Gianfranco Fenzi

Detection of RET/PTC, TRK and BRAF mutations in preoperative diagnosis of thyroid nodules with indeterminate cytological findings

Background  Fine‐needle aspiration biopsy (FNAB) is the primary means to distinguish benign from malignant nodules and select patients for surgery. However, adjunctive diagnostic tests are needed because in 20–40% of cases the FNAB result is uncertain.

Sonic hedgehog-induced type 3 deiodinase blocks thyroid hormone action enhancing proliferation of normal and malignant keratinocytes

The Sonic hedgehog (Shh) pathway plays a critical role in hair follicle physiology and is constitutively active in basal cell carcinomas (BCCs), the most common human malignancy. Type 3 iodothyronine deiodinase (D3), the thyroid hormone-inactivating enzyme, is frequently expressed in proliferating and neoplastic cells, but its role in this context is unknown. Here we show that Shh, through Gli2, directly induces D3 in proliferating keratinocytes and in mouse and human BCCs. We demonstrate that Gli-induced D3 reduces intracellular active thyroid hormone, thus resulting in increased cyclin D1 and keratinocyte proliferation. D3 knockdown caused a 5-fold reduction in the growth of BCC xenografts in nude mice. Shh-induced thyroid hormone degradation via D3 synergizes with the Shh-mediated reduction of the type 2 deiodinase, the thyroxine-activating enzyme, and both effects are reversed by cAMP. This previously unrecognized functional cross-talk between Shh/Gli2 and thyroid hormone in keratinocytes is a pathway by which Shh produces its proliferative effects and offers a potential therapeutic approach to BCC.

Iodide excess induces apoptosis in thyroid cells through a p53-independent mechanism involving oxidative stress.

Thyroid toxicity of iodide excess has been demonstrated in animals fed with an iodide-rich diet; in vitro iodide is cytotoxic, inhibits cell growth, and induces morphological changes in thyroid cells of some species. In this study, we investigated the effect of iodide excess in an immortalized thyroid cell line (TAD-2) in primary cultures of human thyroid cells and in cells of nonthyroid origin. Iodide displayed a dose-dependent cytotoxicity in both TAD-2 and primary thyroid cells, although at different concentrations, whereas it had no effect on cells of nonthyroid origin. Thyroid cells treated with iodide excess underwent apoptosis, as evidenced by morphological changes, plasma membrane phosphatidylserine exposure, and DNA fragmentation. Apoptosis was unaffected by protein synthesis inhibition, whereas inhibition of peroxidase enzymatic activity by propylthiouracil completely blocked iodide cytotoxicity. During KI treatment, reactive oxygen species were produced, and lipid peroxide levels increased mark...

The Primary Occurrence of BRAFV600E Is a Rare Clonal Event in Papillary Thyroid Carcinoma

CONTEXT BRAF(V600E) is considered a primary event, a negative prognostic marker, and a site for pharmacological intervention in papillary thyroid carcinoma (PTC). We asked whether BRAF(V600E) can occur as a subclonal event in PTC and whether this and other oncogenes can coexist in the same tumor. STUDY DESIGN We determined by pyrosequencing the percentage of mutant BRAF, NRAS, and KRAS alleles in a series of conventional PTC. We also analyzed the BRAF mutation status in PTC cell clones in culture. RESULTS BRAF(V600E) alleles were present in 41 of 72 PTC (56.9%) in the range 44.7 to 5.1% of total BRAF alleles. In four PTC samples, mutant BRAF alleles were about 50%, being therefore compatible with a clonal heterozygous mutation. In 27 PTC samples, BRAF(V600E) alleles were in the range of 25 to 5.1%. This finding was confirmed after exclusion of the presence of a large contamination by lymphoreticular cells and by the analysis of PTC cells selected by laser capture. Analysis of clones derived from a single cell confirmed the presence of two distinct PTC populations with wild-type or mutated BRAF. Simultaneous subclonal BRAF and KRAS mutations were demonstrated in two PTC. CONCLUSIONS These data demonstrate that clonal BRAF(V600E) is a rare occurrence in PTC, although frequently this cancer consists of a mixture of tumor cells with wild-type and mutant BRAF. These results suggest that BRAF mutation in PTC is a later subclonal event, its intratumoral heterogeneity may hamper the efficacy of targeted pharmacotherapy, and its association with a more aggressive disease should be reevaluated.

