Metabolic Profiles and Blood Biomarkers to Discriminate between Benign Thyroid Nodules and Papillary Carcinoma, Based on UHPLC-QTOF-ESI+-MS Analysis.

In this study, serum metabolic profiling of patients diagnosed with papillary thyroid carcinoma (PTC) and benign thyroid pathologies (BT) aimed to identify specific biomarkers and altered pathways when compared with healthy controls (C). The blood was collected after a histological confirmation from PTC (n = 24) and BT patients (n = 31) in parallel with healthy controls (n = 81). The untargeted metabolomics protocol was applied by UHPLC-QTOF-ESI+-MS analysis and the statistical analysis was performed using the MetaboAnalyst 5.0 platform. The partial least squares-discrimination analysis, including VIP values, random forest graphs, and heatmaps (p < 0.05), was complemented with biomarker analysis (with AUROC ranking) and pathway analysis, suggesting a model for abnormal metabolic pathways in PTC and BT based on 166 identified metabolites. There were 11 classes of putative biomarkers selected that were involved in altered metabolic pathways, e.g., polar molecules (amino acids and glycolysis metabolites, purines and pyrimidines, and selenium complexes) and lipids including free fatty acids, bile acids, acylated carnitines, corticosteroids, prostaglandins, and phospholipids. Specific biomarkers of discrimination were identified in each class of metabolites and upregulated or downregulated comparative to controls, PTC group, and BT group. The lipidomic window was revealed to be more relevant for finding biomarkers related to thyroid carcinoma or benign thyroid nodules, since our study reflected a stronger involvement of lipids and selenium-related molecules in metabolic discrimination.

High iodine effects on the proliferation, apoptosis, and migration of papillary thyroid carcinoma cells as a result of autophagy induced by BRAF kinase.

Papillary thyroid carcinoma (PTC) is a common endocrine tumor. This study found that different iodine concentrations affected the proliferation, apoptosis, and migration of PTC. For this study, we collected clinical information from PTC patients and monitored the levels of urinary iodine, LC3-II, and caspase-3 in cancer tissue, and BRAF kinase in peripheral blood from PTC patients. We also monitored the proliferation, apoptosis and migration ability of human papillary-thyroid carcinoma (BCPAP) cells at different iodine concentrations and their association with changes in autophagy and BRAF kinase activity of BCPAP cells at high iodine levels (10-3 mol/l). We found that the proportion of tumor diameters ≥ 1 cm in the iodine excess group were lower than that in the iodine non-excess group. The proportion of PTC patients with infiltration in the iodine excess group was higher than that in the iodine non-excess group. Levels of the autophagy-related protein LC3-II and the apoptosis-related protein caspase-3 in cancer tissues, and activity of BRAF kinase in peripheral blood, were positively correlated with urinary iodine concentrations from PTC patients. At high iodine levels, the proliferation rate decreased, and apoptosis percentage and migration rates increased compared with the no-iodine group. At high iodine levels, the frequencies of autophagosomes (Aph) and autophagosome-lysosomes (Apl) in BCPAP cells increased significantly, and activities of LC3-II and BRAF kinase increased, respectively. The activity of LC3-II decreased when BRAF kinase was inhibited. The activity of LC3-II and the proliferation and migration rates of BCPAP cells decreased, and the apoptosis percentage increased when autophagy was inhibited at high iodine concentrations. Our results demonstrated that, in the presence of excessive iodine, the mean tumor size of PTC patients was smaller and easier to invade than tumors in patients not supplied with excessive iodine. The levels of autophagy and apoptosis in PTC cancer tissues, and activities of BRAF kinase in peripheral blood increased with increasing urinary iodine concentrations. High iodine levels inhibited cell proliferation and promoted apoptosis and migration of PTC cells. Autophagy induced by BRAF kinase in PTC cells was involved in anti-apoptosis, and promoted proliferation and migration at high iodine concentrations. This study provides a rationale for iodine supplementation in PTC patients.

Long Noncoding RNA HOXA-AS2 Promotes Papillary Thyroid Cancer Progression by Regulating miR-520c-3p/S100A4 Pathway.

