Detecting thyrotropin receptor mRNA from peripheral blood of patients with differentiated thyroid cancer rules out non-aggressive cases.

BACKGROUND: Early diagnosis of thyroid cancer is hampered by the inability of fine-needle aspiration biopsy (FNAB) to accurately classify ∼30% of cases while preoperative cancer staging detects lymph nodal involvement in only half of cases. Liquid biopsy may present an accurate, non-invasive alternative for preoperative thyroid nodule assessment. Thyrotropin receptor (TSHR) mRNA, a surrogate marker for circulating cancer cells (CTC), may be an option for early detection of malignancy from peripheral blood, but requires methodological improvements. We aimed to investigate if TSHR mRNA can be detected in low sample volumes by employing an ultrasensitive method - droplet digital PCR (ddPCR). METHODS: Less than 5 mL of blood was collected from 47 patients with thyroid nodules (25 benign and 22 malignant). RNA was isolated from the fraction of mononuclear cells where CTCs segregate. Samples were analysed for the presence of TSHR mRNA by ddPCR. RESULTS: Thyrotropin receptor mRNA was detectable in 4 mL sample volumes, with the test having good specificity (80%) but modest diagnostic accuracy (68.1%). Combining TSHR mRNA with ultrasound features and FNAB diagnosis, the test reaches high rule-out performances (sensitivity = 90% and NPV = 88.2%). Strikingly, TSHR mRNA correctly classified all samples with thyroid capsule invasion, lymph node metastasis and extrathyroidal extension. If aggressiveness is defined using these parameters, TSHR mRNA test reaches 100% sensitivity and 100% NPV for detecting high-risk cases. CONCLUSIONS: Employing ddPCR for TSHR mRNA improves its measurement by enabling detection in sample volumes common for laboratory testing. The test displays high prognostic performance, showing potential in preoperative risk assessment.

Challenges and current advances in the methodology of thyroglobulin measurements.

Thyroglobulin (Tg) is a large protein secreted exclusively by the thyroid gland. In a clinical setting, it is measured for the purpose of follow-up of thyroidectomy patients. However, Tg measurements are often impeded by the presence of Tg autoantibodies and/or heterophylic antibodies that interfere with most measuring platforms. This presents a global problem in thyroid cancer patients who need to be postoperatively monitored for recurrent or residual disease. Therefore, in this paper we offer an overview of the existing methodologies and alternative approaches for Tg measurements that are a focus of research worldwide. These include Tg mRNA measurements, exosomal Tg detection, the use of alternative analytes (liquid biopsies) and the development of new approaches for preanalytical sample treatment.

Thyroglobulin (Tg) is a large protein produced only by the thyroid gland. It helps physicians follow-up with patients who have had thyroid surgery. Nevertheless, Tg autoantibodies or other antibodies can occasionally cause Tg tests to malfunction. For those who have thyroid cancer, this makes it difficult to ensure that their disease is not returning. In this article, we examine various approaches of measuring Tg that are being investigated globally. These techniques include analyzing Tg mRNA, identifying Tg in exosomes, employing other components of blood (liquid biopsies), and developing novel approaches to blood sample preparation prior to testing. All of these techniques may aid medical professionals in better monitoring patients with thyroid cancer and preventing issues brought on by interfering antibodies.

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.

MiR-203a-3p, miR-204-3p, miR-222-3p as useful diagnostic and prognostic tool for thyroid neoplasia spectrum.

PURPOSE: The challenge in the diagnosis and treatment of thyroid carcinoma is to correctly classify neoplasias with overlapping features and to identify the high-risk patients among those with a less aggressive form, in order to personalize the treatment of thyroid carcinoma patients accordingly. METHODS: MiR-203a-3p, miR-204-3p, and miR-222-3p levels were determined in 99 cases of thyroid neoplasias (77 papillary thyroid carcinomas (PTC) of diverse variants, 12 follicular thyroid adenomas (FTA) and 10 nodular goiters (NG)) along with 99 adjacent non-malignant thyroid tissues using quantitative RT-PCR. The results were evaluated in comparison with the clinicopathological features of the patients and available TCGA data. RESULTS: Down-regulated miR-203a-3p indicates the presence of thyroid tumor (PTC or FTA) with high sensitivity (75%) and specificity (73%), while its up-regulation indicates NG. If miR-203a-3p is down-regulated, up-regulated miR-204-3p with high sensitivity (83.3%) and specificity (74.4%) indicates FTA presence, while up-regulated miR-222-3p, with high sensitivity (76.6%) and specificity (75.0%), points to PTC. The expression of miR-204-3p and miR-222-3p depends on the PTC subtype (P < 0.05). While the deregulated expression of tested miRs is associated with a long-range of unfavorable clinicopathological parameters of PTC, only abundant expression of miR-222-3p may be used as an independent predictive factor for the presence of extrathyroid invasion and advanced pTNM stage of PTC (P < 0.05). CONCLUSION: Successive evaluation of miR-203a-3p, miR-204-3p, and miR-222-3p expression can help in the differential diagnosis of thyroid neoplasias. A high relative value of miR-222-3p expression is an independent predictive factor for the presence of extrathyroid invasion and advanced pTNM stage of PTC. The panel consisting of miR-203a-3p, miR-204-3p, and miR-222-3p could be used as a diagnostic and prognostic tool for personalizing the treatment of thyroid cancer patients.

