Immunohistochemical detection of epithelialmesenchymal transition associated with stemness phenotype in anaplastic thyroid carcinoma

Anaplastic thyroid carcinoma (ATC) is a highly aggressive neoplasm resistant to radiation and chemotherapy. Epithelial-mesenchymal transition (EMT) generating cells with stem cell characteristics have been reported to be associated with chemoradioresistance in cultured cells. However, EMT and stem cell properties in ATC have not been fully investigated. In this study, we retrieved 2 thyroidectomy specimens of ATC with coexisting well differentiated thyroid carcinomas (DTCs) including one papillary carcinoma (PTC) and one follicular carcinoma (FTC). We used im-munohistochemistry to examine the expression of stem cell markers (nestin, CD133 and CD44) and a marker for EMT (E-cadherin). Intense expressions of nestin, CD133 and CD44, and no expression of E-cadherin were observed in both ATCs. In contrast, the PTC and FTC, and non-neoplastic thyroid tissue in both cases were negative for nestin and positive for E-cadherin. The expressions of CD133 and CD44 were variable in the PTC, FTC, and non-neoplastic thyroid tissue and were at a lower level of expression of these markers in the overall pattern. The results confirmed EMT, demonstrated the stem cell phenotype in ATC, and revealed the difference in expression of these markers between ATC and DTCs/non-neoplastic thyroid tissue. Nestin may be the most specific marker for stemness in ATC by immuno-histochemial staining. The results warrant future studies on a large series of cases in order to gain the understanding of the tumor biology and to provide molecular basis for restoring the sensitivities to clinical therapies.     

Stage-Specific Embryonic Antigen-1 (SSEA-1) Expression in Thyroid Tissues

Stage-specific embryonic antigen-1 (SSEA-1), also known as CD15, is a member of a cluster of differentiation antigens that have been identified in various normal tissues and in different types of cancers including papillary and medullary thyroid carcinoma. SSEA-1 may be expressed in normal stem cells and cancer stem-like cells. To evaluate the potential diagnostic and prognostic utility of SSEA-1 in thyroid tumors, we analyzed the expression of SSEA-1 in normal and neoplastic thyroid tissues by immunohistochemistry (IHC) using a tissue microarray with 158 different tissue cores. To evaluate the potential utility of SSEA-1 as a surface marker, we also assessed the expression of SSEA-1 in thyroid cell lines by flow cytometric analysis. SSEA-1 immunoreactivity was identified in malignant thyroid follicular epithelial cancers but not in the benign thyroid tissues. Anaplastic thyroid (ATC) (80 %) and conventional papillary thyroid carcinoma (PTC) (60.7 %) showed significantly higher percentage of cases that were SSEA-1 immunoreactive than follicular variant of papillary thyroid carcinoma (FVPTC) (20.6 %) and follicular carcinoma (FCA) (32.1 %). Flow cytometric analysis of cultured thyroid cell lines showed that a small subpopulation of ATC and PTC thyroid tumor cells had SSEA-1 immunoreactivity which may represent thyroid cancer stem-like cells. The ATC cells expressed more SSEA-1 immunoreactive cells than the PTC cell lines. Our findings suggest that expression of SSEA-1 immunoreactivity in thyroid neoplasms was associated with more aggressive thyroid carcinomas. SSEA-1 is a marker that detects malignant thyroid neoplasms in formalin-fixed paraffin-embedded thyroid tissue sections and may be a useful marker for thyroid cancer stem-like cells.     

