Matrix metalloproteinase-9 and the Cu/Zn ratio as ancillary diagnostic tools in distinguishing between the classical and follicular variants of papillary thyroid carcinoma.

The most common histological variants of papillary thyroid carcinoma (PTC), classical (CPTC) and follicular (FPTC), have different diagnostic features, molecular biology, and prognosis. Matrix metalloproteinase-9 (MMP-9) endopeptidase which degrades the components of the extracellular matrix is essential in the invasive growth and metastasizing of malignant tumors. The serum copper (Cu)/zinc (Zn) ratios are sensitive diagnostic and prognostic indicators in oncology since Cu- and Zn-dependent enzymes play important roles in the genesis and the progression of tumors. The aim of this study was to examine the expressions of MMP-9 in tissues of CPTC and FPTC, as well as to determine the Cu/Zn ratios in the same samples. MMP-9 was determined immunohistochemically, and the concentrations of copper and zinc in thyroid tissue were determined by means of flame atomic absorption spectrometry. The results obtained revealed significantly higher expressions of MMP-9 in CPTC in comparison with FPTC, as well as higher Cu/Zn ratios in CPTC than in FPTC. Thus, determining MMP-9 activities and the Cu/Zn ratios could improve the accuracy of the standard histopathological diagnosis of these two types of PTC.

Molecular genetics and diagnosis of thyroid cancer.

Thyroid cancer is a common type of endocrine malignancy, and its incidence has been steadily increasing in many regions of the world. Initiation and progression of thyroid cancer involves multiple genetic and epigenetic alterations, of which mutations leading to the activation of the MAPK and PI3K-AKT signaling pathways are crucial. Common mutations found in thyroid cancer are point mutation of the BRAF and RAS genes as well as RET/PTC and PAX8/PPARγ chromosomal rearrangements. The mutational mechanisms seem to be linked to specific etiologic factors. Chromosomal rearrangements have a strong association with exposure to ionizing radiation and possibly with DNA fragility, whereas point mutations probably arise as a result of chemical mutagenesis. A potential role of dietary iodine excess in the generation of BRAF point mutations has also been proposed. Somatic mutations and other molecular alterations have been recognized as helpful diagnostic and prognostic markers for thyroid cancer and are beginning to be introduced into clinical practice, to offer a valuable tool for the management of patients with thyroid nodules.

Expression, localization, and phosphorylation of Akt1 in benign and malignant thyroid lesions.

The serine/threonine protein kinase Akt is a key molecule in the phosphatidyl inositol 3-kinase pathway that is often overactivated in human cancers. Three Akt isoforms (Akt1, Akt2, Akt3) have been identified in human cells and they show different distribution and have non-redundant functions. The aim of this study was to determine whether the expression, phosphorylation, and localization of Akt1 isoform in human thyroid malignant lesions are different from those in benign lesions. Nuclear and cytoplasmic fractions were isolated from tissue samples and Western blot method was used to detect Akt1 presence in both cellular fractions. Akt1 expression was also assessed by ELISA method. To estimate Akt1 phosphorylation, kinase was immunoprecipitated from cell lysates and tested with anti-phospho-Akt antibodies. The Akt1 expression in majority of thyroid cancer samples was significantly higher than in benign lesions (p < 0.05). Akt1 both in differentiated cancers (follicular and papillary) and benign lesions was localized mainly in cytoplasmic fraction. In two of three anaplastic cancer samples Akt1 was predominantly localized in nucleus. The ratio of phosphorylated Akt1 to total Akt1 was lower in cancers than in non-neoplastic lesions and adenomas. Thus, although Akt1 seems to be overexpressed in thyroid neoplasms, its high phosphorylation is not characteristic for thyroid cancers.

Thyroglobulin may affect telomerase activity in thyroid follicular cells.

