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Fumarate is an oncometabolite. However, the mechanism underlying fumarate-exerted tumorigenesis remains unclear. Here, utilizing human type2 papillary renal cell carcinoma (PRCC2) as a model, we show that fumarate accumulates in cells deficient in fumarate hydratase (FH) and inhibits PTEN to activate PI3K/AKT signaling. Mechanistically, fumarate directly reacts with PTEN at cysteine 211 (C211) to form S-(2-succino)-cysteine. Succinated C211 occludes tethering of PTEN with the cellular membrane, thereby diminishing its inhibitory effect on the PI3K/AKT pathway. Functionally, re-expressing wild-type FH or PTEN C211S phenocopies an AKT inhibitor in suppressing tumor growth and sensitizing PRCC2 to sunitinib. Analysis of clinical specimens indicates that PTEN C211 succination levels are positively correlated with AKT activation in PRCC2. Collectively, these findings elucidate a non-metabolic, oncogenic role of fumarate in PRCC2 via direct post-translational modification of PTEN and further reveal potential stratification strategies for patients with FH loss by combinatorial AKTi and sunitinib therapy., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: Phosphatase and tensin homolog (PTEN) hamartoma tumor syndrome (PHTS) is a complex disorder. Carriers develop hamartomatous tumors, with an increased risk for developing malignant tumors in multiple organs. Surveillance to facilitate the early detection and treatment of malignancies is extremely important., Case Report: A 31-year-old male presented with a 10 cm left lobe thyroid gland mass. After fine needle aspiration a left hemithyroidectomy was performed, which demonstrated a minimally invasive follicular thyroid carcinoma (FTC, stage pT3a) and microscopic classical papillary thyroid carcinoma (PTC) in the background of about 50 separate adenomatous nodules (0.2-5 mm). Immunostaining showed loss of PTEN protein in the minimally invasive FTC and in all of the nodules tested, with uninvolved parenchyma serving as an internal control. Kaiser Permanente Northern California (KPNC) Hereditary Cancer Panel, testing for 62 genes, was performed and showed germline mutations in PTEN and RecQ like helicase 4 (RECQL4) genes. Completion thyroidectomy subsequently performed demonstrated about 60 follicular cell-derived adenomatous nodules (0.3-10 mm). Genetic counseling and evaluation documented Cowden syndrome (CS) in the family. Thus, PHTS was confirmed., Conclusion: This report documents synchronous FTC and PTC in a background of multiple follicular adenomatous nodules with a novel RECQL4 mutation in an adult patient with PHTS. As such, documented the loss of PTEN protein in a thyroid gland affected by multiple adenomatous nodules aided in diagnosing PHTS., (Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Evidence regarding intraductal papillary neoplasm of the bile duct (IPNB) as a type of precancerous lesion of cholangiocarcinoma is limited. Moreover, a reproducible in vivo model is lacking, and IPNB pathogenesis remains unclear. Here, we use a doxycycline-inducible tetracycline (Tet)-on mice model to control fibroblast growth factor 10 (FGF10) expression, which regulates branching and tubule formation. FGF10-induced IPNB mimics the multifocal and divergent human IPNB phenotypes via the FGF10-FGF receptor 2 (FGFR2)-RAS-extracellular-signal-regulated kinase (ERK) signaling pathway. A paracrine/autocrine growth factor is sufficient to initiate and maintain IPNB originating from the peribiliary glands, including biliary stem/progenitor cells. With Kras G12D , p53, or p16 mutations or both, Fgf10-induced IPNB shows stepwise carcinogenesis, causing associated invasive carcinoma. Fgf10-induced papillary changes and progression are suppressed by the inhibition of the FGF10-FGFR2-RAS-ERK signaling pathway, demonstrating that the signal is a therapeutic target for IPNB and associated carcinoma., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Thyroid cancer represents a heterogenous disease whose incidence has increased in the last decades. Although three main different subtypes have been described, molecular characterization is progressively being included in the diagnostic and therapeutic algorithm of these patients. In fact, thyroid cancer is a landmark in the oncological approach to solid tumors as it harbors key genetic alterations driving tumor progression that have been demonstrated to be potential actionable targets. Within this promising and rapid changing scenario, current efforts are directed to improve tumor characterization for an accurate guidance in the therapeutic management. In this sense, it is strongly recommended to perform tissue genotyping to patients that are going to be considered for systemic therapy in order to select the adequate treatment, according to recent clinical trials data. Overall, the aim of this article is to provide a comprehensive review on the molecular biology of thyroid cancer focusing on the key role of tyrosine kinases. Additionally, from a clinical point of view, we provide a thorough perspective, current and future, in the treatment landscape of this tumor.

Objective: Antibodies against carbonic anhydrase IX (CAIX) are often part of immunohistochemical panels used to assist renal cell cancer (RCC) subtyping. This study was undertaken to determine, whether assessing CAIX expression levels could provide additional prognostic information., Methods and Materials: More than 1,800 RCCs were analyzed in a tissue microarray (TMA) format for CAIX expression. All tumors had been reviewed and newly classified according to the WHO 2016 classification., Results: Membranous CAIX expression revealed a "black and white" pattern that was strikingly dependent on the RCC subtype. In clear cell RCC, 89.2% of cancers showed strong positivity. The few clear cell RCC with lower CAIX expression levels were more likely to exhibit unfavorable tumor phenotype (p < 0.0001) and poor disease course (p = 0.0036). CAIX was completely absent in 99% of chromophobe RCC and in 100% of oncocytomas. In papillary RCC, 80.2% of cancers showed complete absence of CAIX staining. Papillary RCC with detectable CAIX expression had a less favorable tumor phenotype (p≤0.05) and worse disease outcome (p = 0.0176). These data are consistent with the concept, that "aberrant" CAIX staining - meaning absent or weak staining in a cancer expected to have a high level CAIX expression such as clear cell RCC or detectable CAIX expression in tumors that are typically CAIX negative such as papillary and chromophobe RCC - reflects biologic tumor dedifferentiation., Conclusion: Our data demonstrate that CAIX is a highly useful diagnostic biomarker for RCC providing both diagnostic and prognostic information., (Copyright © 2018 Elsevier Inc. All rights reserved.)

BACKGROUND Approximately 20% of patients with papillary thyroid carcinoma (PTC) will develop cancer recurrence, but no clinically available biomarker has been identified. Our study aimed to evaluate the prognostic value of G protein-coupled receptor kinase 6 (GRK6) in PTCs. MATERIAL AND METHODS We retrospectively enrolled 108 PTC patients in this study, and explored the expression of GRK6 in resected tumor samples by RT-qPCR and immunohistochemistry (IHC). The clinical data were interpreted by chi-square test, univariate analysis, and multivariate analysis. To investigate the functional mechanisms of GRK6 in regulating PTC progression, we also performed overexpression and silencing experiments in TPC-1 cells, a cell line generated from PTC tissues. RESULTS RT-qPCR results showed a higher level of GRK6-mRNA in PTCs than in adjacent thyroid tissues. IHC revealed a distinct protein expression pattern of GRK6 among PTCs. Accordingly, we classified patients into low-GRK6 and high-GRK6 groups. The chi-square test showed that a higher GRK6 was associated with larger tumor size (P=0.045) and advanced TNM stage (P=0.001). Kaplan-Meier survival curve and log rank test demonstrated that higher GRK6 predicted poor disease-free survival (DFS) in PTC patients (P=0.002). Furthermore, Cox regression analysis confirmed that GRK6 was an independent prognostic factor for a higher recurrence risk of PTCs (P=0.047). MTT assay and Transwell assay demonstrated that GRK6 overexpression can significantly enhance tumor proliferation and invasion, which was consistent with clinical findings. CONCLUSIONS Our data show the oncogenic effects of GRK6 in promoting PTC progression.

