with 2 independent algorithms.Results:A total of 23 lncRNA and messenger RNA transcripts were found differentially expressed in the thyroid cancer tissues (fold change ≥2.0, P < .05). This differential lncRNA expression may affect many pathways, including those involved in cysteine and methionine metabolism, Huntington disease, propanoate metabolism, and carcinogenesis.Conclusions:Our study provides a transcriptome-wide screening and analysis of the lncRNA expression profile in thyroid cancer tissues from patients with medical occupational radiation exposure and lays the foundation for further investigation of lncRNAs related to thyroid cancer development and carcinogenic risk of medical occupational radiation exposure.

PFKFB2 Promoter Hypomethylation as Recurrence Predictive Marker in Well-Differentiated Thyroid Carcinomas.

Despite the low mortality rates, well-differentiated thyroid carcinomas (WDTC) frequently relapse. BRAF and TERT mutations have been extensively related to prognosis in thyroid cancer. In this study, the methylation levels of selected CpGs (5-cytosine-phosphate-guanine-3) comprising a classifier, previously reported by our group, were assessed in combination with BRAF and TERT mutations. We evaluated 121 WDTC, three poorly-differentiated/anaplastic thyroid carcinomas (PDTC/ATC), 22 benign thyroid lesions (BTL), and 13 non-neoplastic thyroid (NT) tissues. BRAF (V600E) and TERT promoter (C228T and C250T) mutations were tested by pyrosequencing and Sanger sequencing, respectively. Three CpGs mapped in PFKFB2, ATP6V0C, and CXXC5 were evaluated by bisulfite pyrosequencing. ATP6V0C hypermethylation and PFKFB2 hypomethylation were detected in poor-prognosis (PDTC/ATC and relapsed WDTC) compared with good-prognosis (no relapsed WDTC) and non-malignant cases (NT/BTL). CXXC5 was hypomethylated in both poor and good-prognosis cases. Shorter disease-free survival was observed in WDTC patients presenting lower PFKFB2 methylation levels (p = 0.004). No association was observed on comparing BRAF (60.7%) and TERT (3.4%) mutations and prognosis. Lower PFKFB2 methylation levels was an independent factor of high relapse risk (Hazard Ratio = 3.2; CI95% = 1.1–9.5). PFKFB2 promoter methylation analysis has potential applicability to better stratify WDTC patients according to the recurrence risk, independently of BRAF and TERT mutations.

Review of the histological variants of papillary thyroid carcinoma and assessment of the prognostic factors

The incidence of thyroid cancer has increased in many parts of the world and in Sri Lanka it is the second most common malignancy in females. There are several risk factors of thyroid cancer out of which exposure to radiation, genetic mutations and Hashimoto’s thyroiditis are regarded as the important associations. Papillary carcinoma is the most common cancer of thyroid gland. There are several histological variants of papillary carcinoma with different biological behavior and outcome. Aim of the study was to review the histological variants and assess the prognostic factors in a cohort of papillary carcinoma. A retrospective study done over period of 4 years in 3 specialized surgical centers. There were 91 cases of papillary carcinoma in which the histological diagnosis was reviewed and confirmed. In the study population female to male ratio was 9:1 and the mean age of papillary carcinoma was 41 years. Gross assessment showed that 89% of tumours were solid and 11% were cystic. In 29% of cases the tumour was multifocal. The most common histological types were papillary carcinoma, conventional type (24%) and the follicular variant which accounted for 35% of the cases (p< 0.05). Non-neoplastic thyroid tissue was within normal limits in 47 % and in 36 % there was background chronic thyroiditis. Lymph nodal involvement was seen in 22% and 6% of cases had distant metastasis. It is important to identify the histological variants of papillary thyroid carcinoma as some of the types are associated with a poor outcome. Nodal involvement and distant metastasis showed a statistically significant association with the aggressive patterns (p< 0.05) of papillary carcinoma.

Functional Toll-like Receptor 4 Overexpression in Papillary Thyroid Cancer by MAPK/ERK-Induced ETS1 Transcriptional Activity.

