Three-dimensional reconstruction of vessel distribution in benign and malignant lesions of thyroid

In order to better understand the spatial distribution of thyroid vessels, a series of benign and malignant thyroid lesions were studied with three-dimensional (3D) histological stereomicroscopic reconstruction. Cases consisted of normal autoptic thyroids (n=6), colloid goitres (n=6), Basedows disease (n=2), follicular adenoma (FA) (n=4) one of which with Hurthle cells (HC), minimally invasive, well-differentiated follicular carcinoma (FTC) (n=1), well-differentiated FTC with HC (n=1), poorly differentiated FTC (n=13) with extensive angioinvasion, papillary carcinoma (PTC) (n=8) and medullary carcinoma (MTC) (n=1). From each selected nodule, parallel sections were obtained for 3D reconstruction and for histological and immunohistochemical studies. In normal thyroid, large vessels were located at the periphery of the gland with smaller branches present within the thyroid parenchyma that encircled follicles. The same pattern of vascularisation is maintained in lesions showing a follicular architecture as colloid goitre, Basedows disease, FA, well-differentiated FTC and the follicular variant of PTC. Neoplastic lesions, at variance with non-neoplastic lesions, contained rare anastomoses. Poorly differentiated FTC and MTC contained large intratumoural vessels surrounding avascular areas corresponding to solid neoplastic cellular sheets with necrosis. PTC were more vascularised and contained numerous vascular anastomoses. In conclusion, the present data indicate that the vascular distribution is related to the follicular, papillary or solid type of growth. Vascular anastomoses and intratumoural vessels surrounding solid avascular areas are signs of malignancy.     

Evaluation of coxsackievirus and adenovirus receptor expression in human benign and malignant thyroid lesions

Giaginis C, Zarros A, Alexandrou P, Klijanienko J, Delladetsima I, Theocharis S. Evaluation of coxsackievirus and adenovirus receptor expression in human benign and malignant thyroid lesions. APMIS 2010; 118: 210–21. Coxsackievirus and adenovirus receptor (CAR) expression on tumor cells is associated with sensitivity to adenoviral infection, being considered as a surrogate marker for monitoring and/or predicting adenovirus‐mediated gene therapy. The aim of this study was to evaluate the clinical significance of CAR expression in human benign and malignant thyroid lesions. CAR protein expression was assessed immunohistochemically on paraffin‐embedded thyroid tissues from 107 patients with benign and malignant lesions and was statistically analyzed in relation to histopathologic type; tumor size; lymph node metastasis; capsular, lymphatic and vessel invasion; as well as follicular cells’ proliferative capacity. CAR immunoreactivity was characterized as negative/weak in 53 (49.53%), moderate in 31 (28.97%) and strong in 23 (21.50%) of 107 thyroid cases. CAR immunoreactivity was significantly increased in malignant compared with that in benign thyroid lesions (p = 0.00002). Both malignant and benign thyroid lesions with enhanced follicular cells’ proliferative capacity showed significantly increased CAR immunoreactivity (p = 0.00027). In malignant thyroid lesions, enhanced CAR immunoreactivity was significantly associated with larger tumor size (p = 0.0067). The current data revealed that CAR immunoreactivity could be considered of diagnostic utility in thyroid neoplasia. Further research effort is warranted to delineate whether CAR could be considered clinically important for both diagnosis and future (gene) therapeutic applications in thyroid neoplasia.     

超声造影在甲状腺良恶性结节鉴别诊断中的应用

目的探讨实时灰阶超声造影在甲状腺结节良恶性鉴别诊断中的应用价值。方法 85例甲状腺结节患者(选取85个结节),其中男性29例,女性56例;年龄21～78岁,平均年龄42.5岁。采用声诺维(SonoVue)行超声造影检查,造影过程中观察结节的增强特点,比较增强模式在良恶性鉴别诊断中的价值。结果 85个结节中良性52个,恶性33个。结节超声造影表现为5种增强模式(弥漫性),即无增强、斑点样增强、均匀低增强、均匀高增强、均匀等增强。37个结节性甲状腺肿中,16个为均匀低增强,11个为均匀等增强,3个为均匀高增强,5个为环状增强,2个为斑点样增强中不均匀增强。9个腺瘤中6个为均匀高增强,2个为均匀低增强,1个为均匀等增强。26个甲状腺乳头状癌中15个不均匀增强,8个均匀低增强,1个均匀等增强,2个均匀高增强。6个滤泡性腺癌,4个为不均匀增强,均匀低增强和等增强各1个。不均匀增强特征对甲状腺恶性结节诊断的灵敏度、特异度和准确率分别为57.5%、94.0%和79.5%。环状增强对结节性甲状腺肿有较高的诊断特异度,而均匀高增强对甲状腺腺瘤有较高的诊断特异度。结论甲状腺良恶性结节的超声造影特征存在差异,不均匀增强对甲状腺恶性结节有较高的诊断价值。     