The Role of Epithelial Mesenchymal Transition Markers in Thyroid Carcinoma Progression

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

Papillary Thyroid Carcinoma Emerging from Hashimoto Thyroiditis Demonstrates Increased PD-L1 Expression,Which Persists with Metastasis

There is evidence that programmed death-ligand 1 (PD-L1) is expressed by thyroid follicular epithelium in thyroiditis, but the role of PD-L1 in papillary thyroid carcinoma (PTC) is poorly understood. We aimed to determine whether (1) the presence of background chronic lymphocytic thyroiditis (CLT) or Hashimoto thyroiditis (HT) influenced the expression of PD-L1 in benign follicular epithelium or in PTC and (2) if PD-L1 expression in PTC persisted with lymph node metastasis. We performed immunohistochemistry (IHC) for PD-L1 on formalin-fixed paraffin-embedded tissues. We first studied five cases of unremarkable thyroid, five cases of CLT, and five cases of HT without carcinoma. We subsequently performed PD-L1 IHC on ten cases of PTC arising in normal thyroid, ten cases of PTC arising in CLT, and ten cases of PTC arising in HT. Whenever available, we evaluated corresponding synchronous lymph node metastases from all cases for PD-L1 expression. PD-L1 expression was increased (10–90%) in all five cases of HT, only minimal expressed (1–5%) in two of five cases of CLT, and not expressed in five cases of unremarkable thyroid. PTC arising in normal thyroid or CLT nearly uniformly lacked PD-L1 expression. In contrast, PTC arising in HT demonstrated significant PD-L1 expression, which persisted in corresponding lymph node metastases. Background non-neoplastic follicular epithelium in the HT cases also demonstrated PD-L1 expression. Thyroid follicular epithelium in HT demonstrates increased PD-L1 expression, and PTC arising in a background of HT shows increased PD-L1 expression, which is retained with metastasis.     

Evaluation of Lipocalin-2 and Twist expression in thyroid cancers and its relationship with epithelial mesenchymal transition

ObjectiveTo evaluate the expressions of lipocalin-2 (LCN-2) and Twist in thyroid cancers and examine its relationship with epithelial-to-mesenchymal transition (EMT) and clinicopathological factors.Materials and methodsThe expression of LCN-2 and Twist was immunohistochemically evaluated in a total of 249 cases, including 120 patients with papillary thyroid carcinomas (PTC), 34 with follicular thyroid carcinoma (FTC), 15 with medullary thyroid carcinomas (MTC), 20 with non-invasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP), 47 with follicular adenomas (FA), and 13 with nodular hyperplasia (NH). In addition, the relationship between the expression of EMT markers E-cadherin and vimentin was investigated in malignant cases.ResultsA significant increase was observed in the LCN-2 and Twist expression from NH and FA to NIFTP, MTC, FTC, and PTC (p = 0.001). A high degree of LCN-2 expression was observed in the aggressive variants of PTC (p = 0.002). Twist^high positivity was significantly higher in cases with the EMT-positive mesenchymal phenotype (p = 0.036).ConclusionsLCN-2 and Twist can be helpful diagnostic markers in the differentiation of benign and malignant thyroid nodules. Twist^high expression supports the EMT process in thyroid cancer. LCN-2 and Twist expression may also serve as valuable predictive biomarkers in patients with thyroid cancer. In future, the determination of a LCN-2^high expression in the aggressive variants of PTC may be integrated into targeted treatment strategies.     

LIMD2 Is Overexpressed in BRAF V600E-Positive Papillary Thyroid Carcinomas and Matched Lymph Node Metastases

We previously described that LIM domain containing 2 (LIMD2) overexpression was closely correlated with metastatic process in papillary thyroid carcinoma (PTC). We here evaluated the expression of LIMD2 in a series of non-metastatic and metastatic PTC and their matched lymph node metastases via immunohistochemistry. LIMD2 was expressed in 74 (81%) of primary PTC and 35 (95%) of lymph node metastases. Sub-analysis performed in 37 matched samples demonstrated that in four cases, LIMD2 is expressed in lymph node metastases, while it is not expressed in primary tumors. Moreover, in eight cases, the staining intensity of LIMD2 was stronger in the patient-matched lymph node metastases than in the primary tumors. Next, the expression of LIMD2 was correlated with clinical pathological parameters and BRAF V600E and RET/PTC mutational status. The expression of LIMD2 in primary tumors was correlated with the presence of BRAF V600E mutation (P =?0.0338). Western blot analysis in thyroid cell lines demonstrated that LIMD2 is expressed in two PTC cell lines, while it is not expressed in normal thyroid and follicular thyroid carcinoma cell lines. Importantly, its expression was higher in a PTC cell line that harbors BRAF V600E mutation than in a PTC cell line that harbors RET/PTC1. The available genomic profiling data generated by The Cancer Genome Atlas Research Network confirmed that LIMD2 expression is higher in BRAF-like PTC samples. Our data suggest that LIMD2 may play an important role in the metastatic process of PTC, predominantly in BRAF V600E-positive tumors.     

