Silvia Cantara

Impact of Proto-Oncogene Mutation Detection in Cytological Specimens from Thyroid Nodules Improves the Diagnostic Accuracy of Cytology

CONTEXT Fine-needle aspiration cytology (FNAC) is the gold standard for the differential diagnosis of thyroid nodules but has the limitation of inadequate sampling or indeterminate lesions. OBJECTIVE We aimed to verify whether search of thyroid cancer-associated protooncogene mutations in cytological samples may improve the diagnostic accuracy of FNAC. STUDY DESIGN One hundred seventy-four consecutive patients undergoing thyroid surgery were submitted to FNAC (on 235 thyroid nodules) that was used for cytology and molecular analysis of BRAF, RAS, RET, TRK, and PPRgamma mutations. At surgery these nodules were sampled to perform the same molecular testing. RESULTS Mutations were found in 67 of 235 (28.5%) cytological samples. Of the 67 mutated samples, 23 (34.3%) were mutated by RAS, 33 (49.3%) by BRAF, and 11 (16.4%) by RET/PTC. In 88.2% of the cases, the mutation was confirmed in tissue sample. The presence of mutations at cytology was associated with cancer 91.1% of the times and follicular adenoma 8.9% of the time. BRAF or RET/PTC mutations were always associated with cancer, whereas RAS mutations were mainly associated with cancer (74%) but also follicular adenoma (26%). The diagnostic performance of molecular analysis was superior to that of traditional cytology, with better sensitivity and specificity, and the combination of the two techniques further contributed to improve the total accuracy (93.2%), compared with molecular analysis (90.2%) or traditional cytology (83.0%). CONCLUSIONS Our findings demonstrate that molecular analysis of cytological specimens is feasible and that its results in combination with cytology improves the diagnostic performance of traditional cytology.

Short telomeres, telomerase reverse transcriptase gene amplification, and increased telomerase activity in the blood of familial papillary thyroid cancer patients

BACKGROUND Differentiated papillary thyroid cancer is mostly sporadic, but the recurrence of the familial form has been reported. Short or dysfunctional telomeres have been associated with familial benign diseases and familial breast cancer. OBJECTIVE The aim of our work was to study the telomere-telomerase complex in the peripheral blood of patients with familial papillary thyroid cancer (FPTC), including the measurement of relative telomere length (RTL), telomerase reverse transcriptase (hTERT) gene amplification, hTERT mRNA expression, telomerase protein activity, and search of hTERT or telomerase RNA component gene mutations. PATIENTS Cumulating a series of patients seen at the University of Siena and a series at the University of Rome, the experiments were conducted in 47 FPTC patients, 75 sporadic papillary thyroid cancer (PTC) patients, 20 patients with nodular goiter, 19 healthy subjects, and 20 unaffected siblings of FPTC patients. RESULTS RTL, measured by quantitative PCR, was significantly (P < 0.0001) shorter in the blood of FPTC patients, compared with sporadic PTCs, healthy subjects, nodular goiter subjects, and unaffected siblings. Also by fluorescence in situ hybridization analysis, the results confirmed shorter telomere lengths in FPTC patients (P = 0.01). hTERT gene amplification was significantly (P < 0.0001) higher in FPTC patients, compared with the other groups, and in particular, it was significantly (P = 0.03) greater in offspring with respect to parents. hTERT mRNA expression, as well as telomerase activity, was significantly higher (P = 0.0003 and P < 0.0001, respectively) in FPTC patients, compared with sporadic PTCs. RTL, measured in cancer tissues, was shorter (P < 0.0001) in FPTC patients, compared with sporadic PTCs. No mutations of the telomerase RNA component and hTERT genes were found. CONCLUSION Our study demonstrates that patients with FPTC display an imbalance of the telomere-telomerase complex in the peripheral blood, characterized by short telomeres, hTERT gene amplification, and expression. These features may be implicated in the inherited predisposition to develop FPTC.