BACKGROUND/AIMS: Thyroid cancer is one of the most prevalent endocrine tumors. The present study examined the effects of lncRNA HOXA cluster antisense RNA2 (HOXA-AS2) on the progression of papillary thyroid cancer (PTC), and explored the underlying molecular mechanisms. METHODS: Quantitative real-time PCR was used to detect HOXA-AS2, miR-520c-3p and S100 calcium-binding protein A4 (S100A4) expression. Furthermore, the effects of HOXA-AS2 silencing and overexpression on cell proliferation, migration, and invasion were assessed in PTC in vitro by CCK8 and transwell assay. Furthermore, bioinformatics online programs predicted and luciferase reporter assay were used to validate the association of HOXA-AS2 and miR-520c-3p in PTC. RESULTS: We observed that HOXA-AS2 was up-regulated in PTC tissues. In vitro experiments revealed that HOXA-AS2 knockdown significantly inhibited cell growth in PTC in vitro and in vivo. Further functional assays indicated that HOXA-AS2 significantly promoted PTC cell migration and invasion by promoting EMT. Bioinformatics online programs predicted that HOXA-AS2 sponge miR-520c-3p at 3'-UTR with complementary binding sites, which was validated using luciferase reporter assay. HOXA-AS2 could negatively regulate the expression of miR-520c-3p in PTC cells. MiR-520c-3p was down-regulated in PTC tissues, and S100A4 was predicted as a downstream target of miR-520c-3p, which was confirmed by luciferase reporter assay. CONCLUSION: In summary, our results suggested that the HOXA-AS2/miR-520c-3p/S100A4 axis may play an important role in the regulation of PTC progression, which provides us with new insights into understanding the PTC.

Effects of different extracts of curcumin on TPC1 papillary thyroid cancer cell line.

BACKGROUND: The thyroid gland is one of the largest endocrine glands in the body. The vast majority of TCs (> 90%) originate from follicular cells and are defined as differentiated thyroid cancers (DTC) and the two histological subtypes are the papillary TC with its variants and the follicular TC. Curcumin possesses a wide variety of biological functions, and thanks to its properties, it has gained considerable attention due to its profound medicinal values (Prasad, Gupta, Tyagi, and Aggarwal, Biotechnol Adv 32:1053-1064, 2014). We have undertaken the present work in order to define the possible role of curcumin in modulating the genetic expression of cell markers and to understand the effectiveness of this nutraceutical in modulating the regression of cancer phenotype. METHODS: As a template we used the TPC-1 cells treated with the different extracts of turmeric, and examined the levels of expression of different markers (proliferative, inflammatory, antioxidant, apoptotic). RESULTS: Treatment with the three different curcumin extracts displays anti-inflammatory, antioxidant properties and it is able to influence cell cycle with slightly different effects upon the extracts. Furthermore curcumin is able to influence cell metabolic activity vitality. CONCLUSIONS: In conclusion curcumin has the potential to be developed as a safe therapeutic but further studies are needed to verify its antitumor ability in vivo.

Pilot study demonstrating metabolic and anti-proliferative effects of in vivo anti-oxidant supplementation with N-Acetylcysteine in Breast Cancer.