Overexpression of wild-type c-RET and zero prevalence of RET/PTC rearrangements are associated with papillary thyroid cancer (PTC) in Kuwait

Background

Papillary thyroid carcinoma (PTC) is common in Kuwait. The activation of the RET oncogene by DNA rearrangement (RET/PTC) is known to have an important role in PTC carcinogenesis. However, the real frequency of the RET/PTC expression in PTC is variable between different studies. This study seeks to determine the prevalence of RET/PTC and to analyze the RET oncogene expression associated with PTC in Kuwait.

Methods

RET expression and DNA rearrangements (RET/PTC 1, RET/PTC 2 and RET/PTC 3) were studied by RT–PCR in different thyroid diseases. Results were confirmed by the Southern blot and by immunohistochemistry. Quantitative real-time PCR was used to determine the level of RET mRNA expression in PTCs.

Results

Wild-type (nonrearranged) c-RET oncogene was overexpressed in 60% of PTC cases and absent in follicular thyroid carcinoma (FTC), anaplastic thyroid carcinoma (ATC), follicular adenomas (FA) or normal thyroid. No RET/PTC rearrangement was detected in any sample. The c-RET expression in Hashimoto's thyroiditis and multinodular goiter was limited to follicular cells with PTC-like nuclear changes.

Conclusions

The overexpression of wild-type c-RET is a characteristic molecular event of PTCs in Kuwait. The prevalence of RET/PTC is zero and among the lowest recorded in the world.     

Correlation between calcification and bone sialoprotein and osteopontin in papillary thyroid carcinoma

The correlation between calcification and papillary thyroid carcinoma has received increasing attention. We investigated the ability of bone sialoprotein (BSP) and osteopontin (OPN) protein levels to diagnose papillary thyroid carcinoma (PTC), and explored the correlation between BSP and OPN protein levels and calcification in PTC. Archival PTC specimens from patients with PTC with calcification and lateral cervical lymph node metastasis (LNM) were included in this retrospective immunohistochemical study. The protein levels of BSP and OPN were analysed immunohistochemically using routinely prepared tissue sections. PTC specimens from 66 patients with PTC were reviewed retrospectively (25 patients with histological calcification seen in paraffin sections, 41 patients without calcification; 35 patients with lateral cervical LNM, 31 patients without LNM). The percentage of samples that had cells that demonstrated positive protein staining differed significantly between PTC specimens, benign thyroid nodules, and adjacent normal follicular epithelium (BSP: 87.88%, 55.00%, and 42.50%, respectively; OPN: 83.33%, 70.00% and 50.00%, respectively). There was a significant difference in the immunohistochemical score (IHS) for BSP and OPN protein staining between PTC specimens with and without calcification (P < 0.05). The level of BSP protein staining was found to be significantly correlated with the level of OPN protein staining in PTC specimens. We conclude that the strong correlation between BSP and OPN and PTC suggests a role for BSP and OPN in calcification and tumor progression of PTC. BSP and OPN might be useful tumour markers for the diagnosis of PTC with limited value, because both of them had low specificity.     