Expression of matrix metalloproteinases in benign and malignant follicular thyroid lesions

AIMS: To examine expression of matrix metalloproteinases (MMPs) and related proteins in follicular thyroid lesions (FTLs) and to determine their usefulness for differential diagnosis of FTLs, particularly between minimally invasive carcinoma and adenoma. METHODS AND RESULTS: Six widely invasive follicular carcinomas (WIFCs), 15 minimally invasive follicular carcinomas (MIFCs), 19 follicular adenomas (FAs) and 10 adenomatous goitres (AGs) were analysed immunohistochemically for MMP-1, MMP-2, MMP-7, MMP-9, membrane-type 1-MMP (MT1-MMP) and tissue inhibitor of matrix metalloproteinase-2 (TIMP-2). MMP-1 was positive in all FTLs. MMP-2 and MMP-7 were positive in more than 80% of WIFC and MIFC cases, whereas they were negative in all FA and AG cases except one MMP-2+ FA (P < 0.001). MMP-9 stained positive significantly more in MIFC than FA or AG cases (P < 0.05, respectively). The positivity of MT1-MMP and TIMP-2 was different among some of the FTLs, but with no significant difference between MIFC and FA cases. In-situ hybridization of MMP-2 and MMP-7 mRNA in selected cases demonstrated the expression of these enzymes in the tumour cells as well as in some stromal cells. CONCLUSIONS: Our results confirm MMP expression mainly in malignant FTLs and suggest that MMP-2 and MMP-7 may be useful markers to distinguish MIFC from FA.     

Expression of the sodium iodide symporter and thyroglobulin genes are reduced in papillary thyroid cancer.

Altered expression of the gene encoding the sodium iodine symporter (NIS) may be an important factor that leads to the reduced iodine accumulation characteristic of most benign and malignant thyroid nodules. Both up- and down-regulation of NIS gene expression have been reported in thyroid cancer using several different methods. The goal of the present study was to accurately identify alterations in NIS gene expression in benign and malignant thyroid nodules using an accurate real-time quantitative RT-PCR assay system. Total RNA was prepared from 18 benign thyroid nodules, 20 papillary thyroid cancers, and 23 normal thyroid samples from 38 subjects. Quantitative RT-PCR was used to measure NIS and thyroglobulin (TG) mRNA expression in normal thyroid tissue and in each nodular tissue sample. Papillary thyroid cancer samples had significantly lower NIS mRNA expression (72 +/- 41 picogram equivalents [pg Eq]), than did benign nodules (829 +/- 385 pg Eq), or normal tissues (1907 +/- 868 pg Eq, P = 0.04). Most important, in the paired samples, NIS gene expression was decreased in each papillary thyroid cancer compared with normal tissue (69% median decrease; range, 40-96%; P = .013). Eleven of the 12 benign nodules also demonstrated lower NIS gene expression than the normal tissue (49% decrease; range, 2-96%; P = .04). Analysis of the paired samples demonstrated that Tg mRNA expression was significantly lower in each of the thyroid cancer samples than in corresponding normal tissue (759 +/- 245 pg Eq vs. 1854 +/- 542 pg Eq, P = .03). We have demonstrated a significant decrement in NIS gene expression in all papillary thyroid cancers and in over 90% of benign nodules examined compared with adjacent normal thyroid tissue, using a highly accurate quantitative RT-PCR technique. Similarly, thyroid cancers demonstrated significantly lower TG mRNA expression than corresponding normal thyroid. Reduced NIS expression may be an important factor in the impairment of iodine-concentrating ability of neoplastic thyroid tissues.     

Total thyroidectomy as the single surgical option for benign and malignant thyroid disease: a surgical challenge

Introduction

Total thyroidectomy has been the treatment of choice for patients with malignant thyroid disease. However, the efficacy and safety of this procedure for patients with benign disease is still a matter of debate. The aim of this study is to show that total thyroidectomy can be safely performed for both malignant and benign disease.

