Study of gene expression in thyrotropin-stimulated thyroid cells by cDNA expression array: ID3 transcription modulating factor as an early response protein and tumor marker in thyroid carcinomas.

Induction of cell proliferation by mitogen or growth factor stimulation leads to the specific induction or repression of a large number of genes. To identify genes differentially regulated by the cAMP-dependent transduction pathway, which is poorly characterized so far, we used the cDNA expression array technology. Hybridizations of Atlas human cDNA expression arrays with (32)P-labeled cDNA probes derived from control or thyrotropin (TSH)-stimulated dog thyrocytes in primary culture generated expression profiles of hundreds of genes simultaneously. Among the genes that displayed modified expression, we selected the transcription factor ID3, whose expression was increased by a cAMP-dependent stimulus. ID3 overexpression after TSH stimulation was first verified by Northern blotting analysis, and its mRNA regulation was then investigated in response to a variety of agents acting on thyrocyte proliferation and/or differentiation. We show that: (1) ID3 mRNA induction was stronger after stimulation of the cAMP cascade, but was not restricted to this signaling pathway, as phorbol myristate ester (TPA) and insulin also stimulated mRNA accumulation; (2) in contrast, powerful mitogens for thyroid cells, epidermal growth factor and hepatocyte growth factor, did not significantly modify ID3 mRNA levels; (3) ID3 protein levels closely parallelled mRNA levels, as revealed by immunofluorescence experiments showing a nuclear signal regulated by TSH; (4) in papillary thyroid carcinomas, ID3 mRNA was downregulated. Our results suggest that ID3 expression might be more related to the differentiating process induced by TSH than to the proliferative action of this hormone.

Correlation between BRAF mutation and the clinicopathological parameters in papillary thyroid carcinoma with particular reference to follicular variant

Mutation of the BRAF gene is common in thyroid cancer. Follicular variant of papillary thyroid carcinoma is a variant of papillary thyroid carcinoma that has created continuous diagnostic controversies among pathologists. The aims of this study are to (1) investigate whether follicular variant of papillary thyroid carcinoma has a different pattern of BRAF mutation than conventional papillary thyroid carcinoma in a large cohort of patients with typical features of follicular variant of papillary thyroid carcinoma and (2) to study the relationship of clinicopathological features of papillary thyroid carcinomas with BRAF mutation. Tissue blocks from 76 patients with diagnostic features of papillary thyroid carcinomas (40 with conventional type and 36 with follicular variant) were included in the study. From these, DNA was extracted and BRAF V600E mutations were detected by polymerase chain reaction followed by restriction enzyme digestion and sequencing of exon 15. Analysis of the data indicated that BRAF V600E mutation is significantly more common in conventional papillary thyroid carcinoma (58% versus 31%, P = .022). Furthermore, the mutation was often noted in female patients (P = .017), in high-stage cancers (P = .034), and in tumors with mild lymphocytic thyroiditis (P = .006). We concluded that follicular variant of papillary thyroid carcinoma differs from conventional papillary thyroid carcinoma in the rate of BRAF mutation. The results of this study add further information indicating that mutations in BRAF play a role in thyroid cancer development and progression.

Single nucleotide polymorphisms and mRNA expression of VEGF-A in papillary thyroid carcinoma: Potential markers for aggressive phenotypes

BACKGROUND AND OBJECTIVES Polymorphisms of the VEGF gene are known to affect the biological behaviour of cancers but have seldom been studied in thyroid cancer. The aim of the current study is to evaluate the prevalence and relevance of VEGF-A polymorphisms and mRNA expression in papillary thyroid carcinoma (PTC). MATERIALS AND METHODS Genomic DNA and total RNA were isolated from paraffin-embedded tissue from 91 PTC (51 conventional PTC and 40 follicular variant) and 78 control thyroid tissues. Three DNA polymorphisms (+936C > T, +405C > G and -141A > C) in the 3' and 5' untranslated region (3'-UTR, 5'-UTR) of VEGF-A were studied using PCR and RFLP. Also, the mRNA expression of VEGF-A in these tissues was studied by real-time PCR. RESULTS Distribution of polymorphisms in the 5'-UTR (VEGF-VEGF -141A > C and +405C > G) and 3'-UTR (VEGF +936C > T) were all significantly different in PTC and benign thyroid tissue (p = 0.0001, 0.001 and 0.028 respectively). The VEGF -141 C allele was more common in PTC with lymph node metastases (p = 0.026). VEGF + 405 Galleles andVEGF +936 CC genotype were more common in PTC of advanced pathological staging (p = 0.018 and 0.017 respectively). Also, increased expression of VEGF-A mRNA was noted in PTC compared to control (p = 0.009). Within the group of patients with conventional PTC, those with lymph nodal metastases had a higher level of VEGF-A mRNA expression than other patients (p = 0.0003). CONCLUSION These findings suggest that VEGF polymorphisms and mRNA expression may predict the aggressiveness behaviour of thyroid cancer.