TROP‐2 expression in papillary thyroid carcinoma

TROP‐2 is a type I transmembrane glycoprotein which is over‐expressed in various malignancies, and is related to epithelial cell adhesion molecule (EpCAM), also called TROP‐1, gp40, and KSA. In this study, we evaluated TROP‐2 expression in papillary thyroid carcinoma (PTC) and compared it to other thyroid neoplastic and non‐neoplastic lesions. Immunohistochemical (IHC) evaluation for TROP‐2 was performed on 137 thyroid fine needle aspiration (FNA) cell blocks (CB) which included classic PTC (64), follicular variant PTC (FVPTC) (10), anaplastic thyroid carcinoma (AC) (2), medullary carcinoma (MC) (8), follicular neoplasms (FN) (8), Hurthle cell neoplasms (HCN) (9), follicular lesion of uncertain significance (FLUS) (12), and benign thyroid nodule (BTN) (24). IHC for TROP‐2 expression was also performed on 331 BTN and malignant tumor tissue sections in tissue microarray (TMA). Membranous staining in >5% of tumor cells was considered positive. TROP‐2 stained 61 of 64 PTC CB, 7 of 10 FVPTC CB, and 9 of 12 FLUS CB. All other cases were negative for TROP‐2. TROP‐2 showed a sensitivity of 95.31% and specificity of 89% for classic PTC in FNA CB. In TMA samples, TROP‐2 stained 54 of 60 classic PTC cases and hence showed a high sensitivity and specificity. All BTN in CB and TMA were negative. We conclude that TROP‐2 is a highly sensitive and specific IHC marker for identifying classic PTC. TROP‐2 may play an important role in diagnosing classic PTC, especially in equivocal cases. This study also identifies a strong role for TROP‐2 in separating PTC from BTN. Diagn. Cytopathol. 2016;44:26–31. © 2015 Wiley Periodicals, Inc.     

Real-time quantitative RT-PCR identifies distinct c-RET, RET/PTC1 and RET/PTC3 expression patterns in papillary thyroid carcinoma

RET/PTC1 and RET/PTC3 are the markers for papillary thyroid carcinoma. Their reported prevalence varies broadly. Nonrearranged c-RET has also been detected in a variable proportion of papillary carcinomas. The published data suggest that a wide range in expression levels may contribute to the different frequency of c-RET and, particularly, of RET/PTC detection. However, quantitative expression analysis has never been systematically carried out. We have analyzed by real-time RT-PCR 25 papillary carcinoma and 12 normal thyroid samples for RET/PTC1, RET/PTC3 and for RET exons 10-11 and 12-13, which are adjacent to the rearrangement site. The variability in mRNA levels was marked and four carcinoma groups were identified: one lacking RET/PTC rearrangement with balanced RET exon levels similar to those of the normal samples (7/25 cases, 28%), the second (6/25 cases, 24%) with balanced RET expression and very low levels of RET/PTC1, the third with unbalanced RET exons 10-11 and 12-13 expression, high RET/PTC1 levels but no RET/PTC3 (7/25 cases, 28%), and the fourth with unbalanced RET expression, high RET/PTC1 levels and low levels of RET/PTC3 (5/25 cases, 20%). Papillary carcinomas with high RET/PTC1 expression showed an association trend for large tumor size (P=0.063). Our results indicate that the variability in c-RET and RET/PTC mRNA levels contributes to the apparent inconsistencies in their reported detection rates and should be taken into account not only for diagnostic purposes but also to better understand the role of c-RET activation in thyroid tumorigenesis.     