Telomerase (TA) activity is known to be present in malignant tumor cells, but not in most somatic differentiated cells. TA shows relatively high activity in thyroid cancer cells, but reports vary. This fact prompted us to elucidate whether cell component inhibitors of TA in the thyroid follicles can modulate its activity. The activity of TA extracted from Hela cells was inhibited by mixing with the supernatant fraction of human thyroid tissue extract. To examine the effect of iodine, thyroid hormones (l-T3 and l-T4) and human thyroglobulin (hTg) contained in the thyroid follicles, l-T3, l-T4 and hTg were added to the TRAP assay system in vitro, using TA from Hela cells. Iodine, l-T3 and l-T4 did not affect TA activity, but hTg inhibited the TA activity in a dose-dependent manner (IC(50) of hTg: ca 0.45 microM: inhibiting concentration of hTg was from 0.15 microM to 3.0 microM). The hTg inhibition was not evident in the RT-PCR system, suggesting no effect of hTg on Taq DNA polymerase activity. The hTg inhibition of TA activity was attenuated by dNTP but not significantly by TS primer. These data suggest that hTg contained in thyroid follicular cells of various thyroid diseases may affect the TA activity measured in biopsied thyroid specimens, and that the reduction of the TA activity by hTg may induce slow progression and growth, and low grade malignancy of thyroid cancer, particularly differentiated carcinoma.

Molecular pathology of thyroid cancer: diagnostic and clinical implications.

There is now a reasonably good understanding of the key oncogenic events involved in the initiation and progression of thyroid cancer. Many of these are characteristic of certain tumor types, and their presence conveys diagnostic and prognostic information. It is not yet clear how this information will be applied to clinical practice. Based on preclinical evidence, mutations of genes encoding certain kinases may also predict response to specific tyrosine kinase inhibitors, although this has not yet been explored systematically in clinical trials.

Functional characterization of the novel T599I-VKSRdel BRAF mutation in a follicular variant papillary thyroid carcinoma.

CONTEXT: Mutations in BRAF are rare in the follicular variant of papillary thyroid carcinoma (FV-PTC). OBJECTIVE: We identified and functionally characterized a novel T599I-VKSR(600-603)del BRAF mutation in a FV-PTC patient. We analyzed in vitro the effects of this novel mutation in comparison with other thyroid cancer-associated mutations. DESIGN: Expression vectors for the BRAF mutants were generated and their in vitro kinase activity, signaling along the MAPK pathway, and capability of stimulating transcription from an AP1-responsive reporter evaluated. RESULTS: BRAF kinase and signaling were increased to a similar extent by the T599I-VKSR (600-603)del, V600E, and K601E mutations. Instead, the G474R, a mutation previously found in a FV-PTC, knocked down the BRAF kinase and its intracellular signaling. Some cancer-associated low-activity BRAF mutants stimulate the MAPK cascade via CRAF; however, the G474R protein lacked also this property. CONCLUSION: The T599I-VKSR(600-603)del is a novel gain-of-function mutation that targets BRAF in FV-PTC. Moreover, G474R is the first example of a mutation knocking down enzymatic BRAF activity in a FV-PTC. These findings underscore the importance of functional studies to characterize the role of BRAF mutations associated with thyroid cancer.

Cyclooxygenase-2 in normal, hyperplastic and neoplastic follicular cells of the human thyroid gland.

This study was undertaken to investigate cyclooxygenase-2 (COX-2) expression in follicular cells of the human thyroid. COX-2 expression was studied immunohistochemically in a total of 174 samples. COX-2 immunoreactivity was confined to the cell cytoplasm with the nuclei remaining unlabelled. COX-2 expression was observed in five cases (17.2%) of normal follicular cells and in one case (16.6%) of solid cell nests. Follicular carcinoma expressed COX-2 more frequently than follicular adenoma (93.4% vs 21.1%) (p

Enhanced expression of nicotinamide N-methyltransferase in human papillary thyroid carcinoma cells.