Emerging evidence suggests that unregulated Toll-like receptor (TLR) signaling promotes tumor survival signals, thus favoring tumor progression. Here, the mechanism underlying TLR4 overexpression in papillary thyroid carcinomas (PTC) mainly harboring the BRAFV600E mutation was studied. TLR4 was overexpressed in PTC compared with nonneoplastic thyroid tissue. Moreover, paired clinical specimens of primary PTC and its lymph node metastasis showed a significant upregulation of TLR4 levels in the metastatic tissues. In agreement, conditional BRAFV600E expression in normal rat thyroid cells and mouse thyroid tissue upregulated TLR4 expression levels. Furthermore, functional TLR4 expression was demonstrated in PTC cells by increased NF-κB transcriptional activity in response to the exogenous TLR4-agonist lipopolysaccharide. Of note, The Cancer Genome Atlas data analysis revealed that BRAFV600E-positive tumors with high TLR4 expression were associated with shorter disease-free survival. Transcriptomic data analysis indicated a positive correlation between TLR4 expression levels and MAPK/ERK signaling activation. Consistently, chemical blockade of MAPK/ERK signaling abrogated BRAFV600E-induced TLR4 expression. A detailed study of the TLR4 promoter revealed a critical MAPK/ERK-sensitive Ets-binding site involved in BRAFV600E responsiveness. Subsequent investigation revealed that the Ets-binding factor ETS1 is critical for BRAFV600E-induced MAPK/ERK signaling-dependent TLR4 gene expression. Together, these data indicate that functional TLR4 overexpression in PTCs is a consequence of thyroid tumor-oncogenic driver dysregulation of MAPK/ERK/ETS1 signaling.Implications: Considering the participation of aberrant NF-κB signaling activation in the promotion of thyroid tumor growth and the association of high TLR4 expression with more aggressive tumors, this study suggests a prooncogenic potential of TLR4 downstream signaling in thyroid tumorigenesis. Mol Cancer Res; 16(5); 833-45. ©2018 AACR.

Abstract A13: Gene body methylation could regulate the expression of genes associated to papillary thyroid carcinomas

Abstract Background: Papillary thyroid carcinoma (PTC) is the histological subtype more frequently reported among the endocrine malignancies. Deregulation of DNA methylation has been extensively reported in cancer with hypermethylation usually found in promoter and hypomethylation in nonpromoter regions. The methylation profile of PTC revealed altered genome regions; however, its impact on the gene expression remains poorly understood. DNA methylation profile in PTC patients was performed, aiming to investigate the role of novel genic and intergenic regions on the expression of genes. Patients and Methods: The methylation profile of 41 patients diagnosed with PTC was evaluated using the Methylation 450 Human Infinium®BeadChip platform (Illumina). The data were normalized and analyzed using SVA, watermelon, and LIMMA packages. Delta beta of 0.15 and adjusted p-value &amp;lt;0.05 were used to identify differentially methylated probes between PTC and non-neoplastic thyroid tissue (NT). An integrative analysis was performed using the expression data obtained in 34 matched-cases. The results were submitted to a cross-validation with The Cancer Genome Atlas (TCGA) database. An in silico analysis was performed using genes/probes showing negative correlation and confirmed by TCGA using the Ingenuity Pathways Analysis. ERBB3, FGF1, FGFR2, GABRB2, HMGA2, and RDH5 were validated by pyrosequencing and RT-qPCR methods. Results: Methylation analysis revealed 4,995 differentially methylated probes. A global hypomethylation was found in PTC samples (88% hypomethylated probes) mostly in nonpromoter regions (especially in gene body), distant from CGI and enriched by enhancers. The integrative analysis between gene expression and DNA methylation revealed 185 and 38 genes with negative and positive correlation, respectively. Genes showing negative correlation highlighted FGF and retinoic acid signaling as crucial pathways disrupted by DNA methylation in PTC. The selected genes were confirmed as altered in both methylation and expression levels. Interestingly, four of them (ERBB3, FGF1, GABRB2, and HMGA2) were mapped in gene body, suggesting that methylation in this region regulates the gene expression. Conclusion: Loss of methylation was extensively observed in PTC, especially in nonpromoter, poor CGI, and enhancer-enriched regions. Gene body and promoter regions have the potential to influence the gene expression levels (both repressing and inducing). Genes potentially regulated by DNA methylation were identified from the integrative and cross-validation analyses. ERBB3, FGF1, FGFR2, GABRB2, HMGA2, and RDH5 biomarkers are regulated by methylation in PTC samples. Citation Format: Caroline Moraes Beltrami, Mateus Camargo Barros-Filho, Mariana Bisarro dos Reis, Fábio Albuquerque Marchi, Hellen Kuasne, Skirant Ambatipudi, Zdenko Herceg, Luiz Paulo Kowalski, Silvia Regina Rogatto. Gene body methylation could regulate the expression of genes associated to papillary thyroid carcinomas [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A13.