Follicular variant of papillary thyroid carcinoma : a diagnostic challenge for clinicians and pathologists

The follicular variant of papillary thyroid carcinoma (FVPTC) presents a type of papillary thyroid cancer that has created continuous diagnosis and treatment controversies among clinicians and pathologists. In this review, we describe the nomenclature, the clinical features, diagnostic problems and the molecular biology of FVPTC. It is important for clinicians to understand this entity as the diagnosis and management of this group of patient may be different from other patients with conventional PTC. The literature suggests that FVPTC behaves in a way similar, clinically, to conventional papillary thyroid carcinoma. However, there are some genotypic differences which may characterise this neoplasm. These parameters may account for the phenotypic variation described by some scientists in this type of cancer. Further understanding can only be achieved by defining strict pathological criteria, in-depth study of the molecular biology and long term follow-up of the optional patients with FVPTC.

Association of FOXE1 Polyalanine Repeat Region with Papillary Thyroid Cancer

CONTEXT: Polyalanine tract variations in transcription factors have been identified for a wide spectrum of developmental disorders. The thyroid transcription factor forkhead factor E1 (FOXE1) contains a polymorphic polyalanine tract with 12-22 alanines. Single-nucleotide polymorphisms (SNP) close to this locus are associated with papillary thyroid cancer (PTC), and a strong linkage disequilibrium block extends across this region. OBJECTIVE: The objective of the study was to assess whether the FOXE1 polyalanine repeat region was associated with PTC and to assess the effect of polyalanine repeat region variants on protein expression, DNA binding, and transcriptional function on FOXE1-responsive promoters. DESIGN: This was a case-control study. SETTING: The study was conducted at a tertiary referral hospital. PATIENTS AND METHODS: The FOXE1 polyalanine repeat region and tag SNP were genotyped in 70 PTC, with a replication in a further 92 PTC, and compared with genotypes in 5767 healthy controls (including 5667 samples from the Wellcome Trust Case Control Consortium). In vitro studies were performed to examine the protein expression, DNA binding, and transcriptional function for FOXE1 variants of different polyalanine tract lengths. RESULTS: All the genotyped SNP were in tight linkage disequilibrium, including the FOXE1 polyalanine repeat region. We confirmed the strong association of rs1867277 with PTC (overall P = 1 × 10(-7), odds ratio 1.84, confidence interval 1.31-2.57). rs1867277 was in tight linkage disequilibrium with the FOXE1 polyalanine repeat region (r(2) = 0.95). FOXE1(16Ala) was associated with PTC with an odds ratio of 2.23 (confidence interval 1.42-3.50; P = 0.0005). Functional studies in vitro showed that FOXE1(16Ala) was transcriptionally impaired compared with FOXE1(14Ala), which was not due to differences in protein expression or DNA binding. CONCLUSIONS: We have confirmed the previous association of FOXE1 with PTC. Our data suggest that the coding polyalanine expansion in FOXE1 may be responsible for the observed association between FOXE1 and PTC.

Caracterização imuno-histoquímica de células do infiltrado inflamatório e de citocinas pró e anti-inflamatórias nos carcinomas mamários caninos

Pós-graduação em Medicina Veterinária - FCAV

Vandetanib in locally advanced or metastatic differentiated thyroid cancer: a randomised, double-blind, phase 2 trial

BACKGROUND No effective standard treatment exists for patients with radioiodine-refractory, advanced differentiated thyroid carcinoma. We aimed to assess efficacy and safety of vandetanib, a tyrosine kinase inhibitor of RET, VEGFR and EGFR signalling, in this setting. METHODS In this randomised, double-blind, phase 2 trial, we enrolled adults (aged ≥18 years) with locally advanced or metastatic differentiated thyroid carcinoma (papillary, follicular, or poorly differentiated) at 16 European medical centres. Eligible patients were sequentially randomised in a 1:1 ratio with a standard computerised scheme to receive either vandetanib 300 mg per day (vandetanib group) or matched placebo (placebo group), balanced by centre. The primary endpoint was progression-free survival (PFS) in the intention-to-treat population based on investigator assessment. This study is registered with ClinicalTrials.gov, number NCT00537095. FINDINGS Between Sept 28, 2007, and Oct 16, 2008, we randomly allocated 72 patients to the vandetanib group and 73 patients to the placebo group. By data cutoff (Dec 2, 2009), 113 (78%) patients had progressed (52 [72%] patients in the vandetanib group and 61 [84%] in the placebo group) and 40 (28%) had died (19 [26%] patients in the vandetanib group and 21 [29%] in the placebo group). Patients who received vandetanib had longer PFS than did those who received placebo (hazard ratio [HR] 0·63, 60% CI 0·54-0·74; one-sided p=0·008): median PFS was 11·1 months (95% CI 7·7-14·0) for patients in the vandetanib group and 5·9 months (4·0-8·9) for patients in the placebo group. The most common grade 3 or worse adverse events were QTc prolongation (ten [14%] of 73 patients in the vandetanib group vs none in the placebo group), diarrhoea (seven [10%] vs none), asthenia (five [7%] vs three [4%]), and fatigue (four [5%] vs none). Two patients in the vandetanib group and one in the placebo group died from treatment-related serious adverse events (haemorrhage from skin metastases and pneumonia in the vandetanib group and pneumonia in the placebo group). INTERPRETATION Vandetanib is the first targeted drug to show evidence of efficacy in a randomised phase 2 trial in patients with locally advanced or metastatic differentiated thyroid carcinoma. Further investigation of tyrosine-kinase inhibitors in this setting is warranted. FUNDING AstraZeneca.

