Human herpes simplex viruses in benign and malignant thyroid tumours.

To test the hypothesis that herpes viruses may have a role in thyroid neoplasia, we analysed thyroid tissues from patients with benign (44) and malignant (65) lesions for HSV1 and HSV2 DNA. Confirmatory studies included direct sequencing, analysis of viral gene expression, and activation of viral-inducible signalling pathways. Expression of viral entry receptor nectin-1 was examined in human samples and in cancer cell lines. In vitro experiments were performed to explore the molecular mechanisms underlying thyroid cancer cell susceptibility to HSV. HSV DNA was detected in 43/109 (39.4%) examined samples. HSV capsid protein expression correlated with HSV DNA status. HSV-positive tumours were characterized by activation of virus-inducible signalling such as interferon-beta expression and nuclear NFkappaB expression. Lymphocyte infiltration and oncocytic cellular features were common in HSV-positive tumours. HSV1 was detected with the same frequency in benign and malignant thyroid tumours. HSV2 was significantly associated with papillary thyroid cancer and the presence of lymph node metastases. The expression of HSV entry receptor nectin-1 was increased in thyroid tumours compared to normal thyroid tissue and further increased in papillary thyroid cancer. Nectin-1 expression was detected in all examined thyroid cancer cell lines. Nectin-1 expression in cancer cells correlated with their susceptibility to HSV. Inhibition of PI3K/AKT or MAPK/ERK signalling did not affect the level of nectin-1 expression but decreased thyroid cancer cell susceptibility to HSV. These findings showed that HSV is frequently detected in thyroid cancer. During tumour progression, thyroid cells acquire increased susceptibility to HSV due to increased expression of viral entry mediator nectin-1 and activation of mitogenic signalling in cancer cells.

Clinical and molecular characterization of thyroid cancer when seen as a second malignant neoplasm.

BACKGROUND: Second malignant neoplasms (SMN) are among the most serious long-term adverse health conditions in cancer survivors. The aim of this study was to characterize clinical findings of patients who developed thyroid cancers as SMN, and to examine genomic alterations in thyroid cancer tissue. METHODS: Retrospective analysis of medical records from patients seen for management of thyroid cancer over 10-year period was performed. Clinical and pathologic data were retrieved from their medical charts. Tumor DNA and RNA were extracted from formalin-fixed, paraffin-embedded tissue and subjected to next-generation sequencing (NGS) using Ion Torrent Oncomine Focus Assay. Microfluidic digital polymerase chain reactions (PCRs) were performed using QIAcuity Digital PCR System to identify BRAF V600E mutations and RET/PTC fusions. RESULTS: Sixteen of 620 patients operated for thyroid cancer had history of previously diagnosed malignancy. Eight patients were male and eight patients were female, with a median age at diagnosis of 58.5 years (range, 4-78). Four patients had history of pediatric malignancy (PedCa), and 12 patients had a history of prior malignancy as an adult (AdCa). The latency periods for development of SMN in PedCa and AdCa patients were 10.8 (±5.2) years and 9.5 (±5.2) years, respectively. Histopathology revealed papillary thyroid cancers in 15 cases, and follicular thyroid cancer in 1 case. All tumors were classified as T1 or T2, and there were no patients presenting with metastases at the time of surgery. Genomic alterations were detected in 13/16 (81.2%) tumors including eight gene mutations (BRAF V600E (N = 4), RAS (N = 2), PI3CA (N = 2) and five gene fusions (RET/PTC1 (N = 4) and STRN/ALK (N = 1). In patients with PedCa and AdCa, mutations were detected in 1/4 (25%) and 7/12 (58.3%), respectively, p = 0.56; and fusions were detected in 3/4 (75%) and 2/12 (16.6%), respectively, p = 0.06. In patients with and without history of therapeutic irradiation, mutations were detected with the same frequencies (5/10 (50%), and 3/6 (50%), respectively, p = 1.0). Gene fusions were detected in patients with and without history of irradiation in 5/10 (55.5%) and 0/6 (0%), respectively, p = 0.09. CONCLUSIONS: Monitoring of cancer survivors for thyroid disorders allowed diagnosis of second thyroid cancers at early stages. Second thyroid cancers harbor genomic alterations that are typical for sporadic as well as for radio-induced thyroid cancers.

Cytochrome C Oxidase Subunit 4 (COX4): A Potential Therapeutic Target for the Treatment of Medullary Thyroid Cancer.

