Comprehensive Investigation of Angiogenesis, PASS Score and Immunohistochemical Factors in Risk Assessment of Malignancy for Paraganglioma and Pheochromocytoma.

A challenging task in routine practice is finding the distinction between benign and malignant paragangliomas and pheochromocytomas. The aim of this study is to conduct a comparative analysis of angiogenesis by assessing intratumoral microvascular density (MVD) with immunohistochemical (IHC) markers (CD31, CD34, CD105, ERG), and S100 immunoreactivity, Ki67 proliferative index, succinate dehydrogenase B (SDHB) expressiveness, tumor size with one the most utilized score Pheochromocytoma of Adrenal Gland Scales Score (PASS), using tissue microarray (TMA) with 115 tumor samples, 61 benign (PASS < 4) and 54 potentially malignant (PASS ≥ 4). We found no notable difference between intratumoral MVD and potentially malignant behavior. The group of potentially malignant tumors is significantly larger in size, has lower intratumoral MVD, and a decreased number of S100 labeled sustentacular cells. Both groups have low proliferative activity (mean Ki67 is 1.02 and 1.22, respectively). Most tumors maintain SDHB expression, only 6 cases (5.2%) showed a loss of expression (4 of them in PASS < 4 group and 2 in PASS ≥ 4). PASS score is easily available for assessment and complemented with markers of biological behavior to complete the risk stratification algorithm. Size is directly related to PASS score and malignancy. Intratumoral MVD is extensively developed but it is not crucial in evaluating the malignant potential.

BRAFV600E, BANCR, miR-203a-3p and miR-204-3p in Risk Stratification of PTC Patients.

In order to enhance the risk stratification of papillary thyroid carcinoma (PTC) patients, we assessed the presence of the most common mutation in PTC (BRAFV600E) with the expression profiles of long non-coding RNA activated by BRAFV600E (BANCR) and microRNAs, which share complementarity with BANCR (miR-203a-3p and miR-204-3p), and thereafter correlated it with several clinicopathological features of PTC. BRAFV600E was detected by mutant allele-specific PCR amplification. BANCR and miRs levels were determined by quantitative RT-PCR. Bioinformatic analysis was applied to determine the miRs' targets. The expression profile of miR-203a-3p/204-3p in PTC was not affected by BRAFV600E. In the BRAFV600E-positive PTC, high expression of miR-203a-3p correlated with extrathyroidal invasion (Ei), but the patients with both high miR-203a-3p and upregulated BANCR were not at risk of Ei. In the BRAFV600E-negative PTC, low expression of miR-204-3p correlated with Ei, intraglandular dissemination and pT status (p < 0.05), and the mutual presence of low miR-204-3p and upregulated BANCR increased the occurrence of Ei. Bioinformatic analysis predicted complementary binding between miR-203a-3p/204-3p and BANCR. The co-occurrence of tested factors might influence the spreading of PTC. These findings partially describe the complicated network of interactions that may occur during the development of PTC aggressiveness, potentially providing a new approach for high-risk PTC patient selection.

Expression of pY397-FAK and Its miR Regulators Drive Dedifferentiation in the Thyroid Neoplasia Spectrum.

Thyroid carcinomas are growing malignancies worldwide. They encompass several diagnostic categories with varying degrees of dedifferentiation. Focal adhesion kinase is involved in cellular communication and locomotion. It is regulated on a posttranscriptional level by miR-7, miR-135a, and miR-138 and on a posttranslational level by autophosphorylation at Y397 (pY397-FAK). We related regulators of FAK with histologic dedifferentiation, clinicopathological factors, and differential diagnosis in the thyroid neoplasia spectrum. We classified 82 cases into 5 groups with increasing aggressiveness: healthy tissue, follicular and classical variants of papillary thyroid carcinoma (PTC), dedifferentiated PTC, and anaplastic carcinoma. MiRs were analyzed by RT-qPCR. Protein expression of pY397-FAK was analyzed by immunohistochemistry (separately in the membrane, cytoplasm, and nuclear compartment) and Western blot. All three miRs were upregulated in healthy tissue compared to malignant, while pY397-FAK was downregulated. MiRs and pY397-FAK were not mutually correlated. MiR-135a-5p was decreasing while membranous and cytoplasmic pY397-FAK increased with dedifferentiation. Neither miR correlated with clinicopathological factors. MiR-135a-5p, miR-138-5p, and membranous and cytoplasmic pY397-FAK discriminated the follicular from the classical variant of PTC. Disturbances of FAK regulation on different levels contribute to neoplastic dedifferentiation. pY397-FAK exerts its oncogenic role in the membrane and cytoplasm. Diagnostically, miRs-135a-5p, miR-138-5p, and membranous and cytoplasmic pY397-FAK differentiated between classical and follicular PTC.

