Relationship between Trace Elements and Matrix Metalloproteinases 2 and 9 and their Tissue Inhibitors in Medullary Thyroid Carcinoma.

Medullary Thyroid Carcinoma (MTC) constitutes around 5% of all thyroid cancers. Trace elements assessment has emerged as a useful strategy in the diagnostics of MTC combined with Matrix Metalloproteinases (MMPs) and Tissue Inhibitors of Matrix Metalloproteinases (TIMPs) analysis. The aim of this study was to compare the presence and content of trace elements (i.e., Copper (Cu), Zinc (Zn), Iron (Fe), and Manganese (Mn)) in MTC with respect to control samples and their potential relationship with markers of MTC in tissues. The study included 26 patients who had undergone thyroidectomy, due to the diagnosis of MTC and 17 patients as control. We combined tumour pathology and staging, immunohistochemical analysis of calcitonin, MMPs, and TIMPs, with analytical biochemistry using Inductively Coupled Plasma - Mass Spectrometry (ICP-MS) to determine the levels of trace elements. No differences by MTC type for MMPs and their TIPMs, although strong TIMP-1 and TIMP-2 immunohistochemical expression of MTC were unveiled. Additionally, Zn, Fe, and Mn tended to be decreased, and Cu to be increased in samples presenting MTC with respect to controls. Moreover, Zn was the unique trace element which seemed to be correlated with MMPs and TIMPs. Trace elements such as Zn, Fe, and Mn are decreased in tissues affected by MTC. In addition, Zn may be the trace element which saves more relationship with the proportion and intensity of MMPs, being considered altogether useful biomarkers of MTC. We therefore suggest the analysis of novel and traditional markers of MTC as a novel approach in this pathology.

Matrix Metalloproteinases 2 and 9 and Their Tissue Inhibitors in the Diagnostics of Medullary Thyroid Carcinoma.

Medullary Thyroid Carcinoma (MTC) is a tumor of the neuroendocrine system. In recent years, the need to assess the MTC diagnostic-related parameters has emerged with the aim to elucidate the mechanisms involved in this pathology. The objective of this study was to evaluate the role of Matrix Metalloproteinases (MMPs) 2 and 9, their tissue inhibitors of matrix metalloproteinases (TIMPs), S100 protein, and amyloid in the diagnostic of MTC. Thirty-two samples with MTC (72% women) were included in this cross-sectional study and divided by groups: T category 1 (T1)≤20 mm and T category 2 (T2) 20 to 40 mm of tumor size. MMPs 2 and 9, TIMPs 2 and 1, S100 protein, and calcitonin in tissues were obtained by immunohistochemical techniques. The presence of amyloid in tissue sections was detected on Thioflavin T-stained slides under fluorescent microscope. Percentage of positive cells (P) observed for MMP-2 was higher in those samples presenting T2 MTC with respect to those with T1 MTC ( P <0.05). Moreover, P-MMP-2 showed a direct correlation with higher T category of MTC (Rho=0.439, P < 0.001), whereas P-MPP-9 was directly correlated with S100 protein and the intensity of calcitonin in tissues (Rho=0.419, P =0.017; Rho=0.422, P =0.016, respectively. Therefore, MMPs were directly correlated with some traditional biomarkers of MTC. In this regard, P-MMP-2 was more expressed in type 2 MTC. Combining the analysis of traditional and other useful biomarkers of MTC as MMPs 2 and 9 could be a useful strategy in the diagnostic of MTC.

Focal adhesion kinase splicing and protein activation in papillary thyroid carcinoma progression.

Papillary thyroid carcinoma (PTC), a common endocrine malignancy, presents a challenge from a prognostic standpoint. Molecular alterations underlying PTC progression include deregulation of focal adhesion kinase (FAK) at post-transcriptional and post-translational levels. Searching for candidate markers of PTC progression, we investigated the prognostic significance of FAK alterations on mRNA/protein level. The expression levels and subcellular localisation of auto-phosphorylated FAK (pY397-FAK) were determined by western blot (WB) and immunohistochemistry. The quantity of total FAK mRNA, alternatively spliced FAK-Del26 and FAK-Del33 variants were analysed by RT-qPCR and related to pY397-FAK expression and subcellular distribution. The results were correlated with clinicopathological parameters of the patients. The expression of pY397-FAK was significantly elevated in malignant samples. Active FAK showed predominant cytoplasmic distribution with co-occurrence along the membrane, while nuclear staining was found less frequently. Expression of pY397-FAK in separate cellular compartments correlated with adverse clinicopathological parameters, but the strongest association was found when their mean scores were calculated. Alternatively spliced FAK-Del33 and total FAK transcripts positively correlated to pY397-FAK protein levels as well as to characteristics of PTC advancement. Over-expression of FAK on mRNA (total and Del-33) and activated protein (pY397-FAK) levels is a feature of PTC advanced stages. Of the analysed alterations, the mean pY397-FAK IHC score showed the best predictive performance. Correlation between mRNA FAK-Del33 and pY397-FAK expression implies a regulatory role of alternative splicing in PTC patients.