The nuclear-encoded subunit 4 of cytochrome c oxidase (COX4) plays a role in regulation of oxidative phosphorylation and contributes to cancer progression. We sought to determine the role of COX4 in differentiated (DTC) and medullary (MTC) thyroid cancers. We examined the expression of COX4 in human thyroid tumors by immunostaining and used shRNA-mediated knockdown of COX4 to evaluate its functional contributions in thyroid cancer cell lines. In human thyroid tissue, the expression of COX4 was higher in cancers than in either normal thyroid (p = 0.0001) or adenomas (p = 0.001). The level of COX4 expression correlated with tumor size (p = 0.04) and lymph-node metastases (p = 0.024) in patients with MTCs. COX4 silencing had no effects on cell signaling activation and mitochondrial respiration in DTC cell lines (FTC133 and BCPAP). In MTC-derived TT cells, COX4 silencing inhibited p70S6K/pS6 and p-ERK signaling, and was associated with decreased oxygen consumption and ATP production. Treatment with potassium cyanide had minimal effects on FTC133 and BCPAP, but inhibited mitochondrial respiration and induced apoptosis in MTC-derived TT cells. Our data demonstrated that metastatic MTCs are characterized by increased expression of COX4, and MTC-derived TT cells are vulnerable to COX4 silencing. These data suggest that COX4 can be considered as a novel molecular target for the treatment of MTC.

Detection of BRAFV600E in Liquid Biopsy from Patients with Papillary Thyroid Cancer Is Associated with Tumor Aggressiveness and Response to Therapy.

The detection of rare mutational targets in plasma (liquid biopsy) has emerged as a promising tool for the assessment of patients with cancer. We determined the presence of cell-free DNA containing the BRAFV600E mutations (cfBRAFV600E) in plasma samples from 57 patients with papillary thyroid cancer (PTC) with somatic BRAFV600E mutation-positive primary tumors using microfluidic digital PCR, and co-amplification at lower denaturation temperature (COLD) PCR. Mutant cfBRAFV600E alleles were detected in 24/57 (42.1%) of the examined patients. The presence of cfBRAFV600E was significantly associated with tumor size (p = 0.03), multifocal patterns of growth (p = 0.03), the presence of extrathyroidal gross extension (p = 0.02) and the presence of pulmonary micrometastases (p = 0.04). In patients with low-, intermediate- and high-risk PTCs, cfBRAFV600E was detected in 4/19 (21.0%), 8/22 (36.3%) and 12/16 (75.0%) of cases, respectively. Patients with detectable cfBRAFV600E were characterized by a 4.68 times higher likelihood of non-excellent response to therapy, as compared to patients without detectable cfBRAFV600E (OR (odds ratios), 4.68; 95% CI (confidence intervals)) 1.26-17.32; p = 0.02). In summary, the combination of digital polymerase chain reaction (dPCR) with COLD-PCR enables the detection of BRAFV600E in the liquid biopsy from patients with PTCs and could prove useful for the identification of patients with PTC at an increased risk for a structurally or biochemically incomplete or indeterminate response to treatment.

Mitotane induces mitochondrial membrane depolarization and apoptosis in thyroid cancer cells.

Mitotane is used for the treatment of adrenocortical cancer and elicits its anticancer effects via inhibition of mitochondrial respiration. Targeting mitochondria­dependent metabolism has emerged as a promising strategy for thyroid cancer (TC) treatment. We hypothesized that mitotane targets mitochondria and induces apoptosis in TC cells. Cell lines representative of the major histological variants of TC were chosen: Follicular (FTC­133), poorly differentiated (BCPAP), anaplastic (SW1736 and C643) and medullary (TT) TC cells, and were treated with mitotane (0­100 µM). Mitochondrial membrane potential, cell viability and apoptosis were examined by JC­1 staining and by western blot analysis using an antibody against caspase­3. The expression of mitochondrial molecules and DNA damage markers and the activation of endoplasmic reticulum (ER) stress were determined by western blotting. The expression of mitochondrial ATP synthase subunit ß (ATP5B) was examined by immunostaining in 100 human TC tissue samples. Treatment with mitotane (50 µM for 24 h) decreased the viability of FTC­133, BCPAP, SW1736, C643 and TT cells by 12, 59, 54, 31 and 66%, respectively. Morphological evidence of ER stress and overexpression of ER markers was observed in TC cells following exposure to mitotane. The treatment led to increased expression of histone γH2AX, indicating DNA damage, and to caspase­3 cleavage. Consistent with the results of the cell viability assays, the overexpression of pro­apoptotic genes following treatment with mitotane was more prominent in TC cells harboring mutations in the serine/threonine­protein kinase B­raf gene and proto­oncogene tyrosine­protein kinase receptor Ret. Treatment with mitotane was associated with loss of mitochondrial membrane potential and decreased expression of ATP5B, particularly in the medullary TC (MTC)­derived TT cells. Immunohistochemical analysis of mitochondrial ATP5B in human TC specimens demonstrated its overexpression in cancer compared with normal thyroid tissue. The level of ATP5B expression was higher in MTC compared with the follicular, papillary or anaplastic types of TC. Mitotane elicited pleiotropic effects on TC cells, including induction of ER stress, inhibition of mitochondrial membrane potential and induction of apoptosis. The results of the present study suggest that mitotane could be considered as a novel agent for the treatment of aggressive types of TC.