KMT5A (known as PR-Set7/9, SETD8 and SET8), a member of the SET domain containing methyltransferase family specifically targeting H4K20 for methylation, has been implicated in multiple biological processes. In the present study, we identified that KMT5A was elevated in 50 pairs of papillary thyroid cancer tissue samples and in cell lines K1 and TPC-1 by qRT-PCR and western blotting, as well as by immunohistochemical staining. CCK-8 assay and flow cytometric analysis revealed that inhibition of KMT5A attenuated proliferation and induced apoptosis. Transwell assays revealed that cell migration and invasion were suppressed in KMT5A-knockdown cells. Moreover, the inhibition of KMT5A arrested the cell cycle in the G1/S phase of papillary thyroid cancer cells. The TCGA data revealed that elevated KMT5A expression was significantly correlated with extrathyroidal extension, lymph node metastasis and advanced pathological stage of papillary thyroid cancer. Furthermore, we observed that inhibition of KMT5A suppressed the expression of SREBP1, SCD, FASN and ACC, key molecules involved in lipid metabolism and decreased the level of malondialdehyde in papillary thyroid cancer cells. In conclusion, KMT5A may be a novel oncogenic factor, specifically a regulator for lipid metabolism in papillary thyroid carcinoma.

Thyroid cancer is the most common type of endocrine malignancy and a leading cause of death among endocrine organ-related cancers. Similar to other types of cancers, early diagnosis of thyroid cancer is important to increase the survival and treatment of this disease. Several immunohistochemical markers are used in the differential diagnosis of thyroid papillary carcinoma. Also, increasing evidence indicates that P-element induced wimpy testis like 2 (PIWIL2) is an RNA-binding protein involved in the induction and progression of numerous types of human malignancies such as lung, breast, colon, prostate and cervix cancers. However, the role of PIWIL2 was poorly investigated in thyroid cancers. Accordingly, aim of the present study was to elucidate the relationship between PIWIL2 and thyroid cancers. The expression level of PIWIL2 was determined by analyzing both protein and mRNA levels in papillary and micropapillary carcinoma tissues by using immunohistochemistry and real-time PCR methods, respectively. Immunohistochemical analysis of HBME-1, galectin-3 and CK-19 was also performed. Similar to other immune markers of HBME-1, galectin-3 and CK-19, protein expression levels of PIWIL2 was significantly up-regulated in both papillary and micropapillary thyroid cancers (p < 0.01). Moreover, consistent with protein expression levels, mRNA expression levels of PIWIL2 was elevated in both papillary and micropapillary thyroid cancer tissues. Yet, mRNA expression changes were statistically insignificant. In conclusion, results of the current study suggest that PIWIL2 can be involved in thyroid cancer tumorigenesis and can be used as a novel predictive biomarker and/or therapeutic target., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Rho-associated protein kinase 1 (ROCK1), a serine/threonine kinase, has previously been shown to be over-expressed in various types of human malignant tumors and to play an important role in cancer development and progression. Although ROCK1 has gained growing prominence as an important protein kinase in cancer biology, its potential as a predictive biomarker and a therapeutic target in papillary thyroid carcinoma (PTC) remains unknown. In the present study, ROCK1 expression was examined in 356 formalin-fixed, paraffin-embedded papillary thyroid carcinoma tissues using immunohistochemistry, and its clinical implications and prognostic significance were analyzed. Our results showed that ROCK1 expression was significantly increased in PTC compared with normal tissues, and was significantly associated with tumor size, lymphatic metastasis, distant organ metastasis, extrathyroid invasion, vascular invasion and tumor, node and metastasis (TNM) stage. Patients with strong ROCK1 expression had lower overall survival, disease-free survival, lymph node recurrence-free survival and distant recurrence-free survival rates than those with weak expression. Furthermore, overexpression of ROCK1 in papillary thyroid carcinoma cells was found to increase their invasiveness. Silencing ROCK1 by siRNA, however, caused an inhibition of cell invasion. Knockdown of ROCK1 decreased the volume and weight of the xenograft tumors, while overexpression of ROCK1 showed a proliferative tendency with significantly greater tumor volume and weight in vivo. Moreover, the upregulation of ROCK1 increased the expression of MMP-9, and levels of MMP-9 positively correlated with the ROCK1 levels in PTC tissues, implicating that MMP-9 may be involved in the mechanism of ROCK1 in the development and progression of PTC. These data suggest that ROCK1 might be a potential prognostic marker and therapeutic target for the treatment of PTC.

Tumor stroma-secreted growth factors, cytokines, and reactive oxygen species (ROS) influence tumor development from early stages to the metastasis phase. Previous studies have demonstrated downregulation of ROS-producing extracellular superoxide dismutase (SOD3) in thyroid cancer cell lines although according to recent data, the expression of SOD3 at physiological levels stimulates normal and cancer cell proliferation. Therefore, to analyze the expression of SOD3 in tumor stroma, we characterized stromal cells from the thyroid. We report mutually exclusive desmoplasia and inflammation in papillary and follicular thyroid cancers and the presence of multipotent mesenchymal stem/stromal cells (MSCs) in non-carcinogenic thyroids and papillary thyroid cancer (PTC). The phenotypic and differentiation characteristics of Thyroid MSCs and PTC MSCs were comparable with bone marrow MSCs. A molecular level analysis showed increased FIBROBLAST ACTIVATING PROTEIN, COLLAGEN 1 TYPE A1, TENASCIN, and SOD3 expression in PTC MSCs compared to Thyroid MSCs, suggesting the presence of MSCs with a fibrotic fingerprint in papillary thyroid cancer tumors and the autocrine-paracrine conversion of SOD3 expression, which was enhanced by cancer cells. Stromal SOD3 had a stimulatory effect on cancer cell growth and an inhibitory effect on cancer cell migration, thus indicating that SOD3 might be a novel player in thyroid tumor stroma.

Multicellular three-dimensional (3D) spheroids represent an experimental model that is intermediate in its complexity between monolayer cultures and patients' tumor. In the present study, we characterize multicellular spheroids from papillary (PTC) and follicular (FTC) thyroid cancers and from the corresponding normal tissues. We show that these 3D structures well recapitulate the features of the original tissues, in either the differentiated and "stem-like" components. As a second step, we were aimed to test the effects of a small multikinase inhibitor, SP600125 (SP), previously shown to efficiently induce cell death in undifferentiated thyroid cancer monolayer cultures. We demonstrate the potent effect of SP on cell growth and survival in our 3D multicellular cultures. SP exerts its main effects through direct and highly significant inhibition of the ROCK pathway, known to be involved in the regulation of cell migration and β-catenin turnover. Consistently, SP treatment resulted in a significant decrease in β-catenin levels with respect to basal conditions in tumor but not in normal spheroids, indicating that the effect is promisingly selective on tumor cells.In conclusion, we provide the morphological and molecular characterization of thyroid normal and tumor spheroids. In this 3D model we tested in vitro the effects of the multikinase inhibitor SP and further characterized its mechanism of action in both normal and tumor spheroids, thus making it an ideal candidate for developing new drugs against thyroid cancer.

High-grade papillary urothelial carcinoma with subepithelial connective tissue invasion (T1HG) is an aggressive disease at high risk of progression after transurethral resection/Bacillus Calmette-Guerin standardized therapy. The European Organization for Research and Treatment of Cancer has identified T1HG bladder carcinoma that is single and ≤3 cm in the largest dimension at first diagnosis as a category in which the prognosis cannot be further stratified based on conventional criteria. This category may benefit from biomarker analysis as a valuable tool to determine the patient's outcome. To further the issue of biomarkers in predicting aggressiveness in single T1HG bladder carcinoma ≤3 cm in greatest dimension at first diagnosis, we have conducted a validation study of the biomarker risk score set previously reported by our group. The study set included immunohistochemical detection of galectin-3, CD44, E-cadherin (E-CAD), CD138, p16, survivin, HYAL-1, and topoisomerase-IIα in 92 randomly selected specimens at participating institutions. Topoisomerase-IIα expression was identified as a predictor of disease-free survival. p16, survivin, and E-CAD expression predicted progression-free survival, but p16 and E-CAD also predicted overall survival. The current study validates a panel of immunohistochemical markers with the potential of being implemented in practice and supports the use of biomarkers in predicting aggressiveness in patients with first diagnosis of single T1HG bladder carcinoma ≤3 cm in greatest dimension and therefore in identifying patients who need closer surveillance or earlier aggressive treatment., (Copyright © 2016 Elsevier Inc. All rights reserved.)