BACKGROUND: High oxidative stress as defined by hydroxyl and peroxyl activity is often found in the stroma of human breast cancers. Oxidative stress induces stromal catabolism, which promotes cancer aggressiveness. Stromal cells exposed to oxidative stress release catabolites such as lactate, which are up-taken by cancer cells to support mitochondrial oxidative phosphorylation. The transfer of catabolites between stromal and cancer cells leads to metabolic heterogeneity between these cells and increased cancer cell proliferation and reduced apoptosis in preclinical models. N-Acetylcysteine (NAC) is an antioxidant that reduces oxidative stress and reverses stromal catabolism and stromal-carcinoma cell metabolic heterogeneity, resulting in reduced proliferation and increased apoptosis of cancer cells in experimental models of breast cancer. The purpose of this clinical trial was to determine if NAC could reduce markers of stromal-cancer metabolic heterogeneity and markers of cancer cell aggressiveness in human breast cancer. METHODS: Subjects with newly diagnosed stage 0 and I breast cancer who were not going to receive neoadjuvant therapy prior to surgical resection were treated with NAC before definitive surgery to assess intra-tumoral metabolic markers. NAC was administered once a week intravenously at a dose of 150 mg/kg and 600 mg twice daily orally on the days not receiving intravenous NAC. Histochemistry for the stromal metabolic markers monocarboxylate transporter 4 (MCT4) and caveolin-1 (CAV1) and the Ki67 proliferation assay and TUNEL apoptosis assay in carcinoma cells were performed in pre- and post-NAC specimens. RESULTS: The range of days on NAC was 14-27 and the mean was 19 days. Post-treatment biopsies showed significant decrease in stromal MCT4 and reduced Ki67 in carcinoma cells. NAC did not significantly change stromal CAV1 and carcinoma TUNEL staining. NAC was well tolerated. CONCLUSIONS: NAC as a single agent reduces MCT4 stromal expression, which is a marker of glycolysis in breast cancer with reduced carcinoma cell proliferation. This study suggests that modulating metabolism in the tumor microenvironment has the potential to impact breast cancer proliferation.

Iodine deficiency a persisting problem: assessment of iodine nutrition and evaluation of thyroid nodular pathology in Portugal.

BACKGROUND: The goal of eliminating iodine deficiency (ID) by the year 2000 has still not been achieved in several countries. More than 2 billion people worldwide (over 260 million school age children) remain ID. In Europe, there are still countries, such as Portugal, without national general population data on iodine nutrition (IN). This study aims at evaluating combined complementary data of the IN of the general population through urinary iodine concentration (UIC) and the thyroid histology profile from the inland region of Beira Interior (BI), in Portugal. METHODS: UIC from a population sample of 214 volunteers (131 females and 83 males), with ages ranging from 8 to 97 years (mean 51.5 years ± SD 20.74 years), from BI was determined; the thyroid histology pattern in BI (6-year period) was evaluated; and the iodine content of the largest surface water reservoir of BI, never previously reported, was measured. RESULTS: Median UIC of 62.6 µg/L was measured. Over 92 % of the population had UIC less than 100 µg/L. From 279 histology reports evaluated, the incidence of the different types of thyroid nodular pathology in BI was established. There were 60 histologic diagnoses of malignancy. The observed ratio of papillary to follicular carcinoma relatively close to 1 and the fairly high percentage of anaplastic carcinomas are characteristic of ID areas. CONCLUSIONS: The findings of this first general population study on IN from the inland region of BI, Portugal, document significant ID. This problem, with its serious public health implications, could be corrected by having affordable iodised salt widely and generally available and by promoting a proactive population attitude generated by ample public information and educational programs as to the negative consequences of ID.

Expression of miRNAs and ZEB1 and ZEB2 correlates with histopathological grade in papillary urothelial tumors of the urinary bladder.

Histopathological grading of papillary urothelial tumors (PUTs) of the urinary bladder is subjective and poorly reproducible. We investigated the relationship between the expression of frequently deregulated microRNAs (miRNAs) as well as their target genes (ZEB1/ZEB2) and bladder cancer histopathological grade in an attempt to find a miRNA that might allow more reliable grading of PUTs. We measured the expression levels of four miRNAs (miR-145, miR-205, miR-125b, and miR-200c) in 120 formalin-fixed, paraffin-embedded bladder tumor tissue samples using real-time PCR assays. ZEB1 and ZEB2 expression was assessed in the same bladder tissues by immunohistochemistry. MiR-205 distinguished low-grade papillary urothelial carcinoma (LG) from high-grade papillary urothelial carcinoma (HG), and miR-145 distinguished HG from infiltrating carcinoma (CA) with an area under the receiver operator characteristic curve (AUC) of 0.992 and 0.997, respectively (sensitivity/specificity of 95.8/96.7 % and 100/91.7 %, respectively; p < 0.05). The expression level of miR-125b was significantly lower in LG than in PUNLMP, with an AUC value of 0.870 (93.3 % sensitivity and 84.2 % specificity; p < 0.05). ZEB1 immunoreactivity was more frequently detected in HG than in LG (57 % vs 13 %, p < 0.01) and in HG than in CA (57 % vs 17 %, p < 0.01). ZEB2 immunoreactivity was more frequent in CA than in HG (83 % vs 54 %, p < 0.05). ZEB1/ZEB2 and miRNAs expression seems to reliably distinguish between different grades of PUTs of the urinary bladder. They might well serve as useful complementary diagnostic biomarkers for grading of papillary urothelial tumors.