Microfluidic Droplet Digital PCR Is a Powerful Tool for Detection of BRAF and TERT Mutations in Papillary Thyroid Carcinomas.

We examined the utility of microfluidic digital PCR (dPCR) for detection of BRAF and TERT mutations in thyroid tumors. DNA extracted from 100 thyroid tumors (10 follicular adenomas, 10 follicular cancers, 5 medullary cancers, and 75 papillary thyroid cancer (PTC) were used for detection of BRAF and TERT mutations. Digital PCRs were performed using rare mutation SNP genotyping assays on QuantStudio 3D platform. In PTCs, BRAFV600E was detected by dPCR and Sanger sequencing in 42/75 (56%) and in 37/75 (49%), respectively. BRAFV600E was not detected in other tumors. The ratio of mutant/total BRAF alleles varied from 4.7% to 47.5%. These ratios were higher in classical PTCs (27.1%) as compared to follicular variant PTCs (9.4%) p = 0.001. In PTCs with and without metastases, the ratios of mutant/total BRAF alleles were 27.6% and 18.4%, respectively, (p = 0.03). In metastatic lesions percentages of mutant/total BRAF alleles were similar to those detected in primary tumors. TERTC228T and TERTC250T were found in two and one cases, respectively, and these tumors concomitantly harbored BRAFV600E. These tumors exhibited gross extra-thyroidal extension, metastases to lymph nodes, and pulmonary metastases (one case). Our results showed that dPCR allows quantitative assessment of druggable targets in PTCs and could be helpful in a molecular-based stratification of prognosis in patients with thyroid cancer.

Limited Utility of Circulating Cell-Free DNA Integrity as a Diagnostic Tool for Differentiating Between Malignant and Benign Thyroid Nodules With Indeterminate Cytology (Bethesda Category III).

Background: Analysis of plasma circulating cell-free DNA integrity (cfDI) has emerged as a promising tool in the diagnosis of malignant vs. benign tumors. There is limited data on the role of cfDI in thyroid cancer. The goal of this study was to analyze cfDI as a biomarker of malignancy in patients with cytologically indeterminate thyroid nodules. Methods: The cfDI was measured in the plasma of patients with cytologically indeterminate thyroid nodules. All patients underwent plasma collection within 24-72 h before surgical treatment for thyroid nodules. Additionally, samples were collected from seven patients via the vein draining the thyroid and peripheral vein during surgery. Quantitative real-time PCR was performed on the isolated cell-free DNA using two different primer sets (115 and 247 bp) to amplify consensus ALU sequences. The cfDI was calculated as the ratio of ALU247 to ALU115. Results: All data are given as median [25th-75th percentile]. The study group consisted of 67 patients with 100 nodules, 80.6% (54/67) women, aged 43 [33-60] years. There was no difference in cfDI between 29 patients with benign nodules (0.49 [0.41-0.59]) and 38 patients with malignant lesions (0.45 [0.36-0.57], p = 0.19). There was no difference in cfDI in the vein draining the thyroid (0.47 [0.24-1.05]) and peripheral vein (0.48 [0.36-0.56], p = 0.44). In comparison to thyroid cancer patients, patients with benign nodules were characterized by significantly higher concentrations of ALU115 (1,064 [529-2,960] vs. 411 [27-1,049] ng/ml; p = 0.002) and ALU247 (548 [276-1,894] vs. 170 [17-540] ng/ml; p = 0.0005), most likely because benign tumors were larger (3, [1.8-4.1 cm]) than malignant lesions (0.7 [0.23-1.45], p < 0.0001). Women had significantly lower cfDI (0.45 [0.27-0.54]) than men (0.56 [0.44-0.8], p = 0.011). Conclusion: The cfDI measured in the vein draining the thyroid is similar to the cfDI measured in the antecubital vein, validating cfDI measurements by peripheral liquid biopsy. Analysis of cfDI needs to be stratified by patients gender. In contrast to its diagnostic utility in aggressive cancers, cfDI has limited utility as a biomarker of malignancy in cytologically indeterminate thyroid nodules.