Material and methods

A retrospective study on 216 patients was conducted. Once an indication for surgery was established, our single surgical treatment was total thyroidectomy. Age, sex, nature of thyroid disease, final pathology and postoperative complications were recorded.

Results

For both benign and malignant disease, total thyroidectomy resulted in no permanent laryngeal nerve injury and no permanent hypoparathyroidism. Temporary laryngeal nerve palsy occurred in 0.9% and 3% of patients with benign and malignant disease respectively (p = 0.245). Six percent of patients with benign and 10.0% of patients with malignant thyroid disease suffered temporary hypoparathyroidism (p = 0.280). Immediate reoperation for postoperative hemorrhage was performed in 1.7% of patients with benign disease and in 1.0% of patients with malignancy with an uneventful outcome (p = 0.650).

Conclusions

When performed by surgeons experienced in endocrine surgery, total thyroidectomy may be considered as the treatment of choice for both malignant and benign thyroid disease requiring surgical treatment. Total thyroidectomy virtually eliminates the requirement of completion thyroidectomy for incidentally diagnosed thyroid carcinoma and significantly reduces the rate of reoperation for recurrent disease, as it provides an immediate and permanent cure for all benign thyroid diseases, with a low incidence of postoperative complications.     

Immunohistochemical demonstration of epidermal growth factor and c-myc oncogene product in normal, benign and malignant thyroid tissues

The expression of epidermal growth factor (EGF) and c-myc oncogene product was studied immunohistochemically in 65 benign and malignant human thyroids. Normal thyroid tissues were usually negative with each antibody. Benign and malignant neoplastic thyroid tissues demonstrated a high percentage of stained cells. However, there was no significant difference in positivity between benign and malignant neoplastic tissues. In Graves' disease 25% of the cases were positive for EGF, and 100% for c-myc product. The effect of EGF or c-myc expression on prognosis was also examined in 42 patients with papillary thyroid carcinoma. When the cases were divided into two groups with regard to their EGF staining score (highly-expressed and poorly-expressed groups), the frequency of highly-expressed tumours was significantly higher in the recurrence-positive group compared with the recurrence-free group (86% vs 57%), but there was no significant difference between the groups in respect of c-myc expression.     

Association between microRNA polymorphisms and papillary thyroid cancer susceptibility

Objectives: Papillary thyroid cancer (PTC) is the most common subtype of thyroid cancer, which accounts for 80-90% of all thyroid cancer cases. Though the pathological mechanism hasn’t been fully understood, it is reported that both environmental and genetic factor may contribute to the PTC susceptibility. MicroRNAs (miRNAs) are small non-coding RNA molecules which function as the suppressors to participate in a variety of biological processes. Accumulating evidence suggests that polymorphisms of miRNAs were associated with the tumorigenesis of various cancers, including PTC. In this article, we focus on the association between four common microRNA polymorphisms (miR-146a, miR-608, miR-933, and miR-149) and PTC risk in a Han Chinese population. Methods: In this case-control study, we recruited 1,398 participants in total, including 369 PTC patients, 278 patients with thyroid benign nodules (BN) and 751 normal controls. The miRNAs polymorphisms were genotyped and analyzed by using MALDI-TOF mass spectrometry. The odd ratios and their 95% confidence interval (95% CI) were calculated to evaluate the association between miRNAs polymorphisms and PTC risk. Furthermore, a meta-analysis based on previous studies was conducted to comprehensively assess the diagnostic performance of miR-146a in the PTC diagnosis. Results: The miR-146a polymorphisms were shown to be significantly correlated with elevated risk of PTC under the heterozygous, homozygous, dominant and allelic models by comparing the genotype distribution between PTC cases and healthy controls, as well as between PTC cases and BN cases. However, the result of meta-analysis showed no significant association between miR-146a polymorphisms and PTC risk. Conclusions: Our study indicated that the miR-146a polymorphism was significantly associated with PTC risk. In contrast, meta-analysis revealed no evidence of association between miR-146a variants and PTC risk. Further studies are required to elucidate the role of miR-146a in the etiology of PTC.     

mRNA BRAF expression helps to identify papillary thyroid carcinomas in thyroid nodules independently of the presence of BRAFV600E mutation