We studied the expression and activation of the main effector protein kinase of phosphatidylinositol-3-kinase cascade (PI3K) – Akt in conventionally normal tissues, benign and highly differentiated (with and without metastases) human thyroid tumors. There was a difference in the Akt1 amount in tumor tissue compared with normal tissue in papillary carcinomas and tissue of multinodular goiter. Akt expression both in tumor and conventionally normal tissues of follicular adenoma was significantly lower than in follicular carcinoma. The lowest level of Akt expression was observed in tissues of multinodular goiter. Total activity of all three isoforms of Akt1/2/3 was lower in tumors compared to conventionally normal tissue. Thus, Akt activity (according to Thr308 phosphorylation) is not associated with proliferative processes in the tumor tissue of the thyroid. Apoptosis level detected in these tissues was not associated with the protein kinase activity either. Possible mechanisms of signaling cascade PI3K/Akt inhibition in thyroid tumors are discussed.

Neutrophil gelatinase-associated lipocalin (NGAL), matrix metalloproteinase (MMP)-9, and NGAL/MMP-9 complex have been evaluated as diagnostic markers of several cancers, but results for bladder cancer are scanty. We evaluated these proteins in urine and serum of 89 patients with histologically confirmed bladder cancer and 119 cancer-free controls from a case-control study. Urinary concentrations were standardized on creatinine level. The performance of these proteins as cancer biomarkers was evaluated through the receiver operating characteristic (ROC) analysis. Urinary level of NGAL, MMP-9, and NGAL/MMP-9 complex was higher in current smokers, whereas no impact of dietary habits was observed. After adjusting for tobacco smoking, urinary concentration of MMP-9 was independently associated with cancer invasiveness, grading, and histological subtype, with elevated concentrations among T2-T4 and non-papillary bladder cancers. Conversely, NGAL and NGAL/MMP-9 complex were significantly higher in non-papillary than in papillary subtype. The pattern was less clear in serum, but correlation between urinary and serum concentration was poor, especially for Ta/is-T1 tumors. The ROC analysis confirmed that MMP-9 was the best marker (area under the ROC curve (AUC) = 0.68). Performances were much greater for muscle-invasive bladder cancers (AUC = 0.90), with elevated negative predictive values (97 %). The present study suggests that NGAL/MMP-9 pathway is associated with an aggressive phenotype of bladder cancer. The elevated negative predictive value of MMP-9 and NGAL/MMP-9 complex makes them candidate markers of exclusion test for bladder cancer. These proteins may be integrated in the surveillance of bladder cancer, thus diminishing patients' discomfort and improving compliance.

MicroRNA-29a (miR-29a) has been reported to play important roles in tumor initiation, development, and metastasis in various cancers. However, the biological function and potential mechanisms of miR-29a in papillary thyroid carcinoma (PTC) remain unclear. In the present study, we discovered that miR-29a was frequently downregulated in PTC tissues, and its expression was significantly associated with tumor size, TNM stage, and lymph node metastasis. Functional assays showed that overexpression of miR-29a markedly suppressed PTC cell proliferation, migration, and invasion and promoted PTC apoptosis and cell cycle arrest at G0/G1 phase. In vivo, miR-29a overexpression decreased tumor growth in a xenograft mouse model. Luciferase reporter assay showed that miR-29a can directly bind to the 3' untranslated region (UTR) of AKT3 in PTC cells. Overexpreesion of miR‑29a obviously decreased AKT3 expression, thereby suppressing phosphatidylinositol 3-kinase (PI3K)/AKT pathway activation. We also confirmed that AKT3 expression was increased in PTC tissue and was inversely correlated miR-29a expression in PTC tissues. In addition, downregulation of AKT3 by siRNA mimicked the effects of miR-29a overexpression, and upregulation of AKT3 partially reversed the inhibitory effects of miR-29a. These results suggested that miR-29a could act as a tumor suppressor in PTC by targeting AKT3 and that miR-29a may potentially serve as an anti-tumor agent in the treatment of PTC.

The study of the activity of the constitutive form of nitric oxide synthase (cNOS) revealed that in the papillary thyroid carcinomas it corresponded to that detected in unchanged extratumoral tissue, while the enzyme activity in follicular carcinoma was half lesser. At the same time, the activity of inducible nitric oxide synthase (іNOS) was higher in the papillary and follicular carcinomas. Such changes in the enzyme activity were associated with an increase in its level in papillary carcinomas, and with minor changes in follicular carcinomas. In medullary carcinomas the parameters under study corresponded to those in unchanged tissue, and in the papillary carcinoma metastases without changes in enzyme activity of nitric oxide formation, the level of the latter was much higher. Elevated levels of nitric oxide and іNOS activity in papillary thyroid carcinomas did not depend significantly on the aggression characteristics of the latter, being however absent in tumors of T4 category on a background of reduced cNOS activity and less expressed in tumors surrounded by the tissue in the presence of a chronic thyroiditis. Furthermore, in the papillary carcinomas of papillary or follicular structure nitric oxide level did not differ from the normal range, being slightly higher in tumors of solid or heterogeneous structure with presence of solid areas, whereas in carcinomas of papillary-follicular structure it was twice, and in tissue of solidinsular structure three times higher. іNOS hyperactivity was observed in the carcinomas of different structure, except for tumors of solid structure, in the tumor of which enzyme activity was within the normal range, and in tumor of solid-insular structure where it was significantly higher (as well as cNOS activity) compared with tumors of other structure. Nitric oxide generating system is involved in the transformation of thyroid cells and progression of tumor growth, including through apoptosis regulation, as shown by the results of an analysis of data obtained both in the present study and previously. The nature of such involvement in papillary thyroid carcinomas with different histological structure is different. Key words: nitric oxide; constitutive and inducible nitric oxide synthase; thyroid carcinoma; apoptosis.

Effective adverse event (AE) management is critical to maintaining patients on anticancer therapies. The DECISION trial was a multicenter, randomized, double-blind, placebo-controlled, Phase 3 trial which investigated sorafenib for treatment of progressive, advanced, or metastatic radioactive iodine-refractory, differentiated thyroid carcinoma. Four hundred and seventeen adult patients were randomized (1:1) to receive oral sorafenib (400 mg, twice daily) or placebo, until progression, unacceptable toxicity, noncompliance, or withdrawal. Progression-free survival, the primary endpoint of DECISION, was reported previously. To elucidate the patterns and management of AEs in sorafenib-treated patients in the DECISION trial, this report describes detailed, by-treatment-cycle analyses of the incidence, prevalence, and severity of hand-foot skin reaction (HFSR), rash/desquamation, hypertension, diarrhea, fatigue, weight loss, increased serum thyroid stimulating hormone, and hypocalcemia, as well as the interventions used to manage these AEs. By-cycle incidence of the above-selected AEs with sorafenib was generally highest in cycle 1 or 2 then decreased. AE prevalence generally increased over cycles 2-6 then stabilized or declined. Among these AEs, only weight loss tended to increase in severity (from grade 1 to 2) over time; severity of HFSR and rash/desquamation declined over time. AEs were mostly grade 1 or 2, and were generally managed with dose interruptions/reductions, and concomitant medications (e.g. antidiarrheals, antihypertensives, dermatologic preparations). Most dose interruptions/reductions occurred in early cycles. In conclusion, AEs with sorafenib in DECISION were typically grade 1 or 2, occurred early during the treatment course, and were manageable over time., (© 2015 The authors.)

Warthin-Like tumor of the thyroid is a recently described rare variant of papillary thyroid cancer. The distinct histological feature of this variant is papillary architecture lining oncocytic epithelial cells with nuclear characteristics of papillary carcinoma, accompanied by prominent lymphocytic infiltration in the papillary stalks. Here, we present a case of occult Warthin-like papillary thyroid carcinoma, 0.5-cm in maximum dimension, underwent left thyroid lobectomy in a 65 years old Chinese woman. In this case, there was no extrathyroid extension, vascular invasion and lymphatic metastasis, as well as no complication of lymphocytic thyroiditis. Immunohistochemistry staining revealed that the tumor cells were positive for Leu-M1, HBME-1, 34βE12, and MIB-1 labeling index was low. RET/PTC expression was absent in tumor cells. Furthermore, activated point mutations of BRAF V600E and V600K were concurrently detected by DNA sequencing. Further studies are needed to elucidate the prevalence and role of BRAF(V600K) mutation in papillary thyroid carcinoma, and long-term follow-up for the patient is needed to clarify the biological behavior of this variant with dual BRAF mutations.