Nelfinavir inhibits proliferation and induces DNA damage in thyroid cancer cells.

The HIV protease inhibitor Nelfinavir (NFV) inhibits PI3K/AKT and MAPK/ERK signaling pathways, emerging targets in thyroid cancers. We examined the effects of NFV on cancer cells that derived from follicular (FTC), papillary (PTC) and anaplastic (ATC) thyroid cancers. NFV (1-20 µM) was tested in FTC133, BCPAP and SW1736 cell lines. The effects of NFV on cell proliferation were determined in vitro using real-time microscopy and by flow cytometry. DNA damage, apoptotic cell death and expression of molecular markers of epithelial-mesenchymal transition (EMT) were determined by Western blot and real-time PCR. Real-time imaging demonstrated that NFV (10 µM) increased the time required for the cell passage through the phases of cell cycle and induced DNA fragmentation. Growth inhibitory effects of NFV were associated with the accumulation of cells in G0/G1 phase, downregulation of cyclin D1 and cyclin-dependent kinase 4 (CDK4). NFV also induced the expression of γH2AX and p53BP1 indicating DNA damage. Treatment with NFV (20 µM) resulted in caspase-3 cleavage in all examined cells. NFV (20 µM) decreased the levels of total and p-AKT in PTEN-deficient FTC133 cells. NFV had no significant effects on total ERK and p-ERK in BRAF-positive BCPAP and SW1736 cells. NFV had no effects on the expression of EMT markers (Twist, Vimentin, E- and N-Cadherin), but inhibited the migration and decreased the abilities of thyroid cancer cells to survive in non-adherent conditions. We conclude that NFV inhibits proliferation and induces DNA damage in thyroid cancer cell lines. Our in vitro data suggest that NFV has a potential to become a new thyroid cancer therapeutic agent.

Amifostine does not protect thyroid cancer cells in DNA damaging in vitro models.

BACKGROUND: Amifostine is a potent scavenger of reactive oxygen species that is used for the salivary gland protection during therapy with radioactive iodine for thyroid cancer. There are no data on the potential effect of amifostine on thyroid cancer cells. METHODS: We investigated the effects of the active form of amifostine (WR-1065) on the response of thyroid cancer cells to treatment with DNA-damaging agents. WR-1065 was examined in human thyroid cancer cell lines (FTC133, TPC1, BCPAP and C643) and embryonic fibroblast cells NIH3T3. DNA damage was induced by exposure to H2O2 (0.1 mM), by treatment with the radiomimetic neocarzinostatin (NCS 250 ng/mL) and by γ-radiation (6 Gy). DNA damage, cell viability and apoptosis were examined. RESULTS: We demonstrated the selective action of WR-1065 (0.1 mM), which prevented oxidative stress-induced DNA damage in fibroblasts, but did not protect thyroid cancer cells from DNA damage and apoptosis documented by caspase-3 and PARP cleavage after exposure to H2O2, NCS and γ-radiation. Prolonged exposure to WR-1065 (0.1 mM for 24 h) was toxic for thyroid cancer cells; this treatment decreased the number of viable cells by 8% in C643 cells, 47% in TPC cells, 92% in BCPAP cells and 82% in FTC 133 cells. The cytotoxic effects of WR-1065 were not associated with induction of apoptosis. CONCLUSIONS: Our data show that amifostine has no protective effect on thyroid cancer cells against DNA-damaging agents in vitro and suggest that amifostine will not attenuate the efficacy of radioiodine treatment in patients with thyroid cancer.

Aplidin reduces growth of anaplastic thyroid cancer xenografts and the expression of several angiogenic genes.