Exogenous BH4/Bcl-2 Peptide Reverts Coronary Endothelial Cell Apoptosis Induced by Oxidative Stress

Background: Vascular endothelium undergoes apoptosis when exposed to reactive oxygen species (ROS), including hydrogen peroxide and superoxide radicals. ROS are believed to be the cause of damage to small vessels during ischemia-reperfusion injury and of arterial damage during atherosclerosis. Hydrogen peroxide-induced apoptosis is mediated through the inhibition of Bcl-xl activity and caspase-3 and caspase-9 activation. The BH4 domain of the Bcl-2 family members is responsible for their antiapoptotic activity. The BH4 domains of Bcl-2 and Bcl-xl inhibit cytochrome c release and the loss of mitochondrial membrane potential. Methods and Results: The purpose of this project was to study the antiapoptotic effect of cell-permeant derivative of Bcl-2 (BH4 peptide) on endothelial cells exposed to stress conditions. BH4 peptide was conjugated to the cell-permeable peptide TAT and was applied to endothelial cells under conditions of serum starvation and hydrogen peroxide treatment. TAT-BH4 reduced caspase-3 activity and prevented apoptotic cell death. Conclusion: Our results indicate that TAT-BH4 peptide can protect endothelial cells from ROS-induced apoptosis.

Physiological levels of amyloid peptides stimulate the angiogenic response through FGF-2.

Amyloid β peptides (Aβ) form insoluble aggregates in Alzheimers disease. Accumulation of misfolded amyloid fibrils is generally believed to be a key pathogenic event in several brain disorders. Here we show that small amounts of Aβ peptides activate angiogenesis by promoting endothelial cell proliferation and migration as well as pseudocapillary formation. Aβ peptides functionally synergize with fibroblast growth factor (FGF‐2) to promote c‐Raf and ERK1/2 activation and angiogenesis in vivo. Thus, Aβ peptides at nanomolar concentrations prime FGF‐ 2 effects on the endothelium, enhancing survival and sustaining angiogenesis. The angiogenesis promoted by Aβ peptides via FGF‐2 might have implications for understanding the initial stages of Alzheimers disease and for the design of therapies targeting β amyloid.

Telomere Abnormalities and Chromosome Fragility in Patients Affected by Familial Papillary Thyroid Cancer

INTRODUCTION Genomic instability has been proposed to play a role in cancer development and can occur through different mechanisms including telomere association and telomere loss. Studies carried out in our unit have demonstrated that familial papillary thyroid cancer (fPTC) patients display an imbalance, at the germinal level, in telomere-telomerase complex. AIM We aimed to verify whether familial fPTC patients show an increased spontaneous chromosome fragility. METHODS To this purpose, we compared telomeric fusions and associations as well as other chromosomal fragility features by conventional and molecular cytogenetic analyses, in phytohemagglutinin stimulated T-lymphocytes from fPTC patients, unaffected family members, sporadic papillary thyroid cancer patients, and healthy subjects. RESULTS We demonstrate that fPTC patients have a significant increase in spontaneous telomeric associations and telomeric fusions compared with healthy subjects and sporadic cases in the frame of an otherwise common spontaneous chromosome fragility pattern. A quantitative fluorescence in situ hybridization analysis demonstrates that familial cases display a significant decrease in the telomeric peptide nucleic acid-fluorescence in situ hybridization signal intensity in the metaphase chromosome. Moreover, three copies of the hTERT gene were found only in familial cases, although the result was not statistically significant. CONCLUSIONS These results contribute in defining familial thyroid cancer as a clinical entity characterized by an altered telomere stability, which may be associated with the predisposition to develop the familial form of thyroid cancer.

FGF-2 overexpression opposes the beta amyloid toxic injuries to the vascular endothelium.

Recent evidences suggest that Aβ peptides modulate endothelial cell (EC) functions. At low concentrations, Aβ1–40 enhances the pro-angiogenic activity of FGF-2, whereas deposition of excess Aβ causes EC dysfunction and cerebral amyloid angiopathy (CAA). We investigated whether FGF-2 attenuates EC dysfunction caused by pathological Aβ levels. We studied Aβ1–40 on EC survival, as well as on signals responsible of their angiogenic phenotype. At 5–50 μM Aβ1–40 reduced EC population, caused apoptosis, downregulated FGF-2 production, inhibited FGF-2 binding to heparin, and FGFR1 phosphorylation. Toxic effects were owing to lack of FGF-2 stimulation, as EC overexpressing FGF-2 displayed extraordinary resistance to Aβ1–40 injuries. The FGF-2 mechanism responsible for reversing damages, involves the downstream enhancement of Akt, a pathway independent of eNOS activation. In conclusion, we demonstrate that FGF-2 protects EC from the effects of excess Aβ1–40, suggesting that it may attenuate the consequences of Aβ deposition in pathologies as CAA.