Predictive Significance of Two MMP-9 Promoter Polymorphisms and Acetylated c-Jun Transcription Factor for Papillary Thyroid Carcinoma Advancement.

Papillary thyroid carcinoma represents a challenge from a prognostic standpoint. Molecular alterations responsible for PTC advancement include MMP-9 genetic promoter polymorphisms that bind transcription factors with varying degrees of affinity and, hence, constitute a predisposition for MMP-9 expression. We examined how two promoter polymorphisms (the -1562 C/T transition and -131 (CA)n tandem repeats) as well as levels of the c-Jun transcription factor and its modified form acetylated at Lys271 influence MMP-9 expression and PTC progression. A significant proportion of PTC samples were heterozygous for the (CA)n tandem repeat number, had a transcription-promoting T allele at -1562, and expressed high levels of c-Jun, acetylated c-Jun, and MMP-9 protein. The T allele at the -1562 position accompanied the elevated MMP-9 protein expression, while high acetylated c-Jun levels accompanied the high MMP-9 protein levels on mRNA. The -1562 C/T transition, MMP-9, and acetylated c-Jun were associated with the presence of extra-thyroid invasion and degree of tumor infiltration, while the T allele and acetylated c-Jun also correlated with tumor stage. We conclude that the -1562 MMP-9 polymorphism and levels of acetylated c-Jun affect PTC progression via modulation of MMP-9 levels. Genotyping the MMP-9 at -1562 and estimating the levels of MMP-9 and acetylated c-Jun in PTC may prove beneficial in identifying high-risk patients.

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.

Novel approach to the measurement of antithyroglobulin antibodies in human serum - application of the quartz crystal microbalance sensors.

Measurement of antithyroglobulin antibodies (TgAb) is an inevitable laboratory tool in the management of thyroid gland diseases. Currently available immunoassays still have limitations underlying the necessity of the introduction of fast, sensitive, and label-free technologies. Our aim was to develop a method for TgAb measurement in human serum based on the quartz crystal microbalance (QCM) technology. We immobilized thyroglobulin on the surface of Attana LNB Carboxyl sensor chip®, prepared standard curve covering the range of 1-50000 kIU/L, and established optimal measurement conditions. The validation included determination of the detection limit (LOD), functional sensitivity, linearity, precision, as well as the comparison with the results of the radioimmunoassay (RIA). The LOD and functional sensitivity were 4.2 kIU/L and 4.7 kIU/L, respectively. The method was linear in the range of 20-10000 kIU/L. The regression equation for comparison with RIA was CQCM= 1.0056 • CRIA- 24.2778, whereby no significant proportional or systematic difference was present. There was a good agreement with RIA in the classification of patients according to the clinical significance of the results. The developed method has advantages over currently available assays in terms of better LOQ, a higher upper limit of linearity, and precision. The characteristics of the developed method unambiguously show that the application of the QCM biosensors offers a highly reliable novel approach for the measurement of TgAb in human serum.

Elevated BANCR expression levels have different effects on papillary thyroid carcinoma progression depending on the presence of the BRAFV600E mutation.