BACKGROUND: Anaplastic thyroid cancer (ATC) is one of the most aggressive and highly lethal human cancers. Median survival after diagnosis is 4-6 months despite available radiotherapy and chemotherapy. Additional treatments are needed for ATC. Vascular endothelial growth factor (VEGF) is a potent angiogenic stimulus, which is expressed by ATC. Previously, anti-VEGF antibody was used to block VEGF-dependent angiogenesis in ATC xenografts. This treatment induced partial (56%) but not complete tumor regression. Aplidin (APLD) is a marine derived antitumor agent currently in phase II clinical studies. Multiple activities of this compound have been described which likely contribute to its antiproliferative effect. Notably, APLD has been shown to have antiangiogenic properties which include: inhibition of VEGF secretion, reduction in the synthesis of the VEGF receptor (FLT-1), and blockade of matrix metalloproteinase production by endothelial cells. We hypothesized that Aplidin, with its broad spectrum of action and antiangiogenic properties, would be a potentially effective drug against ATC. METHODS: Thirty BALB/c nu/nu mice were injected with ATC cells (ARO-81, 1 x 10(6)) and allowed to implant for 3 weeks. Animals were randomized to receive daily intraperitoneal injections of vehicle, low dose (0.5 mg/kg/day), or high dose (1.0 mg/kg/day) APLD. After 3 days, the animals were killed and the tumors were removed, weighed, and divided for RNA and protein analyses. RESULTS: APLD significantly reduced ATC xenograft growth (low dose, 20% reduction, P = 0.01; high dose, 40% reduction, P < 0.001). This was associated with increased levels of apoptosis related proteins polyadenosylribose polymerase 85 (PARP-85, 75% increase, P = 0.024) and caspase 8 (greater than fivefold increase, P = 0.03). APLD treatment was further associated with lost or reduced expression of several genes that support angiogenesis to include: VEGF, hypoxia inducible factor 1(HIF-1), transforming growth factor-beta (TGFbeta), TGFbeta receptor 2 (TGFbetaR2), melanoma growth stimulating factor 1 (GRO1), cadherin, and vasostatin. CONCLUSIONS: This data supports the hypothesis that APLD may be an effective adjunctive therapy against ATC. The demonstrated molecular impact against angiogenic related genes specifically supports future strategies combining APLD with VEGF interacting agents.

Metformin Attenuates 131I-Induced Decrease in Peripheral Blood Cells in Patients with Differentiated Thyroid Cancer.

BACKGROUND: 131I treatment (tx) of differentiated thyroid cancer (DTC) is associated with hematopoietic toxicity. It was hypothesized that metformin could have radioprotective effects on bone-marrow function. The objective was to determine whether metformin prevents 131I-induced changes in complete blood counts (CBC) in patients with DTC. METHODS: A retrospective analysis was performed of CBC values in DTC patients who were (40 patients: metformin group) or were not taking metformin (39 patients: control group) at the time of administration of 131I. Repeated measures analysis of variance was used for the analysis of the differences in the averages of CBC that were documented at baseline and at 1, 6, and 12 months post 131I tx. RESULTS: The groups were comparable in terms of age, sex, stage of DTC, 131I dose administered, and baseline CBC values. In the control group, the decrease in white blood cells (WBC) was 35.8% (p < 0.0001) at one month, 21.8% (p < 0.0001) at six months, and 19.4% (p < 0.0001) at 12 months. In the metformin group, the decrease in WBC was 17.1% (p < 0.0001) at one month, and 8.6% at six months (p = 0.01), while at 12 months WBC had returned to baseline values (p = 0.9). Differences between the two groups were highly statistically significant at all time points (p < 0.0001, p = 0.0027, and p < 0.0001, respectively). Lymphocytes were more sensitive to 131I, but metformin's radioprotective properties were more prominent in neutrophils. At 12 months, the decrease in platelets in the control group was 15.5% (p < 0.0001) versus 5.6% (p = 0.056) in the metformin group, while at one and six months the reductions in the two groups were comparable. No statistically significant differences were observed between the two groups in the change from baseline values for hemoglobin. CONCLUSIONS: Metformin attenuated the 131I-induced decrease in CBC parameters, and its radioprotective properties were more prominent in WBC. Patients who were taking metformin during 131I tx also experienced a faster recovery in their blood counts, when compared to the control group. Further study is warranted in order to examine if the radioprotective properties of metformin observed in the current study for 131I tx can also apply to other forms of therapeutic chemo- and radiotherapy.

BRAF mutations are uncommon in papillary thyroid cancer of young patients.

Mortality is low for young patients (younger than 21 years) with papillary thyroid cancer (PTC), and different mutations might contribute to this. Previous studies detected ret/PTC rearrangements more frequently in PTC from children than adults, and recent reports describe a high incidence of BRAF T1796A transversion in adult PTC. However, BRAF mutations have not been adequately studied in PTC from young patients. We amplified and sequenced segments of the BRAF gene spanning the T1796A transversion site in 14 PTC from patients 10-21 years of age (mean, 17.5 +/- 3.5 years). The PTC (7 = class 1; 5 = class 2; 1 = class 3) ranged from 0.7-2.9 cm in diameter (mean, 1.4 +/- 0.75 cm). None of them (0/14) contained BRAF T1796A and none recurred (mean follow-up, 66 +/- 40 months). This incidence of BRAF T1796A is significantly less than that reported for adult PTC (270/699, 38.6%, p = 0.0015) in several series. None of our PTC (0/10) contained ras mutations, but 7/12 (58%) contained ret/PTC rearrangements. We conclude that BRAF mutations are less common in PTC from young patients, and ret/PTC rearrangements were the most common mutation found in these childhood PTC.