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.

Redox metabolism correlates with cellular turnover and clinical phenotype of papillary thyroid carcinoma and colloid goiter.

Introduction: Papillary thyroid carcinoma (PTC) and colloid goiter (CG) represent the most common thyroid malignant and benign diseases, respectively. Oxidative stress is considered to have an important role in the pathogenesis of both diseases, but without sufficient and comprehensive data. The aim was to evaluate the redox profile, its influence on cell survival of PTC, comparing it with CG as a control and its relation with demographic, pathological and clinical parameters. Material and methods: We evaluated for the first time the PTC and CG tissue profile of advanced oxidation protein products (AOPP) and total thiols as parameters of redox metabolism and deoxyribonuclease I (DNase I) and deoxyribonuclease II (DNase II) activity as biomarkers of cell turnover and apoptosis. Tissue levels of biochemical parameters were quantified in PTC and CG tissue using spectrophotometric methods. Study parameters were evaluated in light of different demographic, clinical and pathological features of PTC and CG. Results: Papillary thyroid carcinoma tissue is characterized by increased antioxidant activity and a normal prooxidation level. Biochemical parameters show numerous correlations with demographic and clinical characteristics of PTC and CG patients. DNase I and II activities are dependent upon the AOPP concentration in PTC tissue. The size of CG can be predicted with combined use of AOPP, DNase I and DNase II. AOPP is the most powerful predictor of PTC capsular invasion, multicentric intrathyroid dissemination and lymph node metastasis phenotype. Conclusions: Evaluated parameters can be used for assessment of tumor redox and survival status and the clinical course of PTC and CG.

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.

Prognostic significance of immunohistochemical markers in adrenocortical carcinoma.

Background: To present basic demographic and clinical characteristics of patients with adrenocortical carcinoma (ACC), to determine the overall survival rate and to analyze the results of immunohistochemical staining and its correlation with the length of survival.Material and methods: The study was conducted during the period between 1996 and 2010 and included 30 patients with ACC. Immunohistochemical staining (MMP9, melan A, inhibin, caltretinin, D2-40, synaptophysin and Ki-67) was performed.Results: ACC was diagnosed in 19 females and 11 men (1.7:1). The average age was 50.1 years. The median tumor size was 10 cm, the median weight 400 g. Majority of subjects had positive immunohistochemical staining for the markers of interest. Patients with any negative staining had shorter cancer-specific survival than ones with positive staining. According to the log-rank test results as well as according to the results of the univariate Cox analysis, negative staining for inhibin, D2-40 and synaptophysin and Ki-67 expression ≥7% were associated with poorer prognosis.Conclusions: The results of our study suggest that the absence of staining for some immunohistochemical markers and increased expression of Ki-67 are associated with a poorer prognosis and shorter survival of patients with ACC. Immunohistochemical markers may serve as a prognostic factor for ACC.

Interlaboratory concordance in HER2 testing: Results of a Serbian ring-study.

PURPOSE: The purpose of this study was to assess the immunohistochemistry and chromogenic in situ hybridization (CISH) inter-laboratory consensus between national pathology laboratories in Serbia. METHODS: This study was conducted between 2013 and 2016. In 2013, HER2 results were evaluated using two sets of four different breast cancer specimens in five laboratories. A total of 20 immunohistochemistry and 20 CISH cases were tested. In 2014, there were 6 testing rounds, and a total of 24 specimens were analyzed, whereas in 2015 and 2016, seven testing rounds were conducted, with four additional cases (i.e. a total of 28 specimens). In 2014, 2015 and 2016, all institutions performed immunohistochemical analysis only. RESULTS: We found discrepan¬cies in HER2 immunohistochemical (IHC) results in all four surveys. IHC testing resulted in diagnostic discordance between participating centers in two (2/17) cases in 2013, two (2/24) in 2014, four (4/27) cases in 2015 and three cases (3/27) in 2016. The overall agreement among the centers was 79%, 85.5%, 83.5% and 89.4%, respectively. For CISH analyses, the results for 16 (84.2%) of 19 samples were consistent for all participants. Three results were found to be discordant, indicating a misdiagnosis rate of 15.8%. In all the discrepant cases, interinstitutional discordances were related to technical and evaluation issues. CONCLUSIONS: Our study highlights the difficulty encountered during HER2 testing using immunohistochemistry and CISH. This also emphasizes the need for rigorous quality control procedures for specimen preparation and analysis.