INTRODUCTION: The role of BRAF-activated non-protein coding RNA (BANCR) in papillary thyroid carcinoma (PTC) is controversial, its clinical significance is unclear and no study has correlated the presence of the BRAFV600E mutation in PTC with BANCR expression. METHODS: BANCR levels in PTC and matched nonmalignant thyroid epithelial tissues from 85 patients were determined using quantitative RT-PCR. BRAFV600E was detected by mutant allele-specific PCR amplification. The results were correlated with clinicopathological characteristics of the patients. RESULTS: The presence of BRAFV600E associates with lower relative BANCR expression (RBE) in PTC (p = 0.008). RBE is down-regulated in BRAFV600E positive PTC, while it is unchanged or up-regulated in BRAFV600E negative PTC compared to the levels in paired nonmalignant tissue (p = 0.001). At the cut-off of 31.3%, sensitivity of fold change of BANCR for the presence of BRAFV600E is 68.0% and specificity is 67.2%. In BRAFV600E positive PTC up-regulated BANCR predicts lymph node metastasis (p = 0.001), while in BRAFV600E negative PTCs high RBE predicts thyroid capsule invasion (p = 0.028). CONCLUSIONS: Depending on the presence of BRAFV600E, elevated BANCR levels demonstrated different effects on lymphatic spreading and local PTC invasion. Therefore, BANCR could be a useful prognostic biomarker in risk stratification of PTC patients.

MMP-9-1562 C/T single nucleotide polymorphism associates with increased MMP-9 level and activity during papillary thyroid carcinoma progression.

Papillary thyroid carcinoma (PTC), a common form of thyroid malignancy, displays significant variations in clinical features and outcome. The malignant transformation of the thyroid is driven by altered expression of many matrix-modulating enzymes, including matrix metalloproteinase-9 (MMP-9). A single nucleotide polymorphism in its promotor (-1562 C/T) is suspected to cause overexpression of MMP-9, which in turn contributes to development of a tumour unfavourable phenotype. The aim of this study was to investigate the impact of MMP-9 promotor genotype on MMP-9 expression in PTC samples, and to assess its value as a possible risk factor for developing PTC or its aggressive phenotype. A total of 105 PTC patients and 43 healthy controls were genotyped by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. In order to estimate MMP-9 expression, PTC tissue sections were stained immunohistochemically. Statistical analysis showed that PTC cases and controls did not differ significantly in genotype frequencies (OR = 2.27, CI = 0.854-6.022). In PTC samples, the presence of the T allele was accompanied by elevated MMP-9 expression (p = 0.047) as well as a higher risk of developing extrathyroid extensions (p = 0.037) and high TNM stages (p = 0.009). Moreover, we observed overexpression of MMP-9 in cases presenting with extrathyroid invasion (p = 0.001), lymph node metastasis (p = 0.028), large tumour size (p = 0.031) and advanced stage (p = 0.005) compared to indolent tumours, along with enhanced enzymatic activity demonstrated by in situ zymography. Data suggests that MMP-9 (-1562 C/T) does not facilitate predisposition for PTC but affects the disease course by modulating MMP-9 expression. Genotyping MMP-9 provides important information which may prove beneficial in risk stratification of PTC patients.

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.

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.

Strong expression of HBME-1 associates with high-risk clinicopathological factors of papillary thyroid carcinoma.

Thyroid cancer comprises a heterogeneous group of lesions with great diversity of biological behaviour. Markers which could help clinicians to identify high-risk patients for tailored optimization of clinical management are of crucial importance. HBME-1 protein level was analysed immunohistochemically using routinely prepared archival tissue sections of a broad range of papillary thyroid carcinoma (PTC) variants and in corresponding lymph node metastases (LNM). The results were evaluated in comparison with clinicopathological features of PTC. Positive immunoreaction was noticed in most classical (83/92; 90.2 %), follicular (60/71; 84.5 %) and trabecular (4/5; 80.0 %) variants of PTC. All cases of macrofollicular, Warthin-like and diffuse sclerosing PTC variants were HBME-1 positive (4/4, 3/3, 2/2; 100 % respectively). Tall cell and solid PTC variants showed diversity of staining (2/3; 66.67 % and 13/23; 56.52 % respectively), while PTCs with mixed histological pattern containing insular areas were mainly weakly positive (2/5; 40.0 %). A single case of clear cell PTC variant showed no reaction. Moreover, all matched metastatic PTC into lymph nodes (LNM) were HBME-1 positive (17/17; 100 %) and expressed HBME-1 in a similar pattern to the matched primary tumour. We also found a statistically significant association between high HBME-1 expression and the presence of lymph node metastasis, advanced pT status and pTNM stage (P < 0.05), but only a tendency for association with extrathyroidal invasion of the tumour (P = 0.058). Therefore, we recommend using immunoexpression of HBME-1 as useful mean to increase the likelihood of detecting most PTC variants and to predict some unfavourable clinical parameters in these 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.