The expression profiles of the galectin gene family in primary and metastatic papillary thyroid carcinoma with particular emphasis on galectin-1 and galectin-3 expression

INTRODUCTION: Galectin family members have been demonstrated to be abnormally expressed in cancer at the protein and mRNA level. This study investigated the levels of galectin proteins and mRNA expression in a large cohort of patients with papillary thyroid carcinoma and matched lymph node metastases with particular emphasis on galectin-1 and galectin-3. METHODS: mRNA expression of galectin family members (1, 2, 3, 4, 7, 8, 9, 10 and 12) were analysed by real-time polymerase chain reaction in 65 papillary thyroid carcinomas, 30 matched lymph nodes with metastatic papillary thyroid carcinoma and 5 non-cancer thyroid tissues. Galectin-1 and 3 protein expression was determined by immunohistochemistry in these samples. RESULTS: Significant expression differences in all tested galectin family members (1, 2, 3, 4, 7, 8, 9, 10 and 12) were noted for mRNA in papillary thyroid carcinomas, with and without lymph node metastasis. Galectin-1 protein was more strongly expressed than galectin-3 protein in papillary thyroid carcinoma. Galectin-1 protein was found to be overexpressed in 32% of primary papillary thyroid carcinomas. A majority of lymph nodes with metastatic papillary thyroid carcinoma (53%) had significantly increased expression of galectin-1 protein, as did 47% of primaries with metastases. Galectin-1 mRNA levels were decreased in the vast majority (94%) of primary thyroid carcinomas that did not have metastases present. Galectin-3 protein levels were noted to be overexpressed in 15% of primary papillary thyroid carcinomas. In primary papillary thyroid carcinoma with lymph node metastases, 32% had over expression of galectin-3 protein. Overexpression of galectin-3 mRNA was noted in 58% of papillary thyroid carcinomas and 64% of lymph nodes bearing metastatic papillary thyroid carcinoma. Also, primary papillary thyroid carcinoma with lymph node metastases had significantly higher expression of galectin-3 mRNA compared to those without lymph node metastases. CONCLUSION: Galectin family members show altered expression at the mRNA level in papillary thyroid cancers. Overexpression of galectin-1 and 3 proteins were noted in papillary thyroid carcinoma with lymph node metastases. The results presented here demonstrated that galectin-1 and galectin-3 expression have important roles in clinical progression of papillary thyroid carcinoma.

Expression profile of endothelin 1 and its receptor endothelin receptor A in papillary thyroid carcinoma and their correlations with clinicopathologic characteristics

The endothelin axis is a group of signaling molecules and their receptors that have been implicated in vascularization of cancers, with their expression being observed to change in different cancer types. In this research, we examined the expression of endothelin 1 and endothelin receptor A at the protein and messenger RNA (mRNA) levels in 123 papillary thyroid carcinomas and 40 matched lymph nodes with metastatic papillary thyroid carcinomas. We found altered endothelin axis mRNA expression in several clinicopathologic parameters with increased endothelin 1 expression in thyroid papillary carcinoma showing stromal calcification, cancers in men, and primary cancers with lymph node metastases. Increased endothelin receptor A mRNA expression was noted in the larger cancers. There is a significant correlation between expression of endothelin receptor A and endothelin 1 in papillary thyroid carcinoma. Both endothelin receptor A and endothelin 1 mRNA expressions were significantly higher in metastatic carcinoma in the lymph node than in primary thyroid cancer. The metastatic carcinoma in the lymph node had increased expression compared with matched primary thyroid carcinoma. Expressions of endothelin 1 and endothelin receptor A were also documented as being high at the protein level. Our results indicate that in thyroid cancer, endothelin 1 and endothelin receptor A are associated with growth in advanced stages and lymph node metastases, likely through known angiogenic linkages. Targeting the endothelin axis may be useful in planning angiogenesis therapy for thyroid cancer.