Differences in Chromatin Texture and Nuclear Fractal Dimension Between Hashimoto's and Lymphocytic Thyroiditis Lymphocytes.

Previous evidence suggested that lymphocytic thyroiditis (LT) was a variant of Hashimoto's thyroiditis (HT), thus the aim of the current study is to quantify structural changes in histological specimens taken from HT and LT patients. A total of 600 images containing a single lymphocyte nucleus (300 nuclei per group) were obtained from 20 patients with HT and LT. In order to quantify changes in the nuclear architecture of investigated lymphocytes, the fractal dimension (FD) and some gray-level co-occurrence matrix texture parameters (angular second moment, inverse difference moment, contrast, entropy, and correlation) were calculated for each nucleus. A statistically significant difference in the FD of the "binary-outlined" nucleus and that of the corresponding "black-and-white" nucleus was detected between HT and LT lymphocyte nuclei. In addition, there was also a statistically significant difference in contrast and correlation between HT and LT lymphocyte nuclei. In conclusion, the results of this study suggested that there was a difference in structural complexity between investigated lymphocyte nuclei; additionally, LT lymphocytes possessed probably more complex texture and larger variations as well as more asymmetrical nuclei compared with HT lymphocytes. Accordingly, these findings indicate that LT is probably not a variant of HT; however, more complex studies are necessary to estimate differences between these types of thyroiditis.

Intracerebral hemorrhage as a first sign of pheochromocytoma: case report and review of the literature.

Pheochromocytomas and sympathetic paragangliomas are rare catecholamine-secreting tumours that represent very rare causes of intracerebral haemorrhage in the young, with only a few cases reported. A 32-year-old man presented to our emergency department because of sudden onset of severe headache. He had a six-month history of paroxysmal headache, palpitations, and sweating. During examination he became somnolent and developed left-sided hemiplegia. A computed tomographic (CT) scan of the brain showed a right temporoparietal haematoma. He was admitted to the Clinic for Neurosurgery and the haematoma was evacuated. The patient was comatose, on assisted respiration, with frequent hypertensive crises. An examination for possible secondary causes of hypertension was undertaken. Plasma metanephrine value was elevated (414 pg/mL, reference values < 90 pg/mL). Abdominal CT scans revealed a large mass (6 cm) in the right adrenal gland. After adequate control of the hypertension was achieved with nonselectivealpha- and beta-adrenergic blockers the tumour was excised. The histopathologic findings confirmed the diagnosis of pheochromocytoma. The genetic analysis demonstrated a duplication in exon 1 of the VHL gene. We reported a rare, potentially fatal complication of pheochromocytoma - an intracerebral haemorrhage. This case and review of similar rare cases in the literature illustrate the importance of early recognition of the characteristic symptoms of catecholamine excess in young patients with hypertension.

High expression and localization of ß-catenin and epidermal growth factor receptor identify high risk papillary thyroid carcinoma patients.

We have evaluated the clinical significance of deregulated expression of ß-catenin and epidermal growth factor receptor (EGFR) during papillary thyroid carcinoma (PTC) progression. Immunohistochemical expression of ß-catenin and EGFR was analyzed in 104 archival tissues of PTC and 19 matched lymph node metastases (LNMs). ß-catenin (39/104, 37.5%) and EGFR (58/104, 55.7%) were co-expressed and co-localized in primary PTCs (p < .0001), which was confirmed by double immunofluorescent staining. The high expression of each molecule, as well as their high cytosolic co-expression, correlated with adverse clinicopathological features of the patients (p < .05). High expression of the proteins did not associate with the presence of BRAFV600E mutation (p > .05), tested by mutant allele-specific PCR amplification. Although nuclear localization of ß-catenin was found in a subset of PTC patients (16/104, 15.4%), no ß-catenin mutations were found in exon 3 of the CTNNB1 gene (screened by PCR in combination with denaturing gradient gel electrophoresis and confirmed by next generation sequencing). Cases with additional nuclear ß-catenin staining showed strong association with high EGFR expression (15/16, 93.7%), the presence of capsule invasion (12/16, 81.25%) and regional LNM (9/16, 52.3%). In corresponding LNMs, ß-catenin and EGFR expressions were maintained at high levels or further increased. Co-expression of high levels of ß-catenin and EGFR in association with clinicopathological features implicates their clinical utility in risk stratification of PTC patients, and supports the possibility of crosstalk between Wnt/ß-catenin and EGFR signaling during PTC progression.