Coexistence of malignant struma ovarii and cervical papillary thyroid carcinoma.

CONTEXT: Struma ovarii is an uncommon monodermal teratoma in which thyroid tissue is the predominant element. Malignant transformation of struma ovarii is an even rarer occurrence. CASE PRESENTATION: We describe a 42-year-old woman who underwent a total abdominal hysterectomy and bilateral salpingo-oophorectomy for a symptomatic left pelvic mass. Histology revealed malignant struma ovarii with classical papillary thyroid carcinoma expression. Ultrasonography of the cervical neck showed thyroid micronodules and a dominant 1-cm nodule in the left thyroid lobe. As the ovarian tumor was large, the patient underwent a total thyroidectomy with the intention of administering ¹³¹I therapy in an adjuvant setting. Histology of the cervical thyroid gland revealed bilateral multifocal papillary thyroid carcinoma with extrathyroidal extension and perithyroidal lymph node metastasis. METHODS: Morphological (microscopy), immunohistochemical (Hector Battifora mesothelial cell 1, cytokeratin-19, galectin-3), and molecular (BRAF V600E, RAS, RET-PTC) characteristics and clonality analysis of the cervical thyroid and ovarian tumors were explored to distinguish them as separate malignancies. RESULTS: The thyroid-type tumors from the cervical gland and ovary were discordant in terms of tissue histology and level of cytokeratin-19 expression. The clinical features and tumor profile results supported the independent existence of these two embryologically related, although topographically distinct, malignancies. CONCLUSION: Our findings provided support for synchronous, albeit distinct, primary tumors in the ovary and cervical thyroid. "Field cancerization" and early genomic instability may explain multifocality in all thyroid-type tissue. In this regard, patients with malignant struma ovarii should undergo imaging of their thyroid gland for coexisting disease and thyroidectomy recommended for suspected malignancy or in preparation for radioiodine therapy.

Expression and correlation of sodium/iodide symporter and thyroid stimulating hormone receptor in human thyroid carcinoma.

AIMS: To investigate the expression of sodium/iodide symporter (NIS) and thyroid stimulating hormone receptor (TSHR) in human thyroid cancer. PATIENTS AND METHODS: NIS and TSHR mRNA levels quantified by real-time PCR as well as NIS and TSHR proteins evaluated by immunohistochemistry were examined in surgical specimens including 38 benign nodules, 32 thyroid carcinomas and 36 normal thyroid samples. RESULTS: NIS and TSHR mRNA levels in thyroid carcinomas were significantly lower than in benign nodules and normal thyroid samples (P <0.001). Interestingly, we found that NIS and TSHR mRNA expression in benign nodules had similar levels to those in normal thyroid tissues. However, NIS and TSHR protein expression in benign nodules and thyroid carcinomas was stronger than in normal thyroid samples (P <0.05) but mainly located in cytoplasm. In addition, there was a significant positive correlation between NIS and TSHR in benign nodules and normal thyroid samples (r = 0.551 and 0.667, respectively, P = 0.001 and 0.000, respectively) but there was no such correlation in thyroid carcinomas (r = 0.222, P = 0.376). CONCLUSIONS: In thyroid carcinomas, NIS and TSHR mRNA levels were lower but the proteins were overexpressed. The NIS protein mainly locates in the cytoplasm, which therefore lacks the ability of transporting and absorbing iodine in patients with thyroid carcinoma. In addition, there was no correlation between NIS and TSHR in thyroid cancer, which may explain why, even after TSH stimulation, 10-20% of these malignant tumors are unable to concentrate enough radioiodine for effective therapy.