To gain an understanding of the molecular pathogenesis of thyroid cancer, we used DNA microarray to study the expression profiles of 10 different human thyroid carcinoma cell lines. These included papillary lines BHP 2-7, BHP 7-13, BHP 10-3, BHP 18-21, NPA 87, and TPC1; anaplastic lines ARO 81-1 and DRO 90-1; follicular line WRO 82-1; and medullary line HRO 85-1. Among the genes with increased expression in the cancer cell lines, a gene coding for nicotinamide N-methyltransferase (NNMT) was identified for being highly expressed only in the papillary cell lines. NNMT catalyzes N-methylation of nicotinamide and other structurally related compounds and is highly expressed in the human liver. The results were further confirmed by semiquantitative RT-PCR and Northern blot analysis. NNMT catalytic activities were determined in all of the cells described above and in additional cell lines. Significantly higher NNMT enzyme activities were detected in eight of 10 of the papillary lines and three of six of the follicular cell lines tested. Normal thyroid tissue, thyroid primary cultures, anaplastic cancer cells, and medullary cancer cells showed no or low enzyme activity. Immunohistochemical staining for NNMT of human thyroid specimens showed strong and abundant cytoplasmic reactions in the sections of papillary carcinomas, and weak or scanty reaction in the normal thyroid tissues. These results indicate that NNMT is a potential biomarker for papillary thyroid carcinoma.

The value of thyroid peroxidase immunohistochemistry for preoperative fine-needle aspiration diagnosis of the follicular variant of papillary thyroid cancer.

BACKGROUND: With the use of May-Grunwald-Giemsa staining, cytologic features of the follicular variant of papillary thyroid cancers (FVPTCs) on thyroid fine-needle aspiration (FNA) often resemble those of hyperplastic and adenomatous nodules. Detection of reduced staining after thyroid peroxidase (TPO) immunohistochemistry with monoclonal antibody MoAb47 has been shown to be a helpful diagnostic marker. The purpose of this study was to assess the value of TPO immunohistochemistry for the diagnosis of FVPTC. METHODS: Of 3505 patients with adequate FNA samples, 1576 patients underwent surgical procedures. Histologic examination of the surgical specimen demonstrated papillary thyroid cancer in 227 cases, including 42 FVPTCs (18%). The diagnostic accuracy of standard thyroid FNA and TPO immunohistochemistry, which we use routinely, was compared in these 42 FVPTCs. RESULTS: Standard FNA allowed accurate diagnosis of malignancy in 32 of the 42 FVPTCs and in 170 of the 185 typical papillary thyroid cancers. TPO-immunohistochemistry staining was accurate in all 42 FVPTCs and in 182 of 185 typical PTCs. CONCLUSIONS: FVPTC is a frequent source of false-negative results on standard thyroid FNA. This study shows that TPO immunostaining accurately demonstrates malignancy in these tumors.

Staining for dipeptidyl aminopeptidase IV activity in nodular thyroid diseases.

OBJECTIVE: To determine the effectiveness of staining for dipeptidyl aminopeptidase IV (DAP IV) activity in thyroid nodular diseases. STUDY DESIGN: Imprint smears were made in 76 cases of papillary carcinoma, 10 of follicular carcinoma, 32 of follicular adenoma and 48 of adenomatous goiter after surgery, and staining for DAP IV activity was performed in all cases. RESULTS: All papillary carcinomas stained positively, and most adenomatous goiter cases were negative. Follicular carcinoma and follicular adenoma cases exhibited various degrees of positivity, though the former tended to be stained more than the latter. Of the total 166 cases of thyroid nodules, 53 showed negative staining; of those 53, all except 1 were benign. Follicular carcinoma cases that had vascular invasion tended to show a high DAP IV staining pattern, but no statistically significant difference between it and follicular carcinoma that did not show vascular invasion was found. CONCLUSION: DAP IV activity staining might reveal malignant potential and could have some value in the preoperative diagnosis between thyroid nodular diseases. The DAP IV method is a reliable indicator of benign disease if negative results are obtained.

Expression of MMP-1 in the capsule of thyroid cancer--relationship with invasiveness.

To investigate the invasive activity of thyroid cancer, an in situ hybridization study was carried out in 19 thyroid tumors, including nine papillary carcinomas, five follicular carcinomas and five follicular adenomas, by using a 35S-labeled MMP-1 (matrix metalloproteinase-1) cDNA probe. The MMP-1 gene was expressed not in the cancer cells but in the fibrous capsules of papillary carcinoma. Thyroid cancer is generally circumscribed by a fibrous capsule. We found that types I and III collagen constitute the fibrous capsule, and that the MMP-1 gene was expressed in the outer border of these sites. These findings suggest that MMP-1 plays an important role in the invasion of thyroid cancer.