The roles of microRNA-34b-5p in angiogenesis of thyroid carcinoma.

This study aims to determine the expression of miR-34b-5p in thyroid carcinomas and to investigate the role of miR34b-5p in the modulation of proteins involved in angiogenesis of thyroid carcinoma cells. The expressions of miR-34b-5p levels in five cell lines and 65 tissue samples from thyroid carcinomas were examined by real-time polymerase chain reaction. An exogenous miR-34b-5p (mimic) transiently overexpress miR-34b-5p in theses thyroid carcinoma cells. The effects of miR-34b-5p overexpression on the proteins involved in angiogenesis and cell cycle regulations (VEGF-A, Bcl-2 and Notch1) were investigated by Western blot, immunofluorescence, enzyme-linked immunosorbent assay followed by cell cycle analysis and apoptosis assays. miR-34b-5p is markedly downregulated in all thyroid carcinoma cell lines and tissues samples when compared with non-neoplastic immortalised thyroid cell line and non-neoplastic thyroid tissues, respectively. The expression levels of miR-34b were significantly associated with T-stages of thyroid carcinomas (p = 0.042). Downregulation of VEGF-A, Bcl-2 and Notch1 proteins in thyroid carcinoma cells were noted in cells that transiently transfected with miR-34b-5p mimic. In addition, enzyme-linked immunosorbent assay confirmed the decreased expression of VEGF in thyroid carcinoma cells after transfection with miR-34b-5p mimic. Furthermore, miR-34b-5p mimic transfection induces significant accumulation of cells in G0-G1 of the cell cycle by blocking of their entry into the S transitional phase as well as increasing the total apoptosis. miR-34b-5p functions as a potent regulator of angiogenesis, apoptosis and cell proliferation via modulation of VEGF-A, Bcl-2 and Notch1 proteins. It could be a target for developing treatment strategies of thyroid carcinoma with aggressive clinical behaviour.

Prognostic Classifier Based on Genome-Wide DNA Methylation Profiling in Well-Differentiated Thyroid Tumors.

Context:Even though the majority of well-differentiated thyroid carcinoma (WDTC) is indolent, a number of cases display an aggressive behavior. Cumulative evidence suggests that the deregulation of DNA methylation has the potential to point out molecular markers associated with worse prognosis.Objective:To identify a prognostic epigenetic signature in thyroid cancer.Design:Genome-wide DNA methylation assays (450k platform, Illumina) were performed in a cohort of 50 nonneoplastic thyroid tissues (NTs), 17 benign thyroid lesions (BTLs), and 74 thyroid carcinomas (60 papillary, 8 follicular, 2 Hürthle cell, 1 poorly differentiated, and 3 anaplastic). A prognostic classifier for WDTC was developed via diagonal linear discriminant analysis. The results were compared with The Cancer Genome Atlas (TCGA) database.Results:A specific epigenetic profile was detected according to each histological subtype. BTLs and follicular carcinomas showed a greater number of methylated CpG in comparison with NTs, whereas hypomethylation was predominant in papillary and undifferentiated carcinomas. A prognostic classifier based on 21 DNA methylation probes was able to predict poor outcome in patients with WDTC (sensitivity 63%, specificity 92% for internal data; sensitivity 64%, specificity 88% for TCGA data). High-risk score based on the classifier was considered an independent factor of poor outcome (Cox regression, P < 0.001).Conclusions:The methylation profile of thyroid lesions exhibited a specific signature according to the histological subtype. A meaningful algorithm composed of 21 probes was capable of predicting the recurrence in WDTC.