Combined analysis of galectin-3 and BRAFV600E improves the accuracy of fine-needle aspiration biopsy with cytological findings suspicious for papillary thyroid carcinoma

Ten to fifteen percent of fine-needle aspiration biopsy (FNAB) of thyroid nodules are indeterminate. Galectin-3 (Gal-3) and the oncogene BRAFV600E are markers of malignancy useful to improve FNAB accuracy. The objective of this study was to determine whether the combined analysis of Gal-3 and BRAFV600E expression in thyroid aspirates could improve the diagnosis in FNAB with suspicious cytological findings. Two hundred and sixty-one surgical thyroid tissues and one hundred and forty-four thyroid aspirates were analyzed for the presence of the two markers. In surgical specimens, Gal-3 expression was present in 27.4% benign nodules, 91.9% papillary (PTC) and 75% follicular (FTC) thyroid carcinomas. BRAFV600E was not detected in 127 benign nodules, as well as in 32 FTCs, while was found in 42.9% PTC. No correlation was found between BRAF mutation and Gal-3 expression. Forty-seven consecutive FNAB suspicious for PTC were analyzed for the presence of the two markers. Of these nodules, 23 were benign at histology, 6 were positive for Gal-3, none displayed BRAFV600E, and 17 were negative for both the markers. Twenty suspicious nodules were diagnosed as PTC and four FTCs at histology. Of these 24 carcinomas, 9 resulted positive for BRAFV600E, 17 for Gal-3, and 22 for one or both the markers. The sensitivity, specificity, and accuracy for the presence of Gal-3 and/or BRAFV600E were significantly higher than those obtained for the two markers alone. Notably, the negative predictive value increased from 70.8 to 89.5%. In conclusion, the combined detection of Gal-3 and BRAFV600E improves the diagnosis in FNAB with cytological findings suspicious for PTC and finds clinical application in selected cases.

Cloning, chromosomal localization and identification of polymorphisms in the human thyroid transcription factor 2 gene (TITF2).

The human gene encoding the thyroid transcription factor 2 (TTF-2) was cloned and mapped to human chromosome 9q22. Three polymorphisms were identified in the gene by SSCP and direct sequencing: two consist of a third base substitution in the triplet encoding Leu129 and Ser273, and the third is an alanine stretch that varies from 12 to 17 residues. TTF-2 plays a critical role during thyroid morphogenesis in mice, and in man the TITF2 gene is associated with congenital hypothyroidism and cleft palate with thyroid dysgenesis. The polymorphisms identified in this study can be used as markers to study the role of the TITF2 gene in other cases of thyroid dysgenesis, especially in familial cases.

High Growth Rate of Benign Thyroid Nodules Bearing RET/PTC Rearrangements

CONTEXT Benign thyroid nodules display a broad range of behaviors from a stationary size to a progressive growth. The RET/PTC oncogene has been documented in a fraction of benign thyroid nodules, besides papillary thyroid carcinomas, and it might therefore influence their growth. OBJECTIVE The aim of the present work was to evaluate whether RET/PTC in benign thyroid nodules associates with a different nodular growth rate. STUDY DESIGN In this prospective multicentric study, 125 subjects with benign nodules were included. RET rearrangements were analyzed in cytology samples; clinical and ultrasonographic nodule characteristics were assessed at the start and at the end of the study. RESULTS RET/PTC was present in 19 nodules. The difference between the mean baseline nodular volume of the RET/PTC- and RET/PTC+ nodules was not significant. After 36 months of follow-up, the RET/PTC+ group (n = 16) reached a volume higher than the RET/PTC- group (n = 90) (5.04 ± 2.67 vs. 3.04 ± 2.26 ml; P = 0.0028). We calculated the monthly change of nodule volumes as a percentage of baseline. After a mean follow-up of 36.6 months, the monthly volume increase of nodules bearing a RET rearrangement was 4.3-fold that of nodules with wild-type RET (1.83 ± 1.2 vs. 0.43 ± 1.0% of baseline volume; P < 0.0001). CONCLUSIONS Benign thyroid nodules bearing RET rearrangements grow more rapidly than those with wild-type RET. Searching for RET rearrangements in benign thyroid nodules might be useful to the clinician in choosing the more appropriate and timely therapeutic option.