Overexpression of epidermal growth factor receptor and its downstream effector, focal adhesion kinase, correlates with papillary thyroid carcinoma progression.

Epidermal growth factor receptor (EGFR) and its downstream effector, focal adhesion kinase (FAK), have been shown to be overexpressed frequently in human malignancies and implicated in tumour aggressiveness. We aimed to investigate the relationship between EGFR and FAK expression and their possible correlation with the clinical phenotype of patients with papillary thyroid carcinoma (PTC). Expression profiles of EGFR and FAK were analysed in PTC tissue samples (n = 104) by immunohistochemistry and Western blotting. Additionally, EGFR and FAK were immunohistochemically analysed in 20 primary tumours paired with their metastatic tissue in lymph nodes. High expression of EGFR and FAK was found in 55.77% and 57.69% cases, respectively, with a strong positive association between them (P < 0.0001, Spearman's correlation coefficient = 0.844). Expression of each molecule and their coexpression correlated significantly with the presence of lymph node metastasis (LNM), degree of tumour infiltration, extrathyroid invasion and pT status of the patients. Western blot analysis confirmed that coexpression of high levels of EGFR and FAK correlated with adverse clinicopathological features. When compared to the corresponding primary tumour, increased or maintained high levels of EGFR and FAK were found in LNM, indicating their concordant expression during lymphatic spread. In conclusion, high levels of EGFR and its downstream effector, FAK, in association with lymphatic spread and tumour infiltration indicate their involvement in PTC progression and suggest that both molecules may predict its aggressive behaviour. Furthermore, FAK could be a potential target for anticancer therapy in patients with advanced thyroid cancer.

Concomitant high expression of survivin and vascular endothelial growth factor-C is strongly associated with metastatic status of lymph nodes in papillary thyroid carcinoma.

PURPOSE: Papillary thyroid carcinoma (PTC) has a strong propensity to metastasize to regional lymph nodes which increases the risk of local-regional relapse and affects the course of the disease. Molecular pathogenesis of lymph node metastasis (LNM) is not yet fully understood. Survivin, a multifunctionale molecule involved in apoptosis, proliferation and angiogenesis, and vascular endothelial growth factor-C (VEGF-C) are suggested to be implicated in lymphatic metastases of human malignancies. MATERIALS AND METHODS: Expression of survivin and VEGF-C was examined by immunohistochemistry and Western blot in 75 cases of PTCs in relation to their LNM status. Additionally, survivin and VEGF-C were immunohistochemically analyzed in 15 primary PTCs paired with their metastatic tissue in lymph nodes. RESULTS: High expression of survivin and VEGF-C was found in 62.7% and 64.0% cases, respectively, with a positive correlation to each other (Spearman's correlation co-efficient = 0.878, P < 0.001). Expression levels of both proteins were significantly higher in patients with LNM than in those without LNM (P < 0.001). The rate of concomitant high expression of survivin and VEGF-C in patients with LNM involvement was 88.9% (P < 0.01). Metastatic tissue in lymph nodes expressed survivin and VEGF-C at the same high extent as their primary tumors. CONCLUSION: Concomitant high expression of survivin and VEGF-C is closely associated with LNM status of PTC patients, which suggests their cooperation in the metastatic process. Evaluation of survivin and VEGF-C expression could be clinically significant in predicting the metastatic potential of PTC and subsequent treatment and follow-up of these patients.

Inverse expression of caveolin-1 and EGFR in thyroid cancer patients.