Objective: The aim of this study was to evaluate paraoxonase-1 (PON1) activities and oxidative stress status, and the changes in their levels after total thyroidectomy in patients with papillary thyroid cancer (PTC)., Materials and Methods: Twenty-five patients with PTC and 27 healthy controls were enrolled in the study. Blood samples were obtained from the PTC patients before and 3 months after the operation. Preoperative and postoperative serum samples from PTC patients and healthy controls were analyzed for paraoxonase (PON), arylesterase (ARE) activities, and lipid hydroperoxide (LOOH) and -SH (total free sulfhydryl) levels., Results: The preoperative PON, ARE and -SH levels of the patients with PTC were significantly lower compared to those of the control group (p = 0.033, p < 0.001, p = 0.002, respectively), while LOOH levels were significantly higher (p < 0.001). The levels of PON and ARE decreased significantly in patients with PTC after the operation (p = 0.038, p = 0.023, respectively), while LOOH and -SH levels remained unchanged (p = 0.117, p = 0.487, respectively). PON and ARE levels showed a positive correlation with -SH (r = 0.211, p = 0.065; r = 0.471, p < 0.001, respectively) and a negative correlation with LOOH (r = - 0.391, p < 0.001, r = - 0.486, p < 0.001, respectively)., Conclusion: Serum PON1 activity is decreased in patients with PTC, and serum PON1 is positively correlated with -SH, a well-known antioxidant, and negatively correlated with LOOH, an oxidant. PON1 activity is significantly decreased after total thyroidectomy.

Background: Indoleamine 2,3-dioxygenase (IDO) is overexpressed in many different types of tumor and is associated with activation of FOXP3+ regulatory T cells (Treg cells) and downregulation of cytotoxic cellular immunity in the tumor microenvironment. It has been suggested that IDO inhibitors can be utilized as an effective therapeutic agent against human cancers. However, the expression of IDO and its association with tumor-infiltrating lymphocytes (TILs) remain unclear in papillary thyroid microcarcinoma (PTMC)., Methods: Immunohistochemical staining for IDO expression was performed on 124 PTMC samples. TIL subsets (CD3+, CD8+, and FOXP3+ T cells) were counted in serial sections. The relationships between the expression of IDO and infiltration of TIL subsets, as well as the relationships between these immunomodulating factors and clinicopathologic parameters of PTMCs, were analyzed., Results: There was a significant correlation between IDO expression and reduced CD3+ TIL and increased FOXP3+ TIL. IDO expression was found in 31% of PTMC and was associated with aggressive clinicopathologic features of the tumor such as extrathyroidal extension (ETE) and multifocality. High infiltration of FOXP3+ Treg cells in the tumor was associated with lymph node metastasis, ETE, and multifocality. Furthermore, high FOXP3/CD8+ ratio was associated with multifocality and lymph node metastasis, and high FOXP3+/CD3+ ratio was related to ETE and multifocality. In multivariate analyses, IDO expression was found to be an independent predictive factor for ETE and tumor multifocality., Conclusions: IDO expression and infiltration of FOXP3+ Treg cells were closely related to each other and were associated with aggressive features of PTMC, suggesting that disruption of antitumor immunity by IDO expression, and thus, infiltration of FOXP3+ Treg cells may contribute to tumor progression in PTMC.

Background: The preoperative characterization of thyroid nodules is a challenge for the clinicians. Fine-needle aspiration (FNA) is the commonly used pre-operative technique for diagnosis of malignant thyroid tumor. However, many benign lesions, with indeterminate diagnosis following FNA, are referred to surgery. There is an urgent need to identify biomarkers that could be used with the FNA to distinguish benign thyroid nodules from malignant tumors. The purpose of the study is to examine the level of expression of the helicase-like transcription factor (HLTF) in relation to neoplastic progression of thyroid carcinomas., Methods: The presence of HLTF was investigated using quantitative and semi-quantitative immunohistochemistry in a series of 149 thyroid lesion specimens. Our first clinical series was composed of 80 patients, including 20 patients presenting thyroid adenoma, 40 patients presenting thyroid papillary carcinoma, 12 patients presenting thyroid follicular carcinoma and 8 patients presenting anaplastic carcinoma. These specimens were assessed quantitatively using computer assisted microscopy. Our initial results were validated on a second clinical series composed of 40 benign thyroid lesions and 29 malignant thyroid lesions using a semi-quantitative approach. Finally, the HLTF protein expression was investigated by Western blotting in four thyroid cancer cell lines., Results: The decrease of HLTF staining was statistically significant during thyroid tumor progression in terms of both the percentage of mean optical density (MOD), which corresponds to the mean staining intensity (Kruskall-Wallis: p < 0.0005), and the labelling index (LI), which corresponds to the percentage of immunopositive cells (Kruskall-Wallis: p < 10-6). Adenomas presented very pronounced nuclear HLTF immunostaining, whereas papillary carcinomas exhibited HLTF only in the cytoplasm. The number of HLTF positive nuclei was clearly higher in the adenomas group (30%) than in the papillary carcinomas group (5%).The 115-kDa full size HLTF protein was immunodetected in four studied thyroid cancer cell lines. Moreover, three truncated HLTF forms (95-kDa, 80-kDa and 70-kDa) were also found in these tumor cells., Conclusions: This study reveals an association between HLTF expression level and thyroid neoplastic progression. Nuclear HLTF immunostaining could be used with FNA in an attempt to better distinguish benign thyroid nodules from malignant tumors.

Objective: 1α, 25(OH)2 D3 (calcitriol), the active form of vitamin D, has been shown to exert antiproliferative effects in many cancers. Overexpression of CYP24A1, the primary vitamin D-inactivating enzyme, is also observed in a variety of human cancers, thus potentially neutralizing the antitumour effect of 1α, 25(OH)2 D3. This study investigates the expression of CYP24A1 and the effect of BRAF(V600E) on its expression in thyroid cancer., Methods: We investigated 60 papillary thyroid carcinoma (PTC) specimens for CYP24A1 expression and its association with BRAF mutation and disease progression. CYP24A1 expression was measured by real-time RT-PCR, and BRAF(V600E) mutation was detected by PCR-DNA sequencing analysis. The interaction between BRAF(V600E) and CYP24A1 expression was determined by Western blot analysis and real-time RT-PCR., Results: CYP24A1 expression was increased in PTC as compared to benign multinodular goitre. The expression was further increased in stage III and IV tumours. There is a strong correlation between CYP24A1 overexpression and BRAF(V600E) mutation (P < 0·01). In thyroid cancer cell lines expressing BRAF(V600E) , CYP24A1 expression was significantly higher when compared to those without BRAF(V600E) expression. BRAF(V600E) transgene expression in CAL62 cell line can induce CYP24A1 expression. Furthermore, BRAF(V600E) inhibitor PLX4720 can significantly down-regulate CYP24A1 expression and enhance the antiproliferative effects of calcitriol in thyroid cancer cell lines., Conclusion: CYP24A1 overexpression is a poor prognostic indicator for PTC and may reflect BRAF(V600E) mutation and MARK activation. The crosstalk between vitamin D and MAPK signalling pathways results in resistance to calcitriol-mediated antitumour effects, and the resistance can be reversed by BRAF(V600E) inhibitor PLX4720., (© 2014 John Wiley & Sons Ltd.)

Approximately 90% of non-medullary thyroid malignancies originate from the follicular cell and are classified as papillary or follicular (well-differentiated) thyroid carcinomas, showing an overall favourable prognosis. However, recurrence or persistence of the disease occurs in some cases associated with the presence of loco-regional or distant metastatic lesions that generally become resistant to radioiodine therapy, while glucose uptake and metabolism are increased. Recent advances in the field of tumor progression have shown that CTC (circulating tumour cells) are metabolic and genetically heterogeneous. There is now special interest in unravelling the mechanisms that allow the reminiscence of dormant tumour lesions that might be related to late disease progression and increased risk of recurrence. AMPK (AMP-activated protein kinase) is activated by the depletion in cellular energy levels and allows adaptive changes in cell metabolism that are fundamental for cell survival in a stressful environment; nevertheless, the activation of this kinase also decreases cell proliferation rate and induces tumour cell apoptosis. In the thyroid field, AMPK emerged as a novel important intracellular pathway, since it regulates both iodide and glucose uptakes in normal thyroid cells. Furthermore, it has recently been demonstrated that the AMPK pathway is highly activated in papillary thyroid carcinomas, although the clinical significance of these findings remains elusive. Herein we review the current knowledge about the role of AMPK activation in thyroid physiology and pathophysiology, with special focus on thyroid cancer., (© 2014 The Author(s).)