Thyroid cancers express CD-40 and CD-40 ligand: cancers that express CD-40 ligand may have a greater risk of recurrence in young patients.

The immune response might suppress thyroid cancer recurrence. Although the factors that control this are unknown, CD-40 and CD-40 ligand might be important. To test this, we stained 36 papillary (PTC) and four follicular (FTC) thyroid carcinomas for CD-40 (n = 37) and CD-40 ligand (n = 36) and graded staining from absent (grade 0) to intense (grade 3). Follicular cells of the majority of thyroid tumors expressed CD-40 (30/37, 81%) and CD-40 ligand (15/24, 69%). Cancers from young patients (< or =21 years of age) that expressed CD-40 contained more numerous lymphocytes/high-power field (36 +/- 11) than cancers that failed to express CD-40 (4 +/- 3, p = 0.01), but there was no correlation with clinical outcome. Among young patients, CD-40 ligand expression was more intense in multifocal (1.1 +/- 0.2 vs. 0.45 +/- 0.2, p = 0.037), aggressive (1.14 +/- 0.14 vs. 0.65 +/- 0.2, p = 0.05) and recurrent tumors (1.2 +/- 0.2 vs. 0.65 +/- 0.2, p = 0.05) and associated with reduced disease-free survival (p = 0.03). We conclude that the majority of thyroid cancers express CD-40 and CD-40 ligand. In patients < or =21 years of age, tumors with intense expression of CD-40 ligand are more often multifocal, aggressive, and recurrent.

Glucose-deprivation increases thyroid cancer cells sensitivity to metformin.

Metformin inhibits thyroid cancer cell growth. We sought to determine if variable glucose concentrations in medium alter the anti-cancer efficacy of metformin. Thyroid cancer cells (FTC133 and BCPAP) were cultured in high-glucose (20 mM) and low-glucose (5 mM) medium before treatment with metformin. Cell viability and apoptosis assays were performed. Expression of glycolytic genes was examined by real-time PCR, western blot, and immunostaining. Metformin inhibited cellular proliferation in high-glucose medium and induced cell death in low-glucose medium. In low-, but not in high-glucose medium, metformin induced endoplasmic reticulum stress, autophagy, and oncosis. At micromolar concentrations, metformin induced phosphorylation of AMP-activated protein kinase and blocked p-pS6 in low-glucose medium. Metformin increased the rate of glucose consumption from the medium and prompted medium acidification. Medium supplementation with glucose reversed metformin-inducible morphological changes. Treatment with an inhibitor of glycolysis (2-deoxy-d-glucose (2-DG)) increased thyroid cancer cell sensitivity to metformin. The combination of 2-DG with metformin led to cell death. Thyroid cancer cell lines were characterized by over-expression of glycolytic genes, and metformin decreased the protein level of pyruvate kinase muscle 2 (PKM2). PKM2 expression was detected in recurrent thyroid cancer tissue samples. In conclusion, we have demonstrated that the glucose concentration in the cellular milieu is a factor modulating metformin's anti-cancer activity. These data suggest that the combination of metformin with inhibitors of glycolysis could represent a new strategy for the treatment of thyroid cancer.

Papillary thyroid carcinomas from young adults and children contain a mixture of lymphocytes.

The immune response appears to be important in preventing metastasis and recurrence of thyroid cancer. We previously showed that papillary thyroid carcinoma (PTC) from children and adolescents that contain the most numerous proliferating lymphocytes have the best prognosis. However, the types of lymphocytes involved are not yet known. To further define this, we examined 21 PTCs from patients 21 yr of age or younger (52% were 18-21 yr of age) for the presence of CD4+ (helper), CD8+ (killer), CD19+ (B cells), and CD56+ (natural killer) cells as well as proliferating lymphocytes (Ki-67+). Nearly half the PTCs contained CD4+ (9 of 21, 43%), CD8+ (8 of 21, 38%), or CD19+ (10 of 21, 48%) lymphocytes. Only one PTC (1 of 21, 5%) contained CD56+ lymphocytes, and none contained all four cell types. By dual staining, none of these lymphocytes were proliferating (Ki-67+). However, PTCs containing either CD8+ cells (n = 8) or a combination of CD4+, CD8+, and CD19+ cells (n = 5) contained more numerous proliferating lymphocytes than did PTCs containing any other combination (14.2-fold increase, P = 0.03 and 13.1-fold increase, P = 0.003, respectively). During follow-up, none of the PTCs containing either CD8+ lymphocytes or the combination of CD4+, CD8+, and CD19+ lymphocytes recurred. However, the cohort is too small and the follow-up inadequate to provide accurate information on the clinical impact of these immunological findings. We conclude that the immune response against PTC is important and also complex, involving more than one type of lymphocyte.