Cytological analysis of fine-needle aspiration (FNA) is the first step in evaluation of patients with nodular thyroid disease with the primary goal to exclude thyroid malignancy. Its improvement by combining cytology with molecular markers is still a matter of investigation. In this study, 2 molecular markers were used: caveolin-1 and epidermal growth factor receptor (EGFR), along with the well-established genetic marker BRAF V600E mutation. We set out to determine the expression signatures of EGFR and caveolin-1 in patients with thyroid malignancy as well as to determine their possible association with disease severity. In FNA biopsy samples (n=186), immunocytochemical expression of caveolin-1 and BRAF V600E mutation coincided with malignancy. The patients were sorted according to 3 parameters: final histopathological diagnosis, caveolin-1 expression, and BRAF V600E mutation status before measurement of EGFR mRNA expression. EGFR upregulation was detected in the group of patients with malignant diagnosis, no caveolin-1 expression, and wild-type BRAF. Spearman rank correlation yielded a statistically significant negative correlation of EGFR and caveolin-1. Double immunofluorescence confirmed colocalization and inverse expression of EGFR and caveolin-1. Our data demonstrated that EGFR overexpression is associated with malignancy but not with tumor aggressiveness. Furthermore, high-caveolin-1/low-EGFR cases were associated with an advanced pT status and had a greater degree of neoplastic infiltration than low-caveolin-1/high-EGFR cases. Combining caveolin-1 and BRAF V600E with EGFR might help in recognizing more aggressive thyroid lesions in a pool of relatively indolent tumors in FNA biopsies and thus be useful for early risk stratification of thyroid cancer patients.

Coexpressed High Levels of VEGF-C and Active MMP-9 Are Associated With Lymphatic Spreading and Local Invasiveness of Papillary Thyroid Carcinoma.

OBJECTIVES: Papillary thyroid carcinoma (PTC) usually has a good prognosis, but some patients develop an aggressive course of the disease, leading to a poor outcome. Vascular endothelial growth factor C (VEGF-C) and matrix metalloproteinase 9 (MMP-9) have been shown to play roles in tumor progression in various human malignancies. METHODS: We analyzed VEGF-C and active MMP-9 expression profiles in PTC samples using immunohistochemistry and Western blotting. RESULTS: Immunohistochemistry showed positive staining for VEGF-C and active MMP-9 in 83% and 57% of PTCs, respectively (n = 60), with a positive correlation between their expression levels (Spearman, P < .001). Concomitant high expression of VEGF-C and active MMP-9 correlated with the presence of lymph node metastasis (P = .005), pT status (P = .004), pTNM tumor stage (P = .005), and particularly the degree of tumor infiltration (P < .001, Fisher exact test). Densitometric analysis of Western blot bands confirmed correlation between VEGF-C and active MMP-9 expression (Wilcoxon and Spearman tests) and significant association with the clinicopathologic parameters (Mann-Whitney and Kruskal-Wallis tests). CONCLUSIONS: Association of coexpressed high levels of VEGF-C and active MMP-9 with lymphatic spreading and local invasiveness of PTC suggests their potential usefulness as predictive biomarkers of aggressive PTC behavior.

Defining the value of CD56, CK19, Galectin 3 and HBME-1 in diagnosis of follicular cell derived lesions of thyroid with systematic review of literature.

BACKGROUND: Nodular follicular lesions of thyroid gland comprise benign and malignant neoplasms, as well as some forms of hyperplasia. "Follicular" refers to origin of cells and in the same time to growth pattern - building follicles. Nodular follicular thyroid lesions have in common many morphological features, therefore attempts were made to define additional criteria for distinction between follicular adenoma, follicular carcinoma and follicular variant of papillary carcinoma. Increasing number of immunohistochemical markers is in the continual process of evaluation. METHODS: Tissue microarrays incorporating, total 201 cases, out of which 122 malignant and 79 benign follicular lesions, including neoplastic and non-neoplastic, were constructed and immunostained with antibodies to CD56, CK19, Galectin-3, HBME-1. Tissue cores were exclusively being acquired from tumour/lesion on interface with normal thyroid tissue. A systematic review of literature was done for period from the year 2001 to present time. RESULTS: All analysed markers may make a difference between benign lesions/tumours from differentiated thyroid carcinomas (p = <0.01, for all markers). Expression of all markers is significantly higher in papillary carcinoma than in follicular adenoma (p < 0.01). Statistically significant difference in expression of Galectin-3 and CD56 between follicular carcinoma and follicular adenoma was registered (p = 0.043; p = 0.028, respectively). The only marker which expression showed statistically significant difference between adenoma and carcinoma of Hurthle cells was Galectin 3 (p = 0.041). CK19 and HBME-1 were significantly expressed more in papillary carcinoma as compared to follicular carcinoma. CONCLUSION: Galectin 3 is most sensitive marker for malignancy, while loss of expression of CD56 is very specific for malignancy. Expected co-expression for combination of markers in diagnosis of follicular lesions decreases sensitivity and increases specificity for malignancy.