三叶因子3在甲状腺乳头状癌TPC-1细胞上皮间质转化中的作用及机制

目的 探讨三叶因子3（TFF3）基因沉默对人甲状腺乳头状癌TPC-1细胞迁移、侵袭、克隆形成能力以及对上皮间质转化（EMT）的影响及机制。 方法 免疫组织化学技术检测甲状腺乳头状癌组织芯片中Snail与TFF3的表达。划痕实验、侵袭实验和克隆形成实验分别检测TFF3基因对TPC-1细胞迁移、侵袭和克隆能力的影响;实时定量聚合酶链反应（Real-time PCR）、免疫印迹法和免疫细胞化学染色检测上皮间质转化标志物及丝裂原活化蛋白激酶（MAPK）通路相关蛋白的变化。 结果 31例甲状腺乳头状癌组织中,Snail蛋白于癌细胞胞质表达,31例癌旁组织阴性表达;Snail蛋白与TFF3表达呈正相关(r=0.8450,P<0.05)。TFF3基因沉默后,人TPC-1细胞的细胞迁移、侵袭和克隆形成能力均明显降低(P<0.01);TPC-1细胞上皮间质转化标志物及MAPK通路相关蛋白细胞外调节蛋白激酶（ERK）1/2和p-ERK1/2也显著降低(P<0.05)。 结论 沉默TFF3可能通过MAPK通路相关蛋白ERK1/2抑制上皮间质转化,降低TPC-1细胞迁移、侵袭和增殖能力。     

甲状腺滤泡癌和乳头状癌细胞黏附分子与转移抑制基因nm23-H1蛋白表达的关系

目的 探讨黏附分子CD44、上皮性钙黏附蛋白（E-cad )和转移抑制基因nm23-H1与甲状腺滤泡源性癌分化、浸润转移的关系。方法 采用免疫组织化学SP法和EnVision法检测42例滤泡癌和54例乳头状癌中CD44、E-cad和nm23-h1的表达。结果 CD44主要表达于癌细胞及浸润淋巴细胞膜,低分化滤泡癌和有转移乳头状癌CD44表达分别高于高分化滤泡癌和无转移乳头状癌,E-cad阳性物质和nm23-H1阳性率高于滤泡癌,转移癌的阳性率和表达强度低于原发灶。甲状腺滤泡原性癌CD44检测阳性率高于E-cad和nm23-H1。在滤泡癌中E-cad与nm23-H1的表达之间呈正相关关系,而在乳头状癌中呈正相关趋势。CD44与E-cad的表达、CD44与nm23-H1的表达之间在滤泡癌和乳头状癌均呈负相关趋势。结论 综合检测CD44、E-cad和nm23对甲状腺滤泡源性癌的诊断、预后评估具有一定参考价值。     

Atypical adenoma of the thyroid diagnosed as anaplastic cancer by cytopathology

Atypical adenoma of the thyroid is a rare form of tumor, and its accurate diagnosis prior to surgical resection is difficult as the histological and pathological morphologies are very similar to those of anaplastic thyroid carcinoma (ATC), and its anaplastic transformation remains to be elucidated. We reported a case of a 75‐year‐old female with a thyroid isthmus nodule diagnosed repeatedly by FNAC as anaplastic carcinoma. Both the first and second FNAC specimen slides showed a large number of scattered or aggregated atypical cells consisting of large, pleomorphic nuclei with irregular membranes, chromatin clumps and prominent nucleoli. The morphology of the surgical specimen was similar to that of an anaplastic carcinoma and although it showed signs of transition from a normal follicular epithelium, there was no invasive growth or mitosis. This lesion was diagnosed as an atypical adenoma, and a papillary carcinoma was also present in the right lobe of the thyroid. Here we evaluate the molecular features of atypical adenomas in comparison with 9 ATC samples, and discuss whether or not atypical adenomas represent a form of premalignant lesion. Ki‐67 expression was found to be very low in atypical adenomas whereas all ATC samples showed high levels of Ki‐67 expression. Epithelial–mesenchymal transition (EMT) marker expression suggested that atypical adenomas maintain their epithelial phenotype to a higher degree than do ATCs. Differential diagnosis between ATC and atypical adenoma is difficult by cytological and histological methods alone, and Ki‐67 and EMT marker expression may support the diagnosis.     

敲低长链非编码RNA AFAP1-AS1抑制TPC-1甲状腺乳头状癌细胞上皮间质转化及其侵袭和迁移

目的 检测甲状腺乳头状癌组织长链非编码RNA (lncRNA)肌动蛋白丝相关蛋白1反义RNA1(AFAP1-AS1)的表达,敲低TPC-1甲状腺乳头状癌细胞AFAP1-AS1,研究其对TPC-1细胞上皮间质转化(EMT)的影响及相关分子机制.方法 采用实时定量PCR检测60例甲状腺乳头状癌组织lncRNA AFAP1-...     