Objective: To evaluate c-erbB2 gene amplification in a series of primary uterine serous carcinoma (USC) cell lines. To assess the efficacy of AZD8055, a novel dual mTORC1/2 inhibitor against primary HER2/neu amplified vs HER2/neu not amplified USC cell lines., Methods: Twenty-two primary USC cell lines were evaluated for c-erbB2 oncogene amplification by FISH assays. In vitro sensitivity to AZD8055 was evaluated by flow-cytometry-based viability and proliferation assays. Cell cycle profile and downstream cellular responses to AZD8055 were assessed by measuring the DNA content of cells and by phosphorylation of the S6 protein by flow-cytometry., Results: Nine of 22 (40.9%) USC cell lines demonstrated c-erbB2 gene amplification by FISH. AZD8055 caused a strong differential growth inhibition in USC cell lines, with high HER-2/neu-expressors demonstrating significantly higher sensitivity when compared to low HER-2/neu-expressors (AZD-8055 IC50 mean±SEM=0.27±0.05μM in c-erbB2 amplified versus 1.67±0.68μM in c-erbB2 not amplified tumors, P=0.03). AZD8055 growth-inhibition was associated with a significant and dose-dependent increase in the percentage of cells blocked in the G0/G1 cell cycle phase and a dose-dependent decline in pS6 levels in both c-erbB2 amplified vs c-erbB2 not amplified USC cell lines., Conclusions: AZD8055 may represent a novel targeted therapeutic agent in patients harboring advanced/recurrent/refractory USC. c-erbB2 gene amplification may represent a biomarker to identify USC patients who may benefit most from the use of AZD8055., (© 2013.)

Napsin A is a sensitive and specific marker for pulmonary adenocarcinoma versus squamous cell carcinoma. However, studies have shown that napsin A is also positive in approximately 5% of papillary thyroid carcinomas. The prevalence of napsin A in more aggressive types of thyroid carcinoma is unknown. Napsin A positivity in metastatic thyroid carcinoma, especially in conjunction with thyroid transcription factor-1 (TTF-1), could be misdiagnosed as lung adenocarcinoma. We investigated napsin A, TTF-1, and PAX8 expression in 26 anaplastic, 16 poorly differentiated, and 2 micropapillary pattern thyroid carcinomas. A focal micropapillary component was also present in 3 poorly differentiated and 3 anaplastic thyroid carcinomas. Four of 26 (15%) anaplastic, 2/16 (13%) poorly differentiated, and 2/2 (100%) micropapillary pattern thyroid carcinomas were napsin A positive. Three of the 6 cases (50%) with a focal micropapillary component were napsin A positive (1 of these 3 cases was positive only in the micropapillary component). All napsin A-positive cases were also positive for TTF-1, and all but 1 micropapillary pattern carcinoma were also PAX8 positive. In 1 case, napsin A was positive in the micropapillary component, but PAX8 was only positive in the adjacent poorly differentiated carcinoma. In summary, a minority of anaplastic and poorly differentiated thyroid carcinomas are napsin A positive. More importantly, napsin A expression is more common in carcinomas with a micropapillary component, a pattern shared in common with some lung adenocarcinomas. PAX8 may be diagnostically useful to distinguish these napsin A-positive thyroid carcinomas from lung adenocarcinomas, which are PAX8 negative.

Background: The objective of this study was to evaluate the role of autotaxin (ATX) activity and gene expression compared to soluble intercellular adhesion molecule-1 (sICAM-1) in thyroid carcinoma.
, Patients and Methods: Sixty-five patients with thyroid swelling were included. There were 20 cases of simple multinodular goiter (group I), 15 cases of follicular adenoma (group II) and 30 cases of thyroid cancer (group III). Group III was further subdivided into negative and positive lymph nodes (group IIIa and IIIb; 22 and 8 cases, respectively). sICAM-1 concentration and ATX activity were measured using colorimetric enzyme-linked immunosorbent assay (ELISA), while ATX gene expression was detected by real-time polymerase chain reaction (PCR).
, Results: sICAM-1 level, ATX activity and gene expression were significantly elevated in patients with thyroid carcinoma compared to other groups. The ATX activity showed significantly higher sensitivity and specificity than sICAM-1 (100% and 97.1% vs 93.3% and 88.6%, respectively). Both sICAM-1 and ATX values were significantly higher in patients with positive lymph nodes compared to those without lymph node involvement (p<0.001). Higher levels of ATX activity and gene expression were significantly correlated with larger tumor size and undifferentiated pathological subtype in thyroid carcinoma. In this respect, ATX was superior to sICAM-1.
, Conclusion: Our data suggest that ATX activity and gene expression are reliable diagnostic and prognostic tools in thyroid carcinoma compared to sICAM-1.

Purpose: To investigate the PTEN and p53 gene expression in endometrioid and serous papillary endometrial carcinomas and clarify their prognostic significance by studying the PTEN and p53 expression in relation to tumor stage and grade., Methods: Archival pathological sections of 61 cases with endometrial cancer examined in a 5-year-period (January 2006-December 2010) were retrieved and re-examined. Immunohistochemical investigation was performed by the Ventana system. Anti-PTEN and anti-p53 monoclonal antibodies were used. Disease staging was made according to the FIGO staging system., Results: Forty-nine (80.32%) cases were endometrioid adenocarcinomas. Patient age ranged from 39-75 years (mean 62.5). Grade 1 tumors:19/22 (86.3%) cases had stage Ib, 2/22 (9.09%) stage Ic and 1/22 (4.54%) stage IIIc. Eighteen of 22 (81.8%) cases were PTEN positive and 4/22 (18.2%) p53 positive. Grade 2 tumors: 17/ 23 (73.91%) cases had stage I b, 4/23 (17.39%) stage Ic and 2/23 (8.69%) stage IIIc. Seventeen of 23 (73.91%) cases were PTEN positive and 47sol;23 (17.3%) p53 positive. Grade 3 tumors: 2/4 (50%) cases had stage Ic and 2/4 (50%) stage IIIc. No case was PTEN positive and 2/4 (50%) were p53 positive. Twelve (19.35%) cases were serous papillary carcinomas. Patient age ranged from 63-79 years (mean 76). Five (41.66%) cases had stage Ic and 5 (41.66%) stage IIIc, with nodal metastases and peritoneal involvement. Two (16.66%) cases developed on endometrial polyps with minimal myometrial involvement (stage Ib) and in both cases elements of endometrioid adenocarcinoma were observed as well. Immunohistochemical study showed that 11 (91.66%) cases were p53 positive and 2 (16.66%) PTEN positive., Conclusion: PTEN and p53 immunoexpression helps both in accurate diagnosis and proper therapeutic approach of the various endometrial carcinomas. PTEN and p53 are also prognostic markers for these kind of tumors.

The study analyzes the immunoexpression of EGFR and Her2/neu in 45 cases of papillary urothelial bladder carcinoma. The cases have been investigated through histopathological and immunohistochemistry techniques, the quantification of results considering the number of marked cells and intensity of the reactions. Immunoexpression of EGFR has been observed in 53.3% of cases, the reaction score having high values in moderate carcinomas and poorly differentiated carcinomas with invasion in the muscularis propria or in the entire wall. Immunostain of Her2/neu has been positive in 42.2% of cases, the poorly differentiated carcinomas presenting high scores regardless of tumor stage. The analyzed markers proved useful for identifying urothelial papillary carcinomas with a poorly differentiated grade, which are in an advanced stage.