Transcription factor Nkx-2.5 induces sodium/iodide symporter gene expression and participates in retinoic acid- and lactation-induced transcription in mammary cells

ABSTRACT The sodium/iodide symporter (NIS) is a plasma membrane protein that mediates active iodide transport in thyroid and mammary cells. It is a prerequisite for radioiodide treatment of thyroid cancer and a promising diagnostic and therapeutic tool for breast cancer. We investigated the molecular mechanisms governing NIS expression in mammary cells. Here we report that Nkx-2.5, a cardiac homeobox transcription factor that is also expressed in the thyroid primordium, is a potent inducer of the NIS promoter. By binding to two specific promoter sites (N2 and W), Nkx-2.5 induced the rNIS promoter (about 50-fold over the basal level). Interestingly, coincident with NIS expression, Nkx-2.5 mRNA and protein were present in lactating, but not virgin, mammary glands in two human breast cancer samples and in all-trans retinoic acid (tRA)-stimulated MCF-7 breast cancer cells. A cotransfected dominant-negative Nkx-2.5 mutant abolished tRA-induced endogenous NIS induction, which shows that Nkx-2.5 activity is critical for this process. Remarkably, in MCF-7 cells, Nkx-2.5 overexpression alone was sufficient to induce NIS and iodide uptake. In conclusion, Nkx-2.5 is a novel relevant transcriptional regulator of mammary NIS and could thus be exploited to manipulate NIS expression in breast cancer treatment strategies.

A novel NKX2.1 mutation in a family with hypothyroidism and benign hereditary chorea.

BACKGROUND We studied a boy with congenital hypothyroidism, benign hereditary chorea, and respiratory distress. His mother and his grandfather were affected by hypothyroidism with a late onset and benign hereditary chorea. The aim of this study was to establish the genetic defects that cause that phenotype and study the molecular mechanisms of the pathology. METHODS NKX2.1, PAX8, NKX2.5, and TAZ genes were sequenced. RESULTS Direct sequencing of the NKX2.1 gene showed, in all the affected, a new heterozygous mutation from cytosine to adenine in the second base of the triplet encoding for the amino acid at position 145. The mutation (C609A) is responsible for a change from serine to a stop codon (S145X). We also demonstrated that the mutant protein is predominantly in the cytoplasm and unable to translocate into the nucleus. Of note, the S145X mutation produces variable phenotypes in the affected members of the family. No mutations have been identified in the NKX2.5, PAX8, and TAZ genes. CONCLUSIONS Our study extends the knowledge of the functional effect of NKX2.1 mutations and further highlights the complexities of genotype-phenotype correlation in the NKX2.1 deficiency syndromes.

Prenyltransferase Inhibitors Induce Apoptosis in Proliferating Thyroid Cells through a p53-Independent, CrmA-Sensitive, and Caspase-3-Like Protease- Dependent Mechanism*

The inhibitors of protein prenylation have been proposed for chemotherapy of tumors. Lovastatin, a 3-hydroxy-3-methylglutaryl-Coenzyme A (HMG-CoA) reductase inhibitor, displays proapoptotic activity in tumor cells blocking the synthesis of isoprenoids compounds. To test whether HMG-CoA reductase inhibition can induce apoptosis in proliferating thyroid cells, we studied the effects of lovastatin in normal and neoplastic thyroid cells and in primary cultures from normal human thyroids. In an immortalized human thyroid cell line (TAD-2) and in neoplastic cells, lovastatin induced cell rounding within 24 h of treatment. After 48 h the cells were detached from the plate and underwent apoptosis, as evidenced by DNA fragmentation. Morphological changes and apoptosis did not occur in serum-starved quiescent TAD-2 cells or in primary cultures of normal thyrocytes. Mevalonate, the product of the HMG-CoA reductase enzymatic activity, and the protein synthesis inhibitor cycloheximide completely blocked the effects of...