Prospective, non-randomized phase 2 clinical trial of carboplatin plus paclitaxel with sequential radical pelvic radiotherapy for uterine papillary serous carcinoma

Objective Uterine Papillary Serous Carcinoma (UPSC) is uncommon and accounts for less than 5% of all uterine cancers. Therefore the majority of evidence about the benefits of adjuvant treatment comes from retrospective case series. We conducted a prospective multi-centre non-randomized phase 2 clinical trial using four cycles of adjuvant paclitaxel plus carboplatin chemotherapy followed by pelvic radiotherapy, in order to evaluate the tolerability and safety of this approach. Methods This trial enrolled patients with newly diagnosed, previously untreated patients with stage 1b-4 (FIGO-1988) UPSC with a papillary serous component of at least 30%. Paclitaxel (175 mg/m2) and carboplatin (AUC 6) were administered on day 1 of each 3-week cycle for 4 cycles. Chemotherapy was followed by external beam radiotherapy to the whole pelvis (50.4 Gy over 5.5 weeks). Completion and toxicity of treatment (Common Toxicity Criteria, CTC) and quality of life measures were the primary outcome indicators. Results Twenty-nine of 31 patients completed treatment as planned. Dose reduction was needed in 9 patients (29%), treatment delay in 7 (23%), and treatment cessation in 2 patients (6.5%). Hematologic toxicity, grade 3 or 4 occurred in 19% (6/31) of patients. Patients' self-reported quality of life remained stable throughout treatment. Thirteen of the 29 patients with stages 1–3 disease (44.8%) recurred (average follow up 28.1 months, range 8–60 months). Conclusion This multimodal treatment is feasible, safe and tolerated reasonably well and would be suitable for use in multi-institutional prospective randomized clinical trials incorporating novel therapies in patients with UPSC.

Co-regulatory potential of vascular endothelial growth factor–A and vascular endothelial growth factor–C in thyroid carcinoma

Vascular endothelial growth factor (VEGF) promotes growth of blood or lymphatic vessels. The aim of the current study is to identify relationships between VEGF-A and VEGF-C, and their impact in angiogenesis and metastases in thyroid cancers. VEGF-A and VEGF-C mRNA and protein expression was investigated in 136 thyroid cancers (123 papillary thyroid carcinomas and 13 undifferentiated thyroid carcinomas) and 40 matched lymph node metastases with papillary thyroid carcinoma using reverse transcription polymerase chain reaction and immunohistochemistry. VEGF-A and VEGF-C mRNA expression was significantly different between conventional papillary thyroid carcinoma, follicular variant of papillary thyroid carcinoma, and undifferentiated thyroid carcinomas (P = 1 x 10(-6) and 1 x 10(-5), respectively). In undifferentiated carcinoma, VEGF-A and VEGF-C protein overexpression was noted in all cases. VEGF-A and VEGF-C mRNA overexpression was noted in 51% (n = 62) and 27% (n = 33) of the papillary thyroid carcinomas, whereas VEGF-A and VEGF-C protein overexpression was also identified in 70% (n = 86) and 62% (n = 76) of the carcinomas. VEGF-A mRNA was significantly higher in cancers with lymph node metastases compared with nonmetastatic cancers (P = .001), whereas most metastatic cancers underexpressed VEGF-C (P = .0002), with a similar trend for protein. The expression of VEGF-A and VEGF-C correlated with each other at both mRNA and protein levels (P = .00004 and .003, respectively). In summary, VEGF-A and -C expressions correlate with the pathological parameters and metastatic status of thyroid carcinomas. The significant correlations between the expressions of these genes add weight to hypotheses concerning VEGF-A and -C interaction in cancer progression.

Clonal characteirization of sporadic cribriform-morular variant of papillary thyroid carcinoma by laser microdissection based APC mutation analysis

Cribriform-morular variant (C-MV) of papillary thyroid carcinoma (PTC) is a rare and unusual neoplasm composed of multiple histologic components, including cribriform, papillary, solid, tall columnar, and morular patterns. Analyses of gross C-MV of PTC lesions has linked adenomatous polyposis coli (APC) mutations to its pathogenesis; however, the extent of involvement of mutations in the development Of individual components is unclear We report on bidirectional sequencing of the mutation cluster region (codons 1032-1565) of the APC gene in individually laser-microdissected components of a previously unreported C-MV of PTC. A silent Thr1493Thr gene variant was found in all tumoral components, whereas a 5-base-pair frameshift deletion at codon 1309 was identified only in the morules. Neither variant was observed in matched normal thyroid tissue. These results show the histologic components of C-MV of PTC to have some common mutational background, although additional somatic mutations may be involved in the development of morular structures.