Literature has consistently shown associations of BRAFV600E mutation with papillary thyroid cancer clinical features. However, the clinical utility of BRAF expression has not been clinically explored so far. We studied 67 thyroid nodules (32 benign nodules and 35 PTC cases). BRAF mRNA expression levels measured by a quantitative real-time PCR and a PCR-RFLP were used to identify BRAFV600E mutation. BRAF mRNA expression was significantly higher in malignant (198.2±373.9 AU) than in benign (4.1±6.9 AU) nodules (p<0.0001). BRAF expression identified malignancy with a sensitivity of 80.6%, specificity of 77.1%, positive predictive value of 75.8%, and negative predictive value of 81.8%. A cut-point of 4.712, identified by the ROC curve, was able to sort out malignant nodules with an accuracy of 78.8%. Although we did not find any correlation between the presence of BRAF V600E mutation and clinical or tumor features such as age (p=0.309), gender (p=0.5453), ethnicity (p=0.9820), tumor size (p=1.000), multifocality (p=0.2530) or mRNA levels (p=0.7510), the study power for BRAF expression and diagnosis (99%; FPRP=0.85) indicated that data is noteworthy despite the relative small number of patients investigated. We concluded that BRAF mRNA expression may help to identify PTC among thyroid nodules independently of the presence of BRAFV600E mutation.     

Sodium/iodide‐symporter: distribution in different mammals and role in entero‐thyroid circulation of iodide

The sodium (Na⁺)/iodide (I^–)‐symporter (NIS) is abundantly expressed and accumulates iodide in thyroid follicular cells. The NIS is also found in extrathyroidal tissues, particularly gastric mucosa. Controversies exist on the localization of extrathyroidal NIS. We have studied the presence of both NIS peptide and NIS messenger RNA (mRNA) in the digestive tract and thyroid from different mammals. The role of gastric NIS is enigmatic and we aimed to unravel its possible involvement in iodide transport. Methods: Distribution and expression of NIS were studied using immunocytochemistry and in situ hybridization. Iodide transport in the gastrointestinal tract was measured after oral or intravenous (i.v.) administration of ¹²⁵I to rats with or without ligation of the pylorus. Results: All thyroid follicular cells in rat and mouse expressed NIS, whereas a patchy staining was noted in man, pig and guinea‐pig. Gastric mucosa surface epithelium in all species and ductal cells of parotid gland in guinea‐pig, rat and mouse expressed NIS. In parietal cells and in endocrine cells of intestines and pancreas NIS immunoreactivity but no NIS mRNA was found. Studies of ¹²⁵I uptake showed marked iodide transport from the circulation into the gastric lumen. Conclusions: The localization of NIS varies slightly among mammals. To establish expression of NIS in a particular cell type the need to correlate the presence of both NIS protein by immunocytochemistry and NIS mRNA by in situ hybridization is emphasized. An entero‐thyroidal circulation of iodide mediated principally by gastric NIS, but possibly also by NIS in salivary glands is suggested.     

Human herpes simplex viruses in benign and malignant thyroid tumours

To test the hypothesis that herpes viruses may have a role in thyroid neoplasia, we analysed thyroid tissues from patients with benign (44) and malignant (65) lesions for HSV1 and HSV2 DNA. Confirmatory studies included direct sequencing, analysis of viral gene expression, and activation of viral‐inducible signalling pathways. Expression of viral entry receptor nectin‐1 was examined in human samples and in cancer cell lines. In vitro experiments were performed to explore the molecular mechanisms underlying thyroid cancer cell susceptibility to HSV. HSV DNA was detected in 43/109 (39.4%) examined samples. HSV capsid protein expression correlated with HSV DNA status. HSV‐positive tumours were characterized by activation of virus‐inducible signalling such as interferon‐beta expression and nuclear NFκB expression. Lymphocyte infiltration and oncocytic cellular features were common in HSV‐positive tumours. HSV1 was detected with the same frequency in benign and malignant thyroid tumours. HSV2 was significantly associated with papillary thyroid cancer and the presence of lymph node metastases. The expression of HSV entry receptor nectin‐1 was increased in thyroid tumours compared to normal thyroid tissue and further increased in papillary thyroid cancer. Nectin‐1 expression was detected in all examined thyroid cancer cell lines. Nectin‐1 expression in cancer cells correlated with their susceptibility to HSV. Inhibition of PI3K/AKT or MAPK/ERK signalling did not affect the level of nectin‐1 expression but decreased thyroid cancer cell susceptibility to HSV. These findings showed that HSV is frequently detected in thyroid cancer. During tumour progression, thyroid cells acquire increased susceptibility to HSV due to increased expression of viral entry mediator nectin‐1 and activation of mitogenic signalling in cancer cells. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