Increased lipid peroxidation and impaired enzymatic antioxidant defense mechanism in thyroid tissue with multinodular goiter and papillary carcinoma.

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.

Inhibition of Src with AZD0530 reveals the Src-Focal Adhesion kinase complex as a novel therapeutic target in papillary and anaplastic thyroid cancer.

CONTEXT: Focal adhesion kinase (FAK) and Src are overexpressed and activated in many cancers and have been associated with tumor progression. The role of the Src-FAK complex has not been characterized in papillary and anaplastic thyroid cancer (PTC and ATC). OBJECTIVE: The goal of this study was to determine the role of Src and FAK in the growth and invasion of PTC and ATC. DESIGN: PTC and ATC cells were treated with the oral Src inhibitor, AZD0530, to determine the consequences of Src inhibition using growth and invasion assays. FAK and phospho-FAK levels were analyzed in cell lines as well as in PTC tumor samples. RESULTS: AZD0530 treatment inhibited the growth and invasion in four of five thyroid cancer cell lines, and inhibition did not correlate with basal levels of phospho-Src. Instead, we show for the first time that FAK, a critical substrate and effector of Src, is phosphorylated at tyrosine residue 861 (pY861) in PTC and ATC cells, and high levels of phospho-FAK correlate with AZD0530 sensitivity. We further showed that pY861-FAK phosphorylation is Src-dependent. Sensitivity to AZD0530 was confirmed using a preclinical three-dimensional culture model. Phospho-ERK1/2 was not affected by AZD0530, indicating that Src signaling does not require MAPK. Finally, FAK and pY861-FAK were expressed in 10 of 10 and five of 10 PTC tumors, respectively. CONCLUSIONS: Inhibition of the Src-FAK complex represents a promising therapeutic strategy for patients with advanced thyroid cancer, and phospho-FAK represents a potential biomarker for response.

Iodine content and distribution in extratumoral and tumor thyroid tissue analyzed with X-ray fluorescence and time-of-flight secondary ion mass spectrometry.

BACKGROUND: The thyroid's ability to enrich and store iodine has implications for thyroid cancer genesis, progression, and treatment. The study objective was to investigate thyroid iodine content (TIC) in tumoral and extratumoral tissue in patients with papillary thyroid cancer (PTC) as opposed to thyroid healthy controls using two different techniques: X-ray fluorescence (XRF) and time-of-flight secondary ion mass spectrometry (TOF-SIMS). METHODS: Tissue samples from 10 patients with normal thyroids and 7 patients with PTC were collected. TIC was quantified with XRF, and the iodine stores were located on a histological level with TOF-SIMS. RESULTS: Mean TIC in controls was 0.6 mg/mL (range 0.3-1.2 mg/mL). For the cancer patients, the mean TIC was 0.8 mg/mL (range 0.2-2.3 mg/mL) in extratumoral thyroid tissue, but no iodine was detected in the tumors. TOF-SIMS investigation of the PTC patients showed significantly higher TIC in extratumoral tissue than in tumoral tissue. Iodine in the extratumoral tissue was predominantly located in the follicle lumen with a variation in concentration among follicles. CONCLUSIONS: XRF and TOF-SIMS are two complementary methods for obtaining insight into content and localization of iodine in the thyroid. XRF can be used in vitro or in vivo on a large number of samples or patients, respectively. TOF-SIMS on the other hand provides detailed images of the iodine location. The combined information from the two methods is of value for further studies on iodine metabolism in thyroid malignancy.

Effects of the kava chalcone flavokawain A differ in bladder cancer cells with wild-type versus mutant p53.