Background: Papillary thyroid carcinoma (PTC), follicular thyroid adenoma (FTA), and thyroid nodular hyperplasia (TNH) are the most frequent diseases of the thyroid gland. Previous studies described the involvement of dipeptidyl-peptidase IV (DPPIV/CD26) in the development of thyroid neoplasia and proposed it as an additional tool in the diagnosis/prognosis of these diseases. However, very little is known about the involvement of other peptidases in neoplastic and hyperplastic processes of this gland., Methods: The catalytic activity of 10 peptidases in a series of 30 PTC, 10 FTA, and 14 TNH was measured fluorimetrically in tumour and nontumour adjacent tissues., Results: The activity of DPPIV/CD26 was markedly higher in PTC than in FTA, TNH, and nontumour tissues. Aspartyl aminopeptidase (AspAP), alanyl aminopeptidase (AlaAP), prolyl endopeptidase, pyroglutamyl peptidase I, and aminopeptidase B activities were significantly increased in thyroid neoplasms when compared to nontumour tissues. AspAP and AlaAP activities were also significantly higher in PTC than in FTA and TNH., Conclusions: These data suggest the involvement of DPPIV/CD26 and some cytosolic peptidases in the neoplastic development of PTC and FTA. Further studies will help to define the possible clinical usefulness of AlaAP and AspAP in the diagnosis/prognosis of thyroid neoplasms.

Background: A new modality is necessary to prevent recurrence of superficial bladder cancer after complete transurethral resection because of the high recurrence rate even with current prophylaxis protocols., Methods: In order to analyze the predictive value of cyclooxygenase-2 (COX-2) expression and tumor infiltrating lymphocytes (TILs) in recurrence of this disease tumor specimens from 127 patients with solitary papillary non-muscle invasive bladder cancer (NMIBC), 78 with recurrent disease and 49 without recurrence during follow up of minimum 5 years, were retrieved for tissue microarrays construction and immunohistochemical analysis. COX-2 expression was scored according to Allred's scoring protocol, while presence of TILs was categorized as absent (no) or present (yes) on whole tissue sections., Results: COX-2 immunoreactivity was presented in 70 (71%), weak in 16% and strong in 55% of cases, while 29 (29%) tumors were negative. TILs were present in 64 (58%) NMIBC, while 44 cases (41%) did not reveal mononuclear infiltration in tumoral stroma. Statistical analysis demonstrated a higher proportion of patients with recurrence in the group with the COX-2 score 0, and lower in the group with score 2 (p=0.0001, p=0.0101, respectively). In addition, a higher proportion of recurrent patients in the group with no TILs, and lower proportion in the group with TILs were found (p=0.009, p=0.009, respectively). Univariate and multivariate analysis revealed overexpression of COX-2 and presence of TILs as negative predictors., Conclusion: Patients with lower COX-2 expression and absence of TILs in NMIBC need to be followed up more vigorously and probably selected for adjuvant therapy., Virtual Slide: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1411318819790406.

The incidence of thyroid cancer has remarkably increased in recent years. Epidemiologic data suggest that obesity is associated with an increased incidence of several types of malignancies, including thyroid cancer. Leptin, an adipocyte-derived cytokine, has been shown to be involved in cancer development and progression. We previously demonstrated that papillary thyroid cancer expressing leptin receptor and/or leptin has a higher incidence of lymph node metastasis. In this study, we investigated the effects of leptin on cell migration in K1 and B-CPAP papillary thyroid cancer cells. Expression of leptin receptor was observed in both cell lines. Leptin enhanced the migratory activity significantly in a dose-dependent manner. We showed that leptin induced AKT and extracellular signal-regulated kinase (ERK) phosphorylation. Inhibition of phosphatidylinositol 3-kinase and ERK activation using pharmacological inhibitors effectively blocked leptin-induced migration of K1 and B-CPAP cells. Taken together, this study provides new mechanistic evidence for a role of leptin in the regulation of papillary thyroid cancer progression by stimulating tumor cell migration.

The expression of cyclooxygenase isoform 2 (COX-2) in canine nasal carcinomas has been well documented. COX-2 expression has proven to be a prognostic factor in several human tumours. The aims of this study were to assess the correlation between immunohistochemical COX-2 expression and prognosis using rhinoscopic biopsies from 42 dogs with nasal carcinomas treated with hypofractionated radiotherapy, and to establish a replicable COX-2 scoring system. Ninety per cent of sections evaluated were COX-2 positive with a mean score of 6.6 (median 8.0; range 0-12). Neither COX-2 expression nor tumour type had a significant correlation with survival. There are likely to be many as yet unidentified variants which contribute to length of survival in dogs with nasal carcinomas. Immunohistochemical COX-2 expression appears unlikely to be of prognostic significance for canine nasal carcinoma., (© 2010 Blackwell Publishing Ltd.)

Purpose: The role of the insulin-like growth factor (IGF) system in endometrial cancer has been well established. The IGF-I receptor (IGF-IR) emerged as a promising therapeutic target in a number of cancers. NVP-AEW541 (Novartis Pharma) is a pyrrolo(2,3-d)pyrimidine derivative with specific IGF-IR tyrosine kinase inhibitory activity. NVP-AEW541 has been shown to abrogate IGF-I-mediated IGF-IR autophosphorylation and to reduce activation of the IGF-IR signaling pathways. The aim of the present study was to investigate the anti-proliferative activity of NVP-AEW541 in Type I (endometrioid) and Type II (uterine serous papillary endometrial carcinoma, USPC) endometrial cancer cell lines., Methods: Type I (ECC-1, Ishikawa) and Type II (USPC-1, USPC-2) endometrial cancer cell lines were treated with NVP-AEW541 in the presence of IGF-I, and the following parameters were measured: IGF-IR, AKT and ERK phosphorylation, apoptosis, proliferation, cell cycle progression and IGF-IR internalization., Results: Results obtained showed that NVP-AEW541 abolished the IGF-I stimulated IGF-IR phosphorylation in all of the cell lines investigated, whereas it abolished AKT and ERK phosphorylation preferentially in ECC-1 and USPC-1 cells. Furthermore, the inhibitor prevented from IGF-I from exerting its antiapoptotic effect in ECC-1, USPC-1 and USPC-2 cells. In addition, proliferation assays showed that NVP-AEW541 caused a decrease in proliferation rate in all of the cell lines. NVP-AEW541 had no major effect on the insulin receptor., Conclusion: Our results suggest that specific IGF-IR inhibition by NVP-AEW541 might be a promising therapeutic tool in endometrial cancer., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: A 24-year-old woman presented with a 45 cm complex cystic renal mass, which was resected. The tumor was a type-2 papillary renal cell carcinoma (pRCC-2), and several nodules remained. The patient was treated with mammalian target of rapamycin complex 1 (mTORC1) inhibitors, but after 5 months the tumor had progressed. Genetic testing of the patient revealed a novel heterozygous germline mutation in the gene encoding fumarate hydratase (FH), an enzyme of the tricarboxylic acid (TCA) cycle. As the tumor exhibited loss of heterozygosity for FH and markedly reduced FH activity, and in the absence of other established therapies, treatment with the glycolytic inhibitor 2DG (2-deoxy-D-glucose) was explored., Investigations: CT, histology, immunohistochemistry, genetic studies, 2-deoxy-2-(¹⁸F)fluoro-D-glucose (¹⁸FDG)-PET/CT, FH enzymatic assays, reconstitution experiments and in vitro studies of the effects of 2DG on FH-deficient tumor cells., Diagnosis: pRCC-2 arising in a patient with a novel germline FH mutation and de novo hereditary leiomyomatosis and renal cell cancer (HLRCC) syndrome progressing after mTORC1 inhibitor therapy., Management: Surgical resection of the renal mass, treatment with mTORC1 inhibitors followed by 2DG. Unfortunately, 2DG was not effective, and the patient died several weeks later.

The human protein N(α)-terminal acetyltransferase A complex (hNatA), composed of the catalytic hNaa10p (hArd1) and auxiliary hNaa15p (hNat1/NATH/Tubedown) subunits, was reported to be important for cell survival and growth of various types of cancer. However, little is known about the mechanisms mediating growth inhibition and apoptosis following loss of hNatA function. Here, we have screened 11 different thyroid cell lines for hNAA10 RNAi phenotypes and observed mostly growth inhibition, which was independent of TP53 functional status and developed by several different mechanisms involving (i) downregulation of cyclin D1, (ii) increase in p27/Kip1 and (iii) inactivation of Rb/E2F pathway. hNatA depletion in aggressive thyroid cancer cell lines (8305C, CAL-62 and FTC-133) with mutated TP53 increased sensitivity to drug-induced cytotoxicity, but in a cell type specific manner: 8305C (TRAIL), CAL-62 (daunorubicin) and FTC-133 (troglitazone). Cells harboring wild-type TP53 were also prone to apoptosis via the p53 pathway after hNatA downregulation. Importantly, in hNatA-depleted cells DNA-damage signaling was activated in the absence of exogenous DNA damage independent on TP53 status. Our findings indicate that several mechanisms of growth inhibition and apoptosis may be induced by hNatA knockdown and that hNatA knockdown could be exploited for use in combinatorial chemotherapy., (Copyright © 2010 UICC.)