Telomere Length in Neoplastic and Nonneoplastic Tissues of Patients with Familial and Sporadic Papillary Thyroid Cancer

INTRODUCTION Many studies have found an association between altered telomere length (TL), both attrition or elongation, and cancer phenotype. Recently, we have reported that patients with the familial form of papillary thyroid cancer (FPTC) have short telomeres in blood leucocytes. AIM To evaluate relative TL (RTL) at somatic level in neoplastic and nonneoplastic tissues of patients with FPTC (n = 30) and sporadic PTC (n = 46). METHODS RTL was measured by quantitative PCR in neoplastic thyroid tissues, in the corresponding nontumor thyroid tissues (normal contralateral thyroid), and in other extrathyroidal tissues (lymph nodes, muscles, or buccal mucosa). RTL was also measured in adenomas and hyperplastic nodules. In a subset of samples, telomerase expression was measured by quantitative PCR. RESULTS Mean ± SD RTL of FPTC patients was short in neoplastic thyroid tissues (0.87 ± 0.2) with no difference from the normal contralateral thyroid tissues (0.85 ± 0.11) and extrathyroidal tissues (0.85 ± 0.31). On the contrary, in patients with sporadic PTC, the mean ± SD RTL in the neoplastic tissues (1.73 ± 0.63) was significantly shorter than that found in normal contralateral tissues (2.58 ± 0.89) and extrathyroidal tissues (2.5 ± 0.86). For all tissue samples (cancer, normal thyroid, and nonthyroidal tissues) the mean ± SD RTL of familial cases was shorter (P < 0.0001) than that found in tissues from sporadic PTC. RTL of FPTC was also lower (P < 0.0001) than that of 23 follicular adenomas (1.6 ± 0.7) and 24 hyperplastic nodules (2.2 ± 0.9). CONCLUSIONS Our results demonstrate that short telomeres are a consistent feature of PTC, which in familial cases, is not restricted to the tumor tissue. This finding suggests that FPTC has a distinct, heritable, genetic background.

Telomeres and thyroid cancer.

Telomeres are specialized structures at the ends of chromosomes, consisting of hundreds of repeated hexanucleotides (TTAGGG)n. Genetic integrity is partly maintained by the architecture of telomeres and it is gradually lost as telomeres progressively shorten with each cell replication, due to incomplete lagging DNA strand synthesis and oxidative damage. Telomerase is a reverse transcriptase enzyme that counteracts telomere shortening by adding telomeric repeats to the G-rich strand. It is composed of a telomerase RNA component and a protein component, telomerase reverse transcriptase. In the absence of telomerase or when the activity of the enzyme is low compared to the replicative erosion, apoptosis is triggered. Patients who have inherited genetic defects in telomere maintenance seem to have an increased risk of developing familial benign diseases or malignant diseases. At the somatic level, telomerase is reactivated in the majority of human carcinomas, suggesting that telomerase reactivation is a critical step for cancerogenesis. In sporadic thyroid carcinoma telomerase activity is detectable in nearly 50% of thyroid cancer tissues and some authors proposed that the detection of telomerase activity may be used for differentiating between benign and malignant thyroid tumours. Recently a germline alteration of telomere-telomerase complex has been identified in patients with familial papillary thyroid cancer, characterized by short telomeres and increased expression and activity of telomerase compared to patients with sporadic papillary thyroid cancer. In this report, we will review the role of telomere-telomerase complex in sporadic and familial thyroid cancer.

TAT-BH4 counteracts Aβ toxicity on capillary endothelium

Oxidative stress is one of the factor contributing to blood brain barrier degeneration. This phenomenon is observed during pathological conditions such as Alzheimers disease or cerebral amyloid angiopathy in which brain haemorrhages are very frequent. Both diseases are characterized by beta amyloid peptide deposition either in neurons or in vessels. Oxidative stress leads to impairment of mitochondrial functions and apoptotic cell death subsequent to caspases activation. In this paper we demonstrate that BH4 domain of Bcl‐xl administrated to endothelial cells as the conjugated form with TAT peptide, reverts Aβ‐induced apoptotic cell death by activating a survival programme which is Akt/endothelial nitric oxide synthase dependent.

Reappraisal of the indication for radioiodine thyroid ablation in differentiated thyroid cancer patients

Abstract Radioactive iodine therapy is administered to patients with differentiated thyroid cancer (DTC) for eradication of thyroid remnant after total thyroidectomy or, in patients with metastatic disease, for curative or palliative treatment. In past years, thyroid remnant ablation was indicated in almost every patient with a diagnosis of DTC. Nowadays, careful revision of patients’ outcome has introduced the concept of risk-based selection of patients candidate to thyroid remnant ablation. The present review aims to underline the indications for thyroid remnant ablation and to address methodologies to be employed.