Flavokawain A is the predominant chalcone from kava extract. We have assessed the mechanisms of flavokawain A's action on cell cycle regulation. In a p53 wild-type, low-grade, and papillary bladder cancer cell line (RT4), flavokawain A increased p21/WAF1 and p27/KIP1, which resulted in a decrease in cyclin-dependent kinase-2 (CDK2) kinase activity and subsequent G(1) arrest. The increase of p21/WAF1 protein corresponded to an increased mRNA level, whereas p27/KIP1 accumulation was associated with the down-regulation of SKP2, which then increased the stability of the p27/KIP1 protein. The accumulation of p21/WAF1 and p27/KIP1 was independent of cell cycle position and thus not a result of the cell cycle arrest. In contrast, flavokawain A induced a G(2)-M arrest in six p53 mutant-type, high-grade bladder cancer cell lines (T24, UMUC3, TCCSUP, 5637, HT1376, and HT1197). Flavokawain A significantly reduced the expression of CDK1-inhibitory kinases, Myt1 and Wee1, and caused cyclin B1 protein accumulation leading to CDK1 activation in T24 cells. Suppression of p53 expression by small interfering RNA in RT4 cells restored Cdc25C expression and down-regulated p21/WAF1 expression, which allowed Cdc25C and CDK1 activation, which then led to a G(2)-M arrest and an enhanced growth-inhibitory effect by flavokawain A. Consistently, flavokawain A also caused a pronounced CDK1 activation and G(2)-M arrest in p53 knockout but not in p53 wild-type HCT116 cells. This selectivity of flavokawain A for inducing a G(2)-M arrest in p53-defective cells deserves further investigation as a new mechanism for the prevention and treatment of bladder cancer.

Annexin-I as a potential target for green tea extract induced actin remodeling.

Using a multistep human urothelial model, we previously showed that green tea extract (GTE) selectively modulates actin remodeling in transformed cells (MC-T11), which resulted in increased cell adhesion and reduced cell motility (Lu et al., Clin Cancer Res 2005;11:1675-83). This study further analyzed which actin binding proteins (ABPs) might be involved in this process. Proteomic profiles of GTE treated and untreated MC-T11 cells using two-dimensional gel electrophoresis coupled with liquid chromatography tandem mass spectrometry (LC/MS/MS) and matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) identified 20 GTE-induced proteins. Among them, 3 were ABPs (tropomodulin, cofilin and annexin-I), and only annexin-I showed a dose- and time-dependent expression. The increased annexin-I correlated with actin remodeling, and was the result of transcription level up-regulation, as determined by RT-PCR, pull-down immunoblot and siRNA analyses. 5-Azacytidine, a DNA methylation inhibitor, exhibited no effect on annexin-I expression when used alone, but had an additive effect for GTE-induced annexin-I expression. Immunohistochemistry of bladder cancer tissue array showed a decrease of annexin-I expression in carcinoma in situ and low grade papillary carcinoma (n = 32, 0% positive) compared to nontumor urothelium (n = 18, 89% positive) (p < 0.001 by Fisher exact test), but increased in some (6 of 15, 40%) high-grade tumors. Together, GTE induced annexin-I expression plays a role in regulating actin remodeling and decreased annexin-I expression is a common event in early stage of bladder cancer development.

Unusual case of well-differentiated papillary thyroid carcinoma lacking thyroglobulin expression while still concentrating radioiodine.

We present an unusual case of a well-differentiated papillary thyroid carcinoma with bilateral lung metastases. Despite undetectable serum thyroglobulin (Tg) on thyroid stimulating hormone (TSH) stimulation and no immunohistochemical evidence of Tg expression in the primary tumour, the patient showed significant uptake of radioiodine in both lungs. After five cycles of high dose radioiodine therapy, the patient went into complete remission and therefore had an excellent response to radioiodine treatment. This case is a rare exception to the rule of Tg production as a prerequisite for differentiated thyroid cancers to concentrate radioiodine.

Lithium as adjuvant to radioiodine therapy in differentiated thyroid carcinoma: clinical and in vitro studies.