Focal Adhesion Kinase (FAK) and Src have been reported to regulate tumor growth, invasion, metastasis and angiogenesis. The present study aimed to evaluate by immunohistochemistry the clinical significance of FAK and Src expression in 108 patients with benign and malignant thyroid lesions. Total FAK expression provided a distinct discrimination between malignant and benign (p = 0.00001), as well as between papillary carcinoma and hyperplastic nodules thyroid lesions (p = 0.00005), being also associated with follicular cells' proliferative capacity (p = 0.0003). In malignant thyroid lesions, total FAK expression was associated with tumor size (p = 0.0455), and presence of capsular (p = 0.0102) and lymphatic (p = 0.0173) invasion. Total Src expression was borderline increased in cases of papillary carcinoma compared to hyperplastic nodules (p = 0.0993), being also correlated with tumor size (p = 0.0169). FAK and Src expression was ascribed to a significant extent to the phosphorylated forms of the enzymes, which provided a better discrimination between malignant and benign thyroid lesions. The current data revealed that FAK and to a lesser extent Src expression could be considered of clinical utility in thyroid neoplasia with potential use as therapeutic targets.

Carbonic anhydrase IX (CAIX), a hypoxia-induced protein, is expressed in some renal tumors. We evaluated its immunohistochemical expression in 317 primary and 42 metastatic renal neoplasms (186 clear cell, 52 papillary, 35 chromophobe, 47 unclassified, and 15 Xp11.2 translocation renal cell carcinomas [RCCs]; 26 oncocytomas; 2 metanephric adenomas; 1 urothelial carcinoma; 1 mixed epithelial and stromal tumor; and 1 angiomyolipoma); 7 neoplasms were unknown as to whether they were primary or metastatic. We also correlated expression with tumor type and grade. Variable staining was seen in clear cell, papillary, unclassified, and Xp11.2 translocation carcinomas. One chromophobe carcinoma had focal expression. No staining was seen with other tumors. An association was found between high expression and clear cell vs non-clear cell carcinomas with all cases (P < .01) and primary (P < .01) cases. An association between CAIX expression and grade (P < .01) in primary clear cell carcinomas was found. CAIX expression is more common in clear cell RCC than other renal tumor types and is associated with grade.

Thyroid carcinoma is the most common endocrine malignancy and the first cause of death among endocrine cancers. We show that the tumorigenic capacity in thyroid cancer is confined in a small subpopulation of stem-like cells with high aldehyde dehydrogenase (ALDH(high)) activity and unlimited replication potential. ALDH(high) cells can be expanded indefinitely in vitro as tumor spheres, which retain the tumorigenic potential upon delivery in immunocompromised mice. Orthotopic injection of minute numbers of thyroid cancer stem cells recapitulates the behavior of the parental tumor, including the aggressive metastatic features of undifferentiated thyroid carcinomas, which are sustained by constitutive activation of cMet and Akt in thyroid cancer stem cells. The identification of tumorigenic and metastagenic thyroid cancer cells may provide unprecedented preclinical tools for development and preclinical validation of novel targeted therapies., (©2010 AACR.)

Objective: To compare the in vitro sensitivity/resistance to patupilone versus paclitaxel in uterine serous papillary carcinoma (USPC) with high versus low HER-2/neu expression., Methods: Six primary USPC cell lines, half of which overexpress HER-2/neu at a 3+ level, were evaluated for growth rate and tested for their in vitro sensitivity/resistance to patupilone versus paclitaxel by MTS assays. Quantitative RT-PCR was used to identify potential mechanisms underlying the differential sensitivity/resistance to patupilone versus paclitaxel in primary USPC cell lines., Results: Cell lines overexpressing HER-2/neu showed higher proliferation when compared to low HER-2/neu-expressing cell lines. Compared to low-expressing cell lines, high HER-2/neu expressors were significantly more sensitive to patupilone than to paclitaxel (P<0.0002). In contrast, there was no appreciable difference in sensitivity to patupilone versus paclitaxel in primary USPC cell lines with low HER-2/neu expression. Higher levels of β-tubulin III (TUBB3) and P-glycoprotein (ABCB1) were detected in USPC cell lines with high versus low HER-2/neu expression (P<0.05)., Conclusions: USPC overexpressing HER-2/neu display greater in vitro sensitivity to patupilone and higher levels of the patupilone molecular target TUBB3 when compared to low HER-2/neu expressors. Due to the adverse prognosis associated with HER-2/neu overexpression in USPC patients, patupilone may represent a promising novel drug to combine to platinum compounds in this subset of aggressive endometrial tumors., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Although autophagy is generally considered a prosurvival mechanism that preserves viability, there is evidence that it could drive an alternative programmed cell death pathway in cells with defects in apoptosis. Because the inhibition of autophagic activity promotes resistance to both chemotherapy and external beam radiation in papillary thyroid cancer (PTC), we determined if RAD001, a potent activator of autophagy, improves the efficacy of either therapy. We found that RAD001 increased the expression level of light chain 3-II, a marker for autophagy, as well as autophagosome formation in cell lines and in human PTC ex vivo. RAD001 sensitized PTC to doxorubicin and external beam radiation in a synergistic fashion, suggesting that combination therapy could improve therapeutic response at less toxic concentrations. The effects of RAD001 were abrogated by RNAi knockdown of the autophagy-related gene 5, suggesting that RAD001 acts, in part, by enhancing autophagy. Because the synergistic activity of RAD001 with doxorubicin and external radiation suggests distinct and complementary mechanisms of action, we characterized how autophagy modulates signaling pathways in PTC. To do so, we performed kinome profiling and discovered that autophagic activation resulted in Src phosphorylation and Met dephosphorylation. Src inhibition did not reverse the effects of RAD001, whereas Met inhibition reversed the effects of autophagy blockade on chemosensitivity. These results suggest that the anticancer effects of autophagic activation are mediated largely through Met. We conclude that RAD001 induces autophagy, which enhances the therapeutic response to cytotoxic chemotherapy and external beam radiation in PTC., (© 2010 AACR.)

To evaluate the associations of phosphorylated c-Jun NH2-terminal kinase (p-JNK) expression with clinicopathological features in patients with papillary thyroid carcinoma, p-JNK expression were immunohistochemically measured in 121 thyroid samples. p-JNK was overexpressed in papillary thyroid carcinomas with respect to matched nontumorous tissues (P=0.000), which was supported by western blot analysis. Increased p-JNK expression was significantly associated with the presence of lymph node metastases (P=0.001) and advanced TNM stages (P=0.02). Furthermore, p-JNK expression was positively correlated with osteopontin (OPN) expression (r=0.58, P<0.001). Activation of p-JNK may play a role in the carcinogenesis and lymph node metastasis of papillary thyroid carcinoma, and may be a molecular target for therapeutic intervention., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

Unlabelled: The aim of this study was to evaluate the prognostic information of soluble carbonic anhydrase (CA) IX expression in renal cell carcinoma (RCC)., Patients and Methods: Serum CA IX was analysed in 361 patients. Tumour type, TNM stage, nuclear grade, and RCC-specific survival were assessed. Serum and immunohistochemical expression were compared., Results: Median serum CA IX expression was 141 (range 2-4, 181) pg/ml. Levels were significantly higher in 287 patients with clear cell, compared to 40 papillary (p<0.001) and 22 oncocytoma (p=0.002), but not to 12 chromophobe RCC (p=0.35). Serum CA IX in clear cell RCC was positively correlated to TNM stage (p=0.002). There was a positive trend between serum and immunohistochemical CA IX expression. In a multivariate analysis of clear cell RCC, TNM stage and nuclear grade were independent prognostic factors., Conclusion: Serum CA IX was higher in clear cell RCC compared to other RCC types. In clear cell RCC, serum CA IX correlated to TNM stage, but not survival.