Abstract 3358: Epigenetic signatures associated with patient outcome in thyroid carcinoma

Abstract BACKGROUND: Thyroid cancer (TC) is the most frequent endocrine neoplasia composed essentially by well-differentiated tumors (90%). These tumors generally are indolent and the patients show a favorable outcome. However, a set of TC patients presents aggressive outcome. Global deregulation of DNA methylation has been described as involved in thyroid cancer (TC) development. In this study, DNA methylation profile was performed aiming to identify a prognostic signature in TC. PATIENTS AND METHODS: The methylation profile of 50 non-neoplastic thyroid tissues (NT), 17 benign thyroid lesions and 74 TC (60 papillary, 8 follicular, 2 Hürthle cell, 1 poorly differentiated and 3 anaplastic thyroid carcinomas) were investigated using the Methylation 450 Human Infinium®BeadChip platform (Illumina). The data were normalized and analyzed using SVA, wateRmelon and LIMMA packages. The threshold delta beta of 0.2 and adjusted p-value &amp;lt;0.05 were used to identify differentially methylated probes among the histological subtypes. The delta beta of 0.1 and adjusted p-value &amp;lt;0.05 were used in the prognostic features analysis in WDTC (well differentiated thyroid cancer) cases. An epigenetic classifier according to WDTC was identified using diagonal linear discriminant analysis. The results were compared with The Cancer Genome Atlas (TCGA) database. RESULTS: Methylation analyses revealed a specific epigenetic profile according to the histological subtypes. A global hypermethylation was observed in benign lesions and follicular carcinomas, while papillary and undifferentiated carcinomas were widely hypomethylated compared with NT. An epigenetic signature comprising 21 probes differentially methylated (delta beta 0.1) was able to predict poor outcome in WDTC patients. This classifier reveled 63% of sensitivity and 92% of specificity, which was confirmed by TCGA database (64% of sensitivity and 88% of specificity). Using the established signature, we were able to confirm the involvement of poor prognosis markers with high-risk scores (multivariable analysis; P&amp;lt;0.001). CONCLUSION: Thyroid tumors showed different methylation profile according to the histological subtypes. Genes regulated by methylation in TC and associated with the tumor development were identified and confirmed by TCGA. In addition, a meaningful algorithm was designed and confirmed as capable to predict recurrence in WDTC. FINANCIAL SUPPORT: FAPESP (2015/20548-5) and National Institute of Science and Technology in Oncogenomics (FAPESP 2008/57887-9 and CNPq 573589/08-9), CNPq (302606/2011-4). Note: This abstract was not presented at the meeting. Citation Format: Mariana Bisarro dos Reis, Caroline Moraes Beltrami, Mateus Camargo Barros-Filho, Fabio Albuquerque Marchi, Hellen Kuasne, Skirant Ambatipudi, Zdenko Herceg, Luiz Paulo Kowalski, Silvia Regina Rogatto. Epigenetic signatures associated with patient outcome in thyroid carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3358. doi:10.1158/1538-7445.AM2017-3358

Vitamin D receptor and progesterone receptor protein and gene expression in papillary thyroid carcinomas: associations with histological features.

Vitamin D receptor (VDR) and progesterone receptor (PR) expression has been described in papillary thyroid carcinoma (PTC) but data regarding association with tumor histological characteristics and localization of the protein expression are scarce. Formalin-fixed, paraffin-embedded specimens from 45 patients with PTC (cases) were retrieved and tumor histological data were recorded. We analyzed gene and protein expression of VDR and PR and gene expression of vitamin D-inactivating 24-hyroxylase (CYP24A1) and the activating 1-alpha-hydroxylase (CYP27B1) enzymes in follicular cancer cells and the adjacent non-neoplastic thyroid tissue (NNTT). VDR mRNA and protein expression was higher in PTC compared with NNTT (p<0.05). The protein was globally localized in the cytoplasm and cell membranes of the neoplastic cells in all cases, with differences in intensity. Cytoplasmic positivity was stronger in the majority of cases. Membranous positivity was also evident in cases, whereas in NNTT was generally weak and in a low percentage of the cells. Expression of CYP 24A1, but not CYP27B1, was increased in approximately all PTC specimens and was associated with lymph node metastasis and extrathyroidal extension. PR mRNA was increased in 34% and protein expression was present in 57% of cases, and none of NNTT. PR, but not VDR, mRNA expression was significantly associated with the tumor size (r=0.645, p=0.007). We provide evidence for the expression pattern of VDR, PR and CYP24A1 in the progression of PTC. Rapid anti-tumor responses of vitamin D in PTC may be blocked due to inactivation of local vitamin D metabolism.