Intense expression of the b7-2 antigen presentation coactivator is an unfavorable prognostic indicator for differentiated thyroid carcinoma of children and adolescents.

Previous observations suggest that an immune response against thyroid carcinoma could be important for long-term survival. We recently found that infiltration of thyroid carcinoma by proliferating lymphocytes is associated with improved disease-free survival, but the factors that control lymphocytic infiltration and proliferation are largely unknown. We hypothesized that the antigen presentation coactivators (B7-1 and B7-2), which are important in other immune-mediated thyroid diseases, might be important in lymphocytic infiltration of thyroid carcinoma. To test this, we determined B7-1 and B7-2 expression by immunohistochemistry [absent (grade 0) to intense (grade 3)] in 27 papillary (PTC) and 8 follicular (FTC) thyroid carcinomas and 9 benign thyroid lesions. B7-1 and B7-2 were expressed by the majority of PTC and FTC (78% of PTC and 100% of FTC expressed B7-1; 88% of PTC and 88% of FTC expressed B7-2). B7-1 expression was more intense in PTC (1.4 +/- 0.2; P = 0.01) and FTC (2.6 +/- 0.2; P < 0.001) than in benign tumors (0.57 +/- 0.30) or presumably normal adjacent thyroid (0.07 +/- 0.07) and was more intense in carcinoma that contained lymphocytes (1.95 +/- 0.21) than in carcinoma that did not (1.08 +/- 0.26; P = 0.016). B7-2 expression was of similar intensity in benign and malignant tumors (PTC, 1.6 +/- 0.2; FTC, 2.1 +/- 0.4; benign, 1.86 +/- 0.4), but was more intense than in presumably normal adjacent thyroid (0.64 +/- 0.25; P

Enzyme expression profiles suggest the novel tumor-activated fluoropyrimidine carbamate capecitabine (Xeloda) might be effective against papillary thyroid cancers of children and young adults.

PURPOSE: The fluoropyrimidine carbamate (capecitabine) is converted to 5-fluorouracil (5-FU) by thymidine phosphorylase (TP) inside target tissues. 5-FU interferes with DNA synthesis by blocking thymidylate synthase (TS) but is inactivated by dihydropyrimidine dehydrogenase (DPD). Favorable enzyme profiles (high TP and low DPD) generate high intratumor levels of 5-FU that are effective against many tumors, especially those with low TS. Capecitabine has not been tested against thyroid cancers, and it is not known to what extent thyroid cancers express TP, TS or DPD. METHODS: To test this, we determined TP, TS and DPD in 19 thyroid cancers from young patients (14 papillary, 4 follicular, 1 medullary) by immunohistochemistry. After approval by the Human Use Committee, the intensity of TP, TS, and DPD staining was determined by two independent examiners and graded (absent=0 to intense=3) with >90% concordance. RESULTS: TS was detected in 7/19 cancers (37%), TP in 14/19 cancers (74%) and DPD in 14/19 cancers (74%). In six tumors, TP was more intense that DPD, suggesting capecitabine sensitivity. Only five tumors failed to express TP but four of these expressed DPD, suggesting capecitabine resistance. Overall, 6/19 tumors (32% of the total) had a favorable expression profile, and all of them were papillary cancers. CONCLUSIONS: We conclude that the majority of differentiated thyroid cancers (74%) express TP and low levels of TS (63% undetectable). The results support the hypothesis that capecitabine is activated in the majority of differentiated thyroid cancers and that 32% have favorable expression of all three enzymes (TP, TS, and DPD).

Cytoplasmic localization of the paired box gene, Pax-8, is found in pediatric thyroid cancer and may be associated with a greater risk of recurrence.