微小RNA-497抑制甲状腺乳头状癌细胞活力、侵袭和迁移

目的:研究微小RNA-497(miR-497)抑制甲状腺乳头状癌(PTC)细胞活力、侵袭和迁移的作用机制。方法:采用Target Scan 6. 0软件预测miR-497的靶基因,通过萤光素酶报告基因检测法、RT-qPCR和Western blot进行靶基因验证,再通过RT-qPCR检测PTC组织中miR-497及其靶基因的表达,并通过基因转染研究miR-497及其靶基因对PTC细胞活力、侵袭和迁移的影响。结果:Target Scan 6. 0软件预测、萤光素酶报告基因检测法、RTq PCR和Western blot均证明AKT3是miR-497的直接靶基因。此外,PTC组织中的AKT3表达水平较正常组织高(P 0. 05),且与miR-497表达呈负相关(r=-0. 573 7,P 0. 01)。下调AKT3可以抑制PTC细胞的活力、迁移和侵袭,与miR-497过表达在PTC细胞中具有相似的作用。结论:miR-497通过直接靶向调节AKT3抑制甲状腺乳头状癌细胞的活力、侵袭和迁移。     

The correlation of sodium iodide symporter and BRAFV600E mutation in classical variant papillary thyroid carcinoma

BRAF^V600E mutation was analyzed by real-time polymerase chain reaction in 96 consecutive cases with classical variant papillary thyroid cancer, and immunohistochemical staining of Na +/I − symporter (NIS) protein was evaluated. Localization (intracellular or membranous), density, and the intensity of cytoplasmic staining were characterized semiquantitatively. Extrathyroidal invasion, surgical margin positivity, and lymph node metastasis were compared with BRAF^V600E mutation and NIS expression. Eighty-eight patients who had at least 24-month follow-up were also included in survival analysis. BRAF^V600E mutation was determined in 78.1% (75/96) and functional NIS activity in 74% (71/96) of the cases. There were statistically significant differences in mean ages between BRAF^V600E mutation–positive (48.6) and BRAF^V600E mutation–negative cases (37.3; Levene test, P = .419; Student t test, P = .001). The surgical margin positivity (46.7%) and extrathyroidal extension percentage (54.7%) in the BRAF^V600E mutation–positive group were higher than the negative (28.6% and 33.3%, respectively) group, without statistical significance (P = .138 and P = .084, respectively). Functional NIS activity was higher in BRAF^V600E mutation–positive cases (78.1%) than mutation-negative ones (57.1%; P = .047). The possibility of moderate and intense cytoplasmic staining in BRAF^V600E mutation–positive cases (72%) was 6.3 times higher than the possibility of weak staining (28%) in the mutation-positive cases (95% confidence interval, 2.2-18.8; P = .001). Functional NIS expression is higher in patients with classical variant papillary thyroid cancer with BRAF^V600E mutation. However, the clinical features were not found to be associated with NIS expression. There may be different mechanisms determining the outcome of therapy.     

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.     

Prevalence of papillary microcarcinoma of the thyroid in Brazilian autopsy and surgical series

In order to search for parameters to differentiate patients at low and high risk for development of thyroid cancer, we studied thyroids from 166 consecutive autopsies and 261 thyroids that were surgically resected for thyroid diseases in general. We found 32 papillary microcarcinomas, corresponding to 7.8% of autopsies and 7.2% of surgical material, with a higher incidence between 30 and 49 yr of age. Both genders were similarly affected: 9.3% of the men and 8.8% of the women in autopsy series, and 6.2% of the men and 7.3% of the women in surgical series, suggesting that hormonal factors may favor the subsequent development of clinical lesions in women. Although associated nodular goiter has been observed in 54% of autopsies and 26% of surgical specimens, while Hashimoto's thyroiditis only in surgical material (15% of the cases), we were not able to correlate risk of malignancy with any concomitant lesion. The smallest papillary microcarcinomas presented most frequently as nonencapsulated nonsclerosing tumors without inflammatory infiltrate or fibrosis, suggesting that they may represent the early stages of development. Our data show a relatively high and similar frequency of papillary microcarcinomas in surgical and autopsy series, but do not demonstrate risk factors for clinical evolution.