Enhanced activation of matrix metalloproteinase-9 correlates with the degree of papillary thyroid carcinoma infiltration.

AIM: To determine whether matrix metalloproteinase-9 (MMP-9) may be a useful adjunctive tool for predicting unfavorable biological behavior of papillary thyroid carcinoma (PTC) by evaluating the expression profile and proteolytic activity of MMP-9 in PTC by different techniques and correlating the findings with clinicopathological prognostic factors. METHODS: Immunohistochemical localization of MMP-9 was analyzed with antibodies specific for either total or active MMP-9. Activation ratios of MMP-9 were calculated by quantifying gel zymography bands. Enzymatic activity of MMP-9 was localized by in situ zymography after inhibiting MMP-2 activity. RESULTS: Immunostaining of total and active MMP-9 was observed in tumor tissue and occasionally in non-neoplastic epithelium. Only active MMP-9 was significantly associated with extrathyroid invasion, lymph-node metastasis, and the degree of tumor infiltration (P<0.001, P=0.004, and P<0.001, respectively). Gelatin zymography revealed a correlation between the MMP-9 activation ratio and nodal involvement, extrathyroid invasion, and the degree of tumor infiltration. In situ zymography showed that gelatinases exerted their activity in tumor parenchymal and stromal cells. Moreover, after application of MMP-2 inhibitor, the remaining gelatinase activity, corresponding to MMP-9, was highest in cancers with the most advanced degree of tumor infiltration. CONCLUSIONS: This is the first report suggesting that the evaluation of active MMP-9 by immunohistochemistry and determination of its activation ratio by gelatin zymography may be a useful adjunct to the known clinicopathological factors in predicting tumor behavior. Most important, in situ zimography with an MMP-2 inhibitor for the first time demonstrated a strong impact of MMP-9 activity on the degree of tumor infiltration during PTC progression.

Evaluation of survivin expression and its prognostic value in papillary thyroid carcinoma.

Overexpression of survivin, an inhibitor of apoptosis protein, has been found in a variety of human cancers, and is associated with tumor aggressiveness. In this study, we analyzed the expression of survivin in papillary thyroid carcinoma (PTC) and evaluated its clinical significance for predicting an aggressive course of disease at the time of diagnosis. Survivin expression was determined by immunohistochemistry in 104 tissue specimens of PTC, confirmed by Western blot and correlated with clinicopathological parameters. Of the tumors examined, 74 (71.15%) showed high cytoplasmic survivin expression. There was no association between high survivin expression and age, gender or tumor size. On the other hand, it was closely correlated with the presence of lymph node metastasis (P=0.009), and there was a tendency for correlation with extrathyroidal invasion (P=0.062). The high risk PTC group (TNM stage III-IV) was associated with high levels of survivin (P=0.027). These results indicate that survivin is an unfavorable molecule for PTC prognosis, and that its high expression may indicate a subset of PTC patients with a more aggressive disease course. Evaluation of its expression in fine needle aspiration samples could be a useful tool for the identification of those PTC patients who require more extensive surgery, careful follow-up and therapeutic strategy.

Apoptosis and proliferation related molecules (Bcl-2, Bax, p53, PCNA) in papillary microcarcinoma versus papillary carcinoma of the thyroid.

AIM: To gain a better insight into the differences in biological behaviour between papillary microcarcinoma (PMC) and clinically evident papillary thyroid carcinoma (PTC). METHODS: Immunohistochemical analysis of apoptosis related molecules (Bcl-2, Bax, p53) and proliferation related marker (PCNA) in 39 archival cases of PMC and 46 cases of PTC. RESULTS: Bcl-2 and Bax were expressed in most PMCs and PTCs. The average Bcl-2 staining score did not differ significantly between PMCs and PTCs (p > 0.05), but the average Bax score was significantly lower in PMCs (p < 0.05). The Bcl-2/Bax ratio was significantly higher in PMCs than in PTCs (p < 0.05). The expression of p53 was similar in PMCs and PTCs, without a correlation with clinical data, but was associated with high Bax expression (p < 0.05) in these cases in both groups. Non-malignant tissue expressed only Bcl-2, but not p53 or Bax. PCNA expression was significantly lower (p < 0.05) in PMC than in PTC and positively correlated with tumour size (p < 0.05). CONCLUSIONS: The higher Bcl-2/Bax ratio and lower proliferative activity in PMC suggest differences from PTC in the balance between apoptosis and proliferation. However, the presence of p53 and Bax in PMC indicates malignant potential, and thus PMC should be treated with caution.