The paired box-8 protein (Pax-8) has been observed in the nucleus of normal adult thyroids, follicular adenomas, follicular thyroid cancers, and papillary thyroid cancers (PTC) but not undifferentiated thyroid cancers. To our knowledge, Pax-8 has not been studied in pediatric thyroid cancer. Because of the more favorable prognosis for PTC in children compared to young patients, we hypothesized that Pax-8 expression might be different in pediatric thyroid cancers. To test this, we stained 47 thyroid lesions from children and young patients for Pax-8. Pax-8 was located in the cytoplasm (cPAX) or nucleus (nPAX) in the majority of samples. There was no significant difference in nPAX between benign and malignant lesions. However, cPAX was more commonly seen in PTC than autoimmune diseases (p = 0.01) and the intensity of cPAX staining correlated with tumor size (p = 0.041), metastasis, age, completeness of resection, local invasion, and tumor size (MACIS) scores (p = 0.045), and the presence of invasion, metastasis, recurrence, or persistence (p = 0.012). Disease-free survival was significantly reduced for cancers with intense cPAX staining (p = 0.0003). These data show that cPAX is common in PTC, and although limited by small sample size, suggest an association with higher MACIS scores, an aggressive clinical course, and an increased risk of clinically evident recurrence for children and young patients.

Vascular endothelial growth factor monoclonal antibody inhibits growth of anaplastic thyroid cancer xenografts in nude mice.

BACKGROUND: Anaplastic thyroid cancer (ATC) does not respond well to any treatment and is one of the most aggressive of all human cancers. Based on the importance of angiogenesis in solid tumor growth, we hypothesized that angiogenic blockade might reduce the growth of ATC. METHODS: We tested the in vivo effect of vascular endothelial growth factor monoclonal antibody (VEGF-mAb) and thalidomide against ATC (ARO-81) xenografts in nude mice. Mice were injected subcutaneously with 1 x 10(6) ARO-81 cells, allowed to implant (1 week), and then given daily intraperitoneal injections of vehicle (control; n = 9), VEGF-mAb (200 microg/d; n = 9), or thalidomide (200 mg/kg per day; n = 9). Tumors were removed, sectioned, and stained for routine histology and immunohistochemistry. RESULTS: At 6 weeks, VEGF-mAb-treated tumors were smaller (1603 +/- 296 mm(3)) than either the thalidomide-treated (6007 +/- 1498 mm(3); p = 0.008) or the control groups (4040 +/- 831 mm(3); p = 0.014) and the VEGF-mAb-treated animals maintained greater weight (30.4 +/- 0.84 g at week 6 versus thalidomide-treated, 26.0 +/- 0.94 g, p = 0.003; and control, 25.8 +/- 0.78 g, p = 0.001 animals). Central necrosis was observed in 3 of 9 VEGF-mAb-treated confidence interval (33%; 95% [CI] = 0.12-0.65) but in none of the control or thalidomide-treated tumors (0/18 total; 95% CI = 0.0-0.30; p = 0.029). VEGF staining intensity for VEGF-mAb- (2.0 +/- 0.24; p = 0.012) and thalidomide- (2.1 +/- 0.05; p = 0.052) treated tumors was greater than control (0.89 +/- 0.31) as was p53 staining grade (VEGF-mAb [1.3 +/- 0.37; p = 0.012]; thalidomide [1.0 +/- 0.41; p = 0.05]; and controls [0.11 +/- 0.11]). CONCLUSION: We conclude that systemic VEGF-mAb significantly reduces growth of ATC xenografts and is associated with increased VEGF and p53 expression. Thalidomide has no effect on tumor growth, but is also associated with increased VEGF and p53 expression. These observations provide the first evidence that VEGF-mAb-induced angiogenesis blockade may be of use for the treatment of ATC.

Systemic administration of vascular endothelial growth factor monoclonal antibody reduces the growth of papillary thyroid carcinoma in a nude mouse model.

Papillary thyroid carcinomas (PTC) are the most common thyroid cancers in children. Most are successfully treated with surgery and radioactive iodine, but some persist. PTC express high levels of vascular endothelial growth factor (VEGF) and VEGF receptor (Flt-1). PTC with the most intense expression of VEGF have the greatest recurrence risk. We hypothesized that blockade of VEGF would inhibit PTC growth. To test this, we used systemic VEGF monoclonal antibody (VEGF-MAb) to treat PTC xenografts in nude mice. Treated animals (n = 9) received 200 microg VEGF-MAb by daily i.p. injection for 10 wk, while control animals (n = 9) received vehicle alone. Tumor size was significantly reduced in the treatment group (0.28 +/- 0.06 vs 1.05 +/- 0.25 g, p = 0.008). VEGF immunostaining was more intense (2.57 +/- 0.30 vs 1.75 +/- 0.25, p = 0.06) and the number of p53 positive cells was increased (1.66 +/- 0.24 vs 0.83 +/- 0.31, p = 0.048) in treated tumors. Animal weight was similar in both groups (29.1 +/- 1.1 vs 27.4 +/- 1.1 g, p = 0.30). In conclusion, systemic VEGF-MAb significantly reduced the growth of PTC, suggesting that VEGF-MAb might be useful for treatment of resistant PTC.