Identification of immunohistochemical biomarkers for papillary thyroid carcinoma using gene expression profiling

We report the successful validation of a combined gene expression profiling and tissue microarray approach to papillary thyroid carcinoma (PTC) biomarker identification. Our ultimate goal is the identification of protein biomarkers that can be effectively used in immunocytochemical assays applied to thyroid fine needle aspiration biopsy (FNAB) samples. To that end, we designed our approach to prioritize molecules that were minimally expressed in normal thyroid and highly expressed in PTC. We first generated gene expression profiles from 11 normal thyroid tissue samples and 9 samples of classic PTC. The results were segregated to rank most highly those molecules not expressed in normal thyroid and up-regulated at least 6-fold in PTC. From this list, we chose 2 molecules (P-cadherin and Bax) for immunohistochemical analysis for which commercial antibodies were available. These were compared with 2 other molecules that have been previously studied in thyroid cancer (cytokeratin-19 and galectin-3). For immunohistochemistry, a tissue microarray was generated that contained the following tissues: classic PTC (n = 20), follicular variant of PTC (n = 9), normal thyroid (n = 19), Hashimoto thyroiditis (n = 11), follicular adenoma (n = 15), and follicular carcinoma (n = 14). Immunohistochemical staining was scored and compared with the gene expression profiling. As anticipated, cytokeratin-19 and galectin-3 were highly expressed in PTC and less expressed in other tissues. Bax and P-cadherin were also expressed in PTC, but to a lower level than cytokeratin-19 and galectin-3; however, Bax and P-cadherin demonstrated virtually no staining of normal thyroid, unlike cytokeratin-19 and galectin-3. These results validate our approach for PTC biomarker discovery and identify several candidate biomarkers for further development.     

Immunohistochemical characteristics of diffuse sclerosing variant of papillary carcinoma: comparison with conventional papillary carcinoma

Koo JS, Shin E, Hong SW. Immunohistochemical characteristics of diffuse sclerosing variant of papillary carcinoma: comparison with conventional papillary carcinoma. APMIS 2010; 118: 744–52. Diffuse sclerosing variant of papillary carcinoma (DSVPC) is a rare variant of papillary thyroid carcinoma (PTC). It shows different clinicopathologic features to the conventional PTC, but the immunohistochemical characteristics of DSVPC are yet to be more clearly defined. The purpose of this study was to investigate the immunohistochemical features of DSVPC, which are different from those of PTC. Tissue microarray was constructed from the paraffin‐embedded tissue of 49 DSVPC and 50 conventional PTC samples. Immunohistochemical stains for p63, p53, galectin‐3, cytokeratin 19, β‐catenin, Bcl‐2, EMA, E‐cadherin, CD15, and CD56 were performed on each tissue microarray. Immunohistochemical stain for p63 was negative in all conventional PTCs, but 14 (28.6%) cases of DSVPC showed p63 expression (p = 0.000). p53 was expressed in 38 (76.0%) cases of conventional PTC and 21 (42.9%) cases of DSVPC (p = 0.001). Galectin‐3 was expressed in all 50 cases of conventional PTC, but eight (16.3%) cases of DSVPC did not express galectin‐3 (p = 0.003). EMA was expressed more in DSVPC (40.8%) than in conventional PTC (20.0%, p = 0.024). In univariate analyses, Bcl‐2 positivity (p = 0.016) and EMA negativity (p = 0.036) in DSVPC were associated with shorter time interval to tumor recurrence, but there was no significance for the two in multivariate analyses. DSVPC, a rare variant of PTC, has different immunohistochemical features from the conventional PTC, showing higher expression rate of p63 and lower expression rate of p53. It also shows galectin‐3 negativity and EMA positivity.