Immunohistochemical detection of Cyclooxygenase (Cox)-1 and -2 enzymes in canine mammary tumours (CMT) has recently been described. However, the prognostic value of their expression needs to be established. The aim of this study was to investigate Cox (-1 and -2) prognostic value in malignant CMT by evaluating its correlation with clinicopathological parameters (tumour size, histological type, necrosis, lymph node metastasis) and with Disease Free Survival (DFS) and Overall Survival (OS). Twenty seven female dogs with malignant tumours were included. Cox-2 expression was associated with lymph node metastasis at surgery time, development of distant metastasis during follow-up (p=0.038), DFS (p=0.03) and OS (p=0.04). Multivariate survival analysis showed that Cox-2 did not retain its significance as an independent prognostic factor. For Cox-1 expression, no statistically significant association was observed. Present study suggests the usefulness of testing Cox-2 specific inhibitors as part of an adjuvant therapy in female dogs with malignant mammary neoplasias., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Objectives: We aimed to evaluate the oxidant/antioxidant status of thyroid tissue in patients with multinodular goiter, papillary carcinoma and to compare with their nonpathologic tissues., Methods: We studied 41 patients with multinodular goiter who underwent surgical treatment. The patients were divided into three groups according to clinical diagnosis. Malondialdehyde, selenium, total superoxide dismutase and glutathione peroxidase of thyroid tissue samples were determined in 14 toxic multinodular goiters, 18 non-toxic multinodular goiters, and 9 papillary carcinomas., Result: Superoxide dismutase and glutathione peroxidase and selenium were found lower but malondialdehyde was higher in both nodule and cancerous tissues compared with those of control ones. The level of malondialdehyde in non-toxic multinodular goiters group was higher than toxic multinodular goiters group in nodule tissues., Conclusions: It can be stated that the lipid peroxidation is increased and enzymatic free radical defense system was significantly impaired in patients with both multinodular goiters and papillary carcinomas., (2010 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Background: Renal cancer is one of the ten most common malignant tumours in humans and its histological classification, best clinical management and treatment strategies are continuously debated. Roughly 10% of renal carcinomas are papillary renal cell carcinomas (RCCs), a histologically well characterized tumour subtype that is linked to alterations on chromosomes 7 and 17. Peptidases are proteolytic enzymes known to be involved in oncological processes, although their precise role in renal cancer is poorly understood., Materials and Methods: Eighteen papillary RCCs were selected for the study. Tumour and normal tissue samples were frozen for enzymatic analysis. The catalytic activity for a pool of peptidases (EC numbers: 3.4.11.14; 3.4.11.2; 3.4.11.6; 3.4.11.21; 3.4.11.7; 3.4.14.5; 3.4.24.11; 3.4.21.26; 3.4.19.3) was measured fluorometrically., Results: Statistically significant decreases were observed in the following cell surface activities: EC.3.4.11.2 (six-fold decrease in tumour vs. non-tumour); 3.4.11.6 (five-fold decrease); 3.4.11.7 (eight-fold decrease); 3.4.24.11 (four-fold decrease). No significant alterations were observed in the soluble activities., Conclusion: These data confirm the involvement of cell-surface peptidases in the mechanisms underlying RCC aetiogenesis and suggest that the peptidase activity profile in the RCC may be a diagnostic/prognostic marker.

RET/papillary thyroid carcinoma (PTC), TRK-T, or activating mutations of Ras and BRaf are frequent genetic alterations in PTC, all leading to the activation of the extracellular-regulated kinase (Erk) cascade. The aim of this study was to investigate the role of calmodulin-dependent kinase II (CaMKII) in the signal transduction leading to Erk activation in PTC cells. In normal thyroid cells, CaMKII and Erk were in the inactive form in the absence of stimulation. In primary PTC cultures and in PTC cell lines harboring the oncogenes RET/PTC-1 or BRaf(V600E), CaMKII was active also in the absence of any stimulation. Inhibition of calmodulin or phospholipase C (PLC) attenuated the level of CaMKII activation. Expression of recombinant RET/PTC-3, BRaf(V600E), or Ras(V12) induced CaMKII activation. Inhibition of CaMKII attenuated Erk activation and DNA synthesis in thyroid papillary carcinoma (TPC-1), a cell line harboring RET/PTC-1, suggesting that CaMKII is a component of the Erk signal cascade in this cell line. In conclusion, PTCs contain an active PLC/Ca(2+)/calmodulin-dependent signal inducing constitutive activation of CaMKII. This kinase is activated by BRaf(V600E), oncogenic Ras, and by RET/PTC. CaMKII participates to the activation of the Erk pathway by oncogenic Ras and RET/PTC and contributes to their signal output, thus modulating tumor cell proliferation.

NADPH oxidase 4 (NOX4) belongs to the NOX family that generates reactive oxygen species (ROS). Function and tissue distribution of NOX4 have not yet been entirely clarified. To date, in the thyroid gland, only DUOX1/2 NOX systems have been described. NOX4 mRNA expression, as shown by real-time PCR, was present in normal thyroid tissue, regulated by TSH and significantly increased in differentiated cancer tissues. TSH increased the protein level of NOX4 in human thyroid primary culture and NOX4-dependent ROS generation. NOX4 immunostaining was detected in normal and pathologic thyroid tissues. In normal thyroid tissue, staining was heterogeneous and mostly found in activated columnar thyrocytes but absent in quiescent flat cells. Papillary and follicular thyroid carcinomas displayed more homogeneous staining. The p22(phox) protein that forms a heterodimeric enzyme complex with NOX4 displayed an identical cellular expression pattern and was also positively regulated by TSH. ROS may have various biological effects, depending on the site of production. Intracellular NOX4-p22(phox) localization suggests a role in cytoplasmic redox signaling, in contrast to the DUOX localization at the apical membrane that corresponds to an extracellular H(2)O(2) production. Increased NOX4-p22(phox) in cancer might be related to a higher proliferation rate and tumor progression but a role in the development of tumors has to be further studied and established in the future.

The phosphatidylinositol 3-kinase (PI3K) pathway is a critical signal transduction pathway that regulates multiple cellular functions. Aberrant activation of this pathway has been identified in a wide range of cancers. Several pathway components including AKT, PI3K and mTOR represent potential therapeutic targets and many small molecule inhibitors are in development or early clinical trials. The complex regulation of the pathway, together with the multiple mechanisms by which it can be activated, make this a highly challenging pathway to target. For successful inhibition, detailed molecular information on individual tumours will be required and it is already clear that different tumour types show distinct combinations of alterations. Recent results have identified alterations in pathway components PIK3CA, PTEN, AKT1 and TSC1 in bladder cancer, some of which are significantly related to tumour phenotype and clinical behaviour. Co-existence of alterations to several PI3K pathway genes in some bladder tumours indicates that these proteins may have functions that are not related solely to the known canonical pathway.

Structural chromosome aberrations are known hallmarks of many solid tumors. In the papillary form of thyroid cancer (PTC), for example, activation of the receptor tyrosine kinase (RTK) genes, ret or the neurotrophic tyrosine kinase receptor type I (NTRK1) by intra- or interchromosomal rearrangements have been suggested as a cause of the disease. The 1986 accident at the nuclear power plant in Chernobyl, Ukraine, led to the uncontrolled release of high levels of radioisotopes. Ten years later, the incidence of childhood papillary thyroid cancer (chPTC) near Chernobyl had risen by two orders of magnitude. Tumors removed from some of these patients showed aberrant expression of the ret RTK gene due to a ret/PTC1 or ret/PTC3 rearrangement involving chromosome 10. However, many cultured chPTC cells show a normal G-banded karyotype and no ret rearrangement. We hypothesize that the "ret-negative" tumors inappropriately express a different oncogene or have lost function of a tumor suppressor as a result of chromosomal rearrangements, and decided to apply molecular and cytogenetic methods to search for potentially oncogenic chromosomal rearrangements in Chernobyl chPTC cases. Knowledge of the kind of genetic alterations may facilitate the early detection and staging of chPTC as well as provide guidance for therapeutic intervention.