OBJECTIVE: Lithium has been reported to increase radioactive iodine (RaI) doses in benign thyroid disease and in differentiated thyroid carcinoma (DTC). It is not known whether lithium influences the outcome of RaI therapy in DTC. We therefore studied the clinical effects of RaI without and with lithium carbonate in patients with proven metastatic DTC. Controversy also exists on the mechanism by which lithium increases RaI dose in DTC. We performed an in vitro study specifically aimed at examining the effects of lithium on the sodium iodide symporter (NIS). DESIGN: In a clinical study, 12 patients were selected with metastases of DTC who had received previous RaI therapy without lithium (control) that had not influenced tumour progression, despite RaI accumulation in metastases. The patients received 1200 mg lithium carbonate/day followed by 6000 MBq RaI. Outcome parameters were RaI uptake, serum thyroglobulin (Tg) levels and radiological dimensions of metastases compared between RaI with lithium and control. In an in vitro study, iodide uptake was studied in the benign rat thyroid cell line FRTL-5, in the polarized non-thyroid MDCK cell line, stably transfected with human sodium iodide symporter (hNIS) to study the effects of lithium on NIS in a non-thyroid background, and the human follicular thyroid carcinoma cell line FTC133-hNIS to study lithium effects in a background of DTC. Lithium chloride (LiCl) was added in concentrations up to 2 mM for 0-48 h. Both steady-state iodide uptake (30 min) and initial rate (2 min) were studied using a specific activity of 100 mCi/mmol I, the latter experiment to determine lithium effects on substrate dependency. Iodide efflux studies were performed as well. RESULTS: Despite an increased uptake of RaI in seven patients, no beneficial effect of RaI with lithium was observed on the clinical course as assessed by serum Tg measurements and radiographically. In the in vitro studies, no effects of LiCl on iodide uptake or efflux were observed. CONCLUSIONS: The addition of lithium to RaI did not have any beneficial effects on the clinical course in 12 patients with metastatic DTC. No beneficial effects of lithium on iodide uptake were observed in vitro. Therefore, the clinical value of lithium in DTC remains subject to debate.

Gene expression in poorly differentiated papillary thyroid carcinomas.

We used cDNA microarrays to study gene expression in fresh frozen papillary thyroid carcinoma (PTC) specimens. Seven clinically aggressive carcinomas were included, comprising poorly differentiated PTC and tumors with extensive local invasion or synchronous distant metastases. Ten differentiated (classic) papillary thyroid carcinomas (PTC) and non-neoplastic thyroid tissues were also investigated. TaqMan quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), in situ hybridization, and immunohistochemistry verified the differential gene expression. The B-Raf gene was mutated with a T-->A transversion at nucleotide 1799 (V600E) in 8 of 10 differentiated PTC, and in 4 of 7 aggressive carcinomas. Among genes markedly and equally over-expressed in carcinomas of both the aggressive and classic PtC groups, compared to normal thyroid tissue, were CBP/p300 transactivator (CItED1), fibronectin, growth/differentiation factor 15, potassium inwardly rectifying channel KCNJ2, glutaminyl peptide cyclotransferase, WNT7A, and dipeptidyl peptidase IV. A marked upregulation in carcinomas of P-cadherin mRNA and protein concomitant with E-cadherin downregulation, indicates a possible P-E cadherin "switch" in PTC. The growth factor homologue Nel-like 2, dual specificity phosphatase 5, the serine protease kallikrein 10, and also the tight junction genes claudin 1 and claudin 16, were upregulated in classic PTC but not in aggressive tumors, which may be consistent with altered cell polarity in the dedifferentiated PtC. The aggressive, poorly differentiated PtC group was specifically characterized by marked upregulation of several genes related to cell proliferation such as cell division cycle 2 (CDC2), CDC7, kinesin-like 5, ubiquitin conjugating enzyme E2C, and topoisomerase IIalpha, and by upregulation of genes encoding extracellular matrix proteins such as seprase, extracellular matrix protein 1, and several collagens. These aggressive tumors were also characterized by overexpression of the integrin ligand periostin, and in some biopsies also of osteopontin and of the upstream Rac-regulator dedicator of cytokinesis 10 (DOCK10). These data are interpreted to be consistent with altered cell motility, extracellular matrix remodeling and increased cell proliferation, as important processes in PTC tumor progression.