Malignant risk stratification of thyroid FNA specimens with indeterminate cytology based on molecular testing.

BACKGROUND: Fine-needle aspiration (FNA) has been employed for many years for examining thyroid nodules, and the cytology of aspirates is the primary determinant for whether thyroidectomy is indicated. Fifteen to thirty percent of thyroid nodules, not being clearly benign or malignant, fall into an indeterminate category. The main goals of molecular diagnostics for thyroid nodules are to prevent unnecessary surgery in patients with benign nodules and to stop patients with malignant nodules from being subjected to repeated operations. METHODS: This study was designed to evaluate the diagnostic utility of 4 markers in thyroid FNA cytology via testing for the BRAF V600E mutation and the expression of microRNA-221, microRNA-222, and galectin-3 protein in FNA samples with indeterminate cytology. RESULTS: A predictor model distinguishing benign samples from malignant samples on the basis of the 4 aforementioned markers was formulated. This decision model provided a sensitivity of 73.5%, a specificity of 89.8%, and a diagnostic accuracy of 75.7%. The positive predictive value was 80.6%, and the negative predictive value was 85.5%; this suggested that the prediction had good reliability. CONCLUSIONS: One hundred twenty FNA samples were examined, and 62 nodules were classified as benign with the proposed diagnostic algorithm. This resulted in a reduction of the initial 120 patients to 58 and thus decreased by half the number of persons undergoing surgery.

The relationship between proliferation activity and parathyroid hormone levels in parathyroid tumors.

PURPOSE: This article examines as to whether the Ki-67 index may be useful as a marker for cell proliferation, as well as to whether Ki-67 immunohistochemical expression and parathyroid hormone (PTH) levels are useful in distinguishing between parathyroid carcinoma (PC) and adenoma. METHODS: A retrospective analysis of 50 patients (10 with PC and 40 with adenoma) who had been previously diagnosed with primary hyperparathyroidism (PHPT) was conducted. Normal parathyroid glands served as the control group. Immunostaining of Ki-67 was estimated through image analysis and the results were statistically analyzed. RESULTS: Ki-67 was higher in PC patients (median 785.15) compared to adenoma patients (median 297.41; Mann-Whitney U-test p<0.001). ROC analysis confirmed that Ki-67 has a positive predictive marker in diagnosing cancer. Mann-Whitney U-test confirmed a highly statistically significant difference in the preoperative PTH levels between the PC and adenoma group (p <0.001). The PTH serum preoperative level was higher in PC patients (median 1721) than in those with adenoma (median 189.5). A highly significant correlation was also found between Ki-67 and preoperative PTH levels (p <0.001). CONCLUSION: A higher rate of cellular proliferation was noted in malignant tumors as compared to benign tumors. Moreover, the expression profile of Ki-67 and high PTH levels in this study indicates a role for them as potential markers of malignancy.

Survival and prognostic factors for adrenocortical carcinoma: a single institution experience.

BACKGROUND: Adrenocortical carcinoma (ACC) is aggressive, but rare tumours that have not been sufficiently studied. The aim of our study was to present the demographic and clinical characteristics of patients with ACC, to determine the overall survival rates, analyse the effect of prognostic factors on survival, as well as to identify favorable and unfavourable predictors of survival. METHOD: The study included 72 patients (42 women and 30 men) with ACC. We analysed the prognostic value of the demographic and clinical characteristics of the patients, tumour characteristics, therapy administered and survival rates. Kaplan-Meier survival curves and the log-rank test were used to estimate the overall and specific survival probabilities and the Cox regression model was used to identify independent prognostic factors for survival. RESULTS: The patients had mean age of 50 years. The 1-, 5-, and 10-year probabilities of survival in patients with ACC were 52.5 %, 41.1 %, and 16.4 %, respectively. The median survival time was 36 months. The results of multivariate Cox regression analysis showed that the presence of lymphatic metastases (HR = 7.37, 95 % CI = 2.31-23.48, p = 0.001) and therapy with mitotane (HR = 0.11, 95 % CI = 0.04-0.27, p = 0.001) were independent prognostic factors for survival. CONCLUSION: The presence of lymphatic metastasis is an unfavourable prognostic factor, while postoperative therapy with mitotane is a favorable prognostic factor for survival in patients with ACC.