Changes in the expression pattern of apoptotic molecules (galectin-3, Bcl-2, Bax, survivin) during progression of thyroid malignancy and their clinical significance.

BACKGROUND: Papillary carcinoma of the thyroid (PTC) is generally a slow growing tumor with favorable prognosis, while anaplastic thyroid carcinoma (ATC) is highly aggressive malignancy. Genetic defects in apoptotic pathways may contribute to differences in their biological behavior. METHODS: In this study, we analyzed immunohistochemically the expression of apoptosis-related molecules: galectin-3, Bcl-2, survivin (antiapoptotic), and Bax (pro-apoptotic), in archival tissue sections of PTC (n = 69) and ATC (n = 30) and correlated the results with clinicopathological parameters of these tumors. RESULTS: Galectin-3 and Bcl-2 showed a similar trend of down-regulation from high levels of both in PTC to low levels in ATC (p < 0.05). Bax was expressed at high levels in both type of thyroid carcinoma. Expression of survivin increased from PTC to ATC (p < 0.05), which may, at least in part, further facilitate the ability of malignant thyroid cell of ATC to escape programmed cell death despite high Bax expression. Only survivin, but not galectin-3, Bcl-2, or Bax, correlated significantly with lymph node metastasis presence and advanced stages of malignancy. CONCLUSIONS: In conclusion, this study documented down-regulation of galectin-3 and Bcl-2 (antiapoptotic molecules) and stepwise increase of survivin (inhibitor of apoptosis), during thyroid tumor progression from PTC to ATC. Correlation of high survivin expression with aggressive behavior implies its role in progression of thyroid tumor malignancy and suggests that survivin could be a useful tool in the prediction of aggressiveness of a subset of papillary carcinomas and a possible target for molecular therapy for ATC patients.

Copper as ancillary diagnostic tool in preoperative evaluation of possible papillary thyroid carcinoma in patients with benign thyroid disease.

Preoperative diagnosis of papillary thyroid carcinoma (PTC) comprises numerous diagnostic procedures which are mostly applicable in tertiary institutions. Normal thyroid function depends on the presence of many trace elements and copper (Cu) and zinc (Zn) are some of those. The study is based on retrospective review of 118 patients with preoperatively diagnosed benign thyroid disease (BTD) and 12 with PTC, who underwent thyroid surgery at the Center for Endocrine Surgery Clinical Center of Serbia, Belgrade, between 2010 and 2012. The objective was to evaluate concentrations of Cu and Zn in serum as possible prediction markers for PTC in patients who underwent surgery for preoperatively diagnosed BTD. Concentrations of Cu and Zn ions in serum were measured using atomic absorption spectrophotometer. Data were analyzed using methods of descriptive statistics, Anova and t-test (p < 0.05 was considered statistically significant). Definitive pathohistological findings revealed PTC in 23 (19.5%) and papillary microcarcinoma-mPTC in 13 (11.0%) of BTD patients. The concentrations of Cu ions in serum of PTC patients as well as in serum of patients with mPTC were significantly higher than in serum of BTD patients (p < 0.05). The concentrations of Zn ions and Cu/Zn ratio in serum of PTC and mPTC patients were not significantly higher than in serum of BTD patients. The concentration of Cu ions in serum of patients before thyroid surgery can be useful, easy available, and a low-cost tool in prediction of preoperatively undiagnosed PTC in patients with BTD.

Activity of superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase in different stages of colorectal carcinoma.