BRAF is a therapeutic target in aggressive thyroid carcinoma.

PURPOSE: Oncogenic conversion of BRAF occurs in approximately 44% of papillary thyroid carcinomas and 24% of anaplastic thyroid carcinomas. In papillary thyroid carcinomas, this mutation is associated with an unfavorable clinicopathologic outcome. Our aim was to exploit BRAF as a potential therapeutic target for thyroid carcinoma. EXPERIMENTAL DESIGN: We used RNA interference to evaluate the effect of BRAF knockdown in the human anaplastic thyroid carcinoma cell lines FRO and ARO carrying the BRAF V600E (V600EBRAF) mutation. We also exploited the effect of BAY 43-9006 [N-(3-trifluoromethyl-4-chlorophenyl)-N'-(4-(2-methylcarbamoyl pyridin-4-yl)oxyphenyl)urea], a multikinase inhibitor able to inhibit RAF family kinases in a panel of six (V600E)BRAF-positive thyroid carcinoma cell lines and in nude mice bearing ARO cell xenografts. Statistical tests were two sided. RESULTS: Knockdown of BRAF by small inhibitory duplex RNA, but not control small inhibitory duplex RNA, inhibited the mitogen-activated protein kinase signaling cascade and the growth of ARO and FRO cells (P < 0.0001). These effects were mimicked by thyroid carcinoma cell treatment with BAY 43-9006 (IC50 = 0.5-1 micromol/L; P < 0.0001), whereas the compound had negligible effects in normal thyrocytes. ARO cell tumor xenografts were significantly (P < 0.0001) smaller in nude mice treated with BAY 43-9006 than in control mice. This inhibition was associated with suppression of phospho-mitogen-activated protein kinase levels. CONCLUSIONS: BRAF provides signals crucial for proliferation of thyroid carcinoma cells spontaneously harboring the (V600E)BRAF mutation and, therefore, BRAF suppression might have therapeutic potential in (V600E)BRAF-positive thyroid cancer.

Is [18F]-2-fluoro-2-deoxy-d-glucose (FDG) scintigraphy with non-dedicated positron emission tomography useful in the diagnostic management of suspected metastatic thyroid carcinoma in patients with no detectable radioiodine uptake?

OBJECTIVE: Dedifferentiation of thyroid cancer leads to an inability of thyroid cells to concentrate iodine. In these cases, imaging methods that allow an accurate detection of recurrence and/or metastases at an early stage are essential for an adequate management of patients. Positron emission tomography using [18F]-2-fluoro-2-deoxy-d-glucose and a dedicated (dPET-FDG) or non-dedicated (nPET-FDG) camera has been suggested as a potential tool for the detection of tumour foci. DESIGN AND METHODS: This prospective study was undertaken to evaluate nPET-FDG in 51 consecutive patients (18 men, 33 women) with differentiated thyroid cancer (33 papillary, 11 follicular, four insular and three oncocytic (Hurthle-cell) thyroid carcinomas). Selection criteria were high thyroglobulin (Tg) levels (>10 ng/ml off-levothyroxine treatment) and no detectable radioiodine uptake, on a whole body scan performed with a high dose, in the absence of iodine contamination. RESULTS: Results were interpreted in terms of assumed presence of tumoral tIssue. Sensitivity of nPET-FDG was similar to that of conventional imaging modalities (67%). False negative nPET-FDG (n=16) were observed mostly in cases of micro-lesions (lymph nodes or lung metastases). Conversely, nPET-FDG identified new tumoral sites in 11 cases. Better sensitivity was found for nPET-FDG in patients with Tg levels higher than 15 microg/l (P<0.05). On a patient basis, results of nPET-FDG were equivalent to that of dPET-FDG. Finally, nPET-FDG changed treatment strategy in seven patients. CONCLUSIONS: nPET-FDG has a high sensitivity for the detection of tumour sites in patients when pathological iodine uptake cannot be demonstrated and appears to be a useful method in patients with elevated Tg levels, especially when dedicated PET is either unavailable or impractical.