Expression of GLP-1 receptor and CD26 in human thyroid C-cells: The association of thyroid C-cell tumorigenesis with incretin-based medicine

Recent reports have demonstrated that long-term and high dosage treatments with incretin-based medicine, such as hormone glucagon-like peptide-1 (GLP-1) may induce thyroid C-cell pathological changes in rodents, rather than in humans. Doubts regarding the tumorigenic potential of GLP-1 analogues in human thyroid C-cells remain. The present study aimed to determine the expression levels of GLP-1 receptor (GLP-1R) and cluster of differentiation 26 (CD26) in the C-cells of thyroid tissues from non-neoplastic, medullary carcinoma and hyperplasia subjects, and to explore the potential clinical significance. The following cases were analyzed: Medullary thyroid carcinoma (n=62, including 59 paraffin-embedded samples and 3 fresh frozen samples), C-cell hyperplasia (n=20, paraffin-embedded samples) and non-neoplastic thyroid tissue samples (n=7, paraffin-embedded samples). GLP-1R and CD26 expression was detected using immunohistochemical staining and western blotting. There were significant differences in the expression levels of the two markers between medullary thyroid carcinoma and C-cell hyperplasia, in addition to between medullary thyroid carcinoma and non-neoplastic thyroid tissue following immunohistochemical staining. Similar significant differences in the expression of GLP-1R and CD26 were detected using western blot analysis in the medullary thyroid carcinoma compared with non-neoplastic thyroid tissue sectioned from the aforementioned fresh frozen samples. There was a significant negative correlation between GLP-1R and CD26 expression. In addition, the present data indicated that GLP-1R expression was associated with the age of the patients with medullary thyroid carcinoma. These results suggested that GLP-1R and CD26 may be closely associated with the development of thyroid C-cell hyperplasia and medullary thyroid carcinoma, and indicated the importance of being aware of the side effects of incretin medicine.

PAX8 Expression in Thyroid Tumors: Comparison with PAX2, TTF-1, and Thyroglobulin

Objective: Diagnostic markers for thyroid differentiation remain in development. Paired box (PAX8) is a member of a transcription factor family instrumental for fetal development and probably neoplastic transformation of the kidney, mullerian organs, and thyroid. Expression of PAX8 in thyroid tissue is evaluated and compared with traditional thyroid markers which are thyroglobulin and thyroid transcription factor-1 (TTF-1). Materials and Methods: Consecutive tissue sections of non-neoplastic thyroid tissue (n = 131), adenomatous nodule (n = 26), follicular neoplasms (n = 25), papillary carcinoma (n = 13), medullary carcinoma (n = 6), poorly differentiated carcinoma (n = 16), undifferentiated carcinoma (n = 6), and benign parathyroid tissue (n = 15) were submitted for PAX8, PAX2, TTF-1, and thyroglobulin immunostain. Staining extent (% of cells stained) and intensity (score 0-3) were evaluated. Results: PAX2 was not seen in any specimens. Strong (intensity score 3) (and diffuse 100% of cells) nuclear staining for PAX8 was noted in every case of non-neoplastic thyroid tissue and differentiated thyroid tumors. Staining for TTF-1 was similar to that of PAX8 in term of frequency, but the extent and intensity were less for some variants of papillary carcinoma or less differentiated follicular neoplasms. Thyroglobulin was noted in every case of non-neoplastic thyroid tissue and differentiated thyroid tumors, but the staining (which is cytoplasmic) was weak and focal in 66 cases of them, and this staining was often masked by strong staining of the adjacent colloid. For undifferentiated carcinoma, PAX8 was the only expressed marker, but in only 1/6 cases. For medullary carcinoma, PAX8 was not seen in any case, but TTF-1 and thyroglobulin were noted in 67% and 33% of cases, respectively. For parathyroid tissue, PAX8 was noted in 80% of cases, but the staining was weak and focal in each; TTF-1 and thyroglobulin were not seen. Conclusions: (1) PAX8 is a very sensitive marker for thyroid differentiation, regardless of diagnoses, (2) PAX8 is the only available marker, albeit of limited sensitivity, for undifferentiated thyroid carcinoma, (3) Both PAX8 and TTF-1 are sensitive markers for thyroid differentiation; with a diagnostic advantage for PAX8; and both are superior to thyroglobulin, (4) PAX8 may be the only marker needed for evaluating thyroid differentiation, and (5) In spite of an ontogenic similarity with PAX8, PAX2 is not expressed by thyroid tissue

Follicular Variant of Papillary Carcinoma Arising in Struma Ovarii.