BACKGROUND: Reactive oxygen species are involved in the pathogenesis of colorectal carcinoma. Clarification of oxidative/antioxidant specificities of different stages of colorectal carcinoma is of special importance. AIM: To determine oxidative/antioxidant status in plasma of patients with different stages of colorectal carcinoma using malondialdehyde concentration, superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase activities and distribution of superoxide dismutase isoforms. METHODS: Lipid peroxidation and antioxidant enzymes activity were estimated using spectrophotometric methods. Reverse zymography was applied for characterization of superoxide dismutase isoforms. RESULTS: Lipid peroxidation is increased in all groups compared to the control, but without differences between different stages of colorectal carcinoma. Total superoxide dismutase activity is lower in all colorectal carcinoma groups than in control, and there is a significant increase in tumor stage IV when compared with tumor stage II. Manganese superoxide dismutase isoform is dominant in all groups and its relative activities are significantly higher than activities of a copper/zinc isoform. Total peroxidase potential reflected in catalase and glutathione peroxidase activity is increased when compared to the control, but without any significant differences between colorectal carcinoma groups. Glutathione reductase activity is lower in all colorectal carcinoma groups than in control, and a significant decrease in glutathione reductase activity was obtained between patients in tumor stage II and III compared to tumor stage IV. CONCLUSIONS: Colorectal carcinoma is characterized by increased oxidative stress and antioxidant disbalance. Progression of disease is followed by an increase in redox disbalance.

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.

Matrix metalloproteinase-2 (MMP-2) and -9 (MMP-9) in preoperative serum as independent prognostic markers in patients with colorectal cancer.

Colorectal cancer is one of the leading causes of cancer related death in developed countries. One of the reasons is the absence of tumor specific diagnostic and prognostic markers. The aim of this study was to examine the correlation of matrix metalloproteinase-2 (MMP-2) and -9 (MMP-9) expressions in serum and clinicopathological features of the colorectal adenocarcinoma. Another aim was to examine expression of MMP-9 in the tissue of the colorectal carcinoma in MMP-9 serum positive patients. In addition, we tried to establish the correlation between preoperative levels of serum markers (CEA and CA 19-9) and presence of MMP-2 or MMP-9. The study was performed on 32 patients with colorectal adenocarcinoma who underwent surgery and 11 patients in a control group who were operated for benign diseases. The samples were analyzed by SDS-PAGE to determine the molecular mass and SDS-PAGE zymography to determine levels of MMP-2 and MMP-9. Expression of MMP-9 was determined immunohistochemically in the tissue of the colorectal carcinoma of MMP-9 serum positive patients. MMP-2 and MMP-9 levels were increased in the serum of the patients with colorectal cancer compared to the control group. There was significant correlation in MMPs levels among the patients with tumor stage I and II and the patients with tumor stage III and IV. Obtained results did not demonstrate correlation between levels of CEA, CA 19-9 and presence of MMP-2 or MMP-9. MMP-9 expression was positive in 85% of MMP-9 serum positive patients with colorectal carcinoma. The overexpression of MMP-2 and MMP-9 strongly suggests its association with colorectal adenocarcinoma. Detection of MMP-2 and MMP-9 in serum might be useful for identification of patients with higher risk for colorectal cancer recurrence.

Galectin-3 expression in papillary thyroid carcinoma: relation to histomorphologic growth pattern, lymph node metastasis, extrathyroid invasion, and tumor size.

BACKGROUND: Galectin-3 has been recently recognized as a promising presurgical marker of thyroid malignancy. METHODS: Galectin-3 expression was examined immunohistochemically in 202 specimens of papillary thyroid carcinoma (PTC) in relation to histomorphologic subtypes and clinicopathologic data. RESULTS.: The sensitivity of galectin-3 immunostaining versus conventional histology was 98% (100 of 102) for classical PTC, 85.2% (46 of 54) for follicular variant, and 50% (23 of 46) for follicular/solid variant of PTC. All cases (n = 36) involving lymph node metastases and 42 of 45 cases with extrathyroid invasion expressed galectin-3. However, among the galectin-3-positive cases (n = 169), 133 were without lymph node metastases, and 127 were without extrathyroid invasion. Galectin-3 expression was not related to the size of intrathyroid PTC. CONCLUSIONS: Galectin-3 immunohistochemical expression itself is not an indicator of local metastatic spread or extrathyroid invasion of PTC, thus being irrelevant clinically from this aspect. Galectin-3 is an excellent marker for classical PTC but must be used with caution in diagnosing unconventional variants of PTC because of the possibility of false-negative results.