Sir, A 47-year-old female presented with pain in the right iliac fossa and excess menstrual bleeding since five months. Pain was non-radiating, gradual in onset and of moderate intensity. She was obese and anaemic. Abdominal examination revealed a single, soft to cystic, slightly mobile and non-tender mass in the right iliac fossa measuring 12x10cm. Examination of other systems were nil remarkable. Her haemoglobin was 11 gm/dl, TC, DC, Platelet count and urine examination was with in normal limits. Viral markers (VDRL, H.I.V and HBsAg) were nonreactive. Ultrasound abdomen revealed bulky anteverted uterus with fibroid in the anterior wall near the fundus and large right ovarian cyst. She was posted for total abdominal hysterectomy and right salpingoopherectomy. During surgery, there were no signs of capsular invasion or metastasis. Postoperative recovery is good. Gross examination revealed a 4cm size fibroid in the body of the uterus and right ovarian cyst of size 15x15x12cm. Cut section of right ovarian cyst showed grey brown solid area measuring 7x7x5cm and a unilocular cystic area filled with light brown colour fluid. Histopathology revealed follicular variant of papillary thyroid carcinoma arising from pre-existing struma ovarii of right ovarian cyst and interstitial leiomyoma of uterus [Table/Fig-1a-c] [Table/Fig-1a-d]: (a) Struma ovarii (HE (b-c) Thyroid type of papillary carcinoma (H&E, X100), (H&E, X400) - Microfollicles lined by cuboidal follicular ... To rule out occult teratocarcinomas, alpha-fetoprotein (2.02ng/ml) and alpha-1-antitrypsin (1.56g/dl) levels were estimated postoperatively, they were with in normal limits. Thyroid profile was within normal limits. To rule out occult primary, ultrasound neck and FNAC of thyroid was done and it revealed adenomatous goitre [Table/Fig-1d] Serum thyroglobulin levels were estimated after 6 months of the surgery they were within normal limits (2.8ng/ml). Struma ovarii is a monodermal variant of ovarian teratoma, which was composed of more than 50% of thyroid tissue. Malignant transformation of struma ovarii occurs rarely. Papillary and follicular thyroid carcinomas are the most frequent types of malignancy to occur in struma ovarii, whereas anaplastic and medullary carcinomas occur rarely [1]. A new variant of follicular carcinoma i.e. highly differentiated follicular carcinoma of ovarian origin (HDFCO) has been described by Roth and Karseladze which has an innocuous appearance, resembling that of nonneoplastic thyroid tissue in both the ovary and sites of dissemination [2]. Strumal carcinoid is a distinctive form of ovarian teratoma characterized by a mixture of thyroid tissue and carcinoid. Thyroid-type carcinoma can occur in strumal carcinoid [3]. Follicular variant of papillary thyroid carcinoma arising in struma ovarii exhibit diffuse positivity for CK19 and HBME-1(hector battifora mesothelial cell-1) which is similar to follicular variant of papillary carcinoma in cervical thyroid. Molecular analysis has revealed activating mutations of BRAF, NRAS, KRAS, RET/PTC and NTRK1 in approximately 70% of all follicular cell–derived thyroid carcinomas [1]. Surgical treatment includes total abdominal hysterectomy, plus bilateral salpingo-oophorectomy with omentectomy in postmenopausal women, or in premenopausal women who have completed childbearing [1]. Young women, with unilateral lesion without evidence of capsular invasion or gross metastasis, unilateral oophorectomy or strumectomy (cystectomy), should be performed for preservation of fertility [1]. Adjuvant therapy includes radioactive iodine ablation, external radiotherapy and chemotherapy. Radioactive iodine is the first-line therapy for treatment of recurrent disease [1]. Prognostic factors such as tumour size, extensive adhesions, capsular invasion, peritoneal dissemination, lymph nodal metastasis and solid histologic architecture were associated with an increased incidence of recurrence or metastasis. Anaplastic carcinoma is the most aggressive tumour type, followed by follicular, papillary and HDFCO is the least aggressive [4].