Estrogen and thyroid cancer is a stem affair: A preliminary study.

Gender influences Papillary Thyroid Cancer (PTC) with an incidence of 3:1 when comparing women to men with different aggressiveness. This gender discrepancy suggests some role of sex hormones in favoring the malignant progression of thyroid tissue to cancer. Estrogens are known to promote Stem Cell self-renewal and, therefore, may be involved in tumor initiation. The goals of these studies are to investigate the underlying causes of gender differences in PTC by studying the specific role of estrogens on tumor cells and their involvement within the Cancer Stem Cell (CSC) compartment. Exposure to 1nmoll-1 Estradiol for 24h promotes growth and maintenance of PTC Stem Cells, while inducing dose-dependent cellular proliferation and differentiation following Estradiol administration. Whereas mimicking a condition of hormonal imbalance led to an opposite phenotype compared to a continuous treatment. In vivo we find that Estradiol promotes motility and tumorigenicity of CSCs. Estradiol-treated mice inoculated with Thyroid Cancer Stem Cell-enriched cells developed larger tumor masses than control mice. Furthermore, Estradiol-pretreated Cancer Stem cells migrated to distant organs, while untreated cells remained circumscribed. We also find that the biological response elicited by estrogens on Papillary Thyroid Cancer in women differed from men in pathways mediated. This could explain the gender imbalance in tumor incidence and development and could be useful to develop gender specific treatment of (PTC).

Estrogens and stem cells in thyroid cancer.

Recent discoveries highlight the emerging role of estrogens in the initiation and progression of different malignancies through their interaction with stem cell (SC) compartment. Estrogens play a relevant role especially for those tumors bearing a gender disparity in incidence and aggressiveness, as occurs for most thyroid diseases. Although several experimental lines suggest that estrogens promote thyroid cell proliferation and invasion, their precise contribution in SC compartment still remains unclear. This review underlines the interplay between hormones and thyroid function, which could help to complete the puzzle of gender discrepancy in thyroid malignancies. Defining the association between estrogen receptors' status and signaling pathways by which estrogens exert their effects on thyroid cells is a potential tool that provides important insights in pathogenetic mechanisms of thyroid tumors.

Prevalence, tumorigenic role, and biochemical implications of rare BRAF alterations.

BACKGROUND: Papillary thyroid carcinoma (PTC) is the most common malignant tumor of the thyroid gland, accounting for 74-80% of all thyroid cancers. The 1799T>A transversion is an activating mutation of the BRAF oncogene that is common in and specific to conventional PTC. We studied the prevalence, tumorigenic role, and biochemical implications of rare BRAF variants in a large cohort of patients. METHODS: A total of 2131 fine-needle aspiration biopsy samples were collected and subjected to BRAF mutation analysis. BRAF genetic variants were analyzed by Western blot, immunofluorescence, and in silico analysis. RESULTS: BRAF mutations were found in 50% (347/700) of thyroid cancers (644 PTCs, 22 anaplastic thyroid carcinomas, 34 follicular thyroid carcinomas). They were the classic (c.1799T>A, p.V600E) mutation in 96.8% (336/347) and rare genetic variants in 3.2% (11/347). In all, five infrequent BRAF alterations were detected: (i) c.1795_1797dupACA (p.T599dup); (ii) c.1801A>G (p.K601E); (iii) c.1799_1801delTGA (p.V600_K601>E); (iv) c.1799_1814>A (p.V600_S605>D); and (v) c.1798_1810delinsA (p.V600_W604>R). The last BRAF variant has never been described in the literature. Western blot analysis and immunofluorescence both revealed a variegated reactivity pattern, again emphasizing the peculiar role of every specific BRAF genetic alteration. In silico analysis of the samples studied revealed a stabilization of the "active" geometrical conformation of the B-raf enzyme associated with the activated and productive state of the kinase domain. CONCLUSIONS: Rare BRAF variants were found in 1.6% of all thyroid malignancies, all clustered around the codon V600, in the binding pocket named A-loop, confirming its crucial role in the enzymatic activation of the B-Raf protein. These mutations were associated mainly with the activation of key effectors in the mitogen-activated protein kinase pathway, but a simultaneous stimulation of the PI3k/Akt cascade was demonstrated in some cases. The rare BRAF variants were not generally associated with an aggressive behavior of the PTC. To our knowledge, this is the largest series of thyroid cancers analyzed to identify and functionally characterize rare BRAF variants.

Incidental medullary thyroid microcarcinoma revealed by mild increase of preoperative serum calcitonin levels: therapeutic implications.

To investigate whether further, diagnostic procedures should be recommended in patients with slight increase of preoperative serum basal calcitonin (bCT) levels in whom surgical treatment can be recommendable. Fourteen consecutive patients with nodular thyroid disease underwent thyroidectomy in our center for suspected medullary thyroid microcarcinoma (MTC) because their serum bCT levels were slightly higher than the upper limit of normal range. Serum bCT was measured by radioimmunoassay, normality range = 0-20 ng/L. Surgical specimens were examined by the same pathologist using histologic and immunohistochemistry techniques. An extensive search for parafollicular C-cell hyperplasia (CCH) and/or microscopic MTC foci was performed. At preoperative ultrasound, a single thyroid nodule was depicted in three patients while a multinodular goiter in 11. The bCT values ranged between 24.4 and 94.6 ng/L, median 42.2 ng/L while the pentagastrin-stimulated CT (sCT) values by pentagastrin test ranged between 61.5 and 1,262 ng/L, median 245.0 ng/L. Total thyroidectomy was performed in 13 patients, and lobectomy in the other one; central node dissection was also performed in eight cases. At histology, MTC was diagnosed in nine patients (64.3 %), showing a median maximum diameter of 6.1 mm (range, 1.5-17 mm); CCH was diagnosed in the other five patients (35.7 %). The pentagastrin stimulation test was obtained in all patients. It is worth noting that a very high increase of sCT >100 ng/mL was observed in 5/9 patients with MTC and in 2/4 patients with HCC, therefore suggesting the absence of a relationship between the entity of response to pentagastrin test with a specific pathology (MTC vs. HCC). In six patients, the MTC was the nodule on which preoperative FNAC had been performed, while in other three patients preoperative FNAC had been performed on a different nodule from the MTC. Based on our experience, in case of the pentagastrin stimulation test with sCT <100 ng/L and a single nodule, the CT assay on FNAC may be useful, subsequently lobectomy with definitive histological diagnosis is recommended. In case of the Pg test with sCT <100 ng/L and bilateral goiter, total thyroidectomy with histological diagnosis is recommended. In this way, as for the surgical procedure, total thyroidectomy is recommended in cases of bilateral goiter, while lobectomy can be offered for cases with single nodes with serum dosage of bCT in the strict follow up. In case of the pentagastrin stimulating test with sCT <100 ng/L and bilateral goiter, total thyroidectomy with histological diagnosis is recommended.

Circulating cell-free DNA, SLC5A8 and SLC26A4 hypermethylation, BRAF(V600E): A non-invasive tool panel for early detection of thyroid cancer.

PURPOSE: In the latest years, high levels of circulating cell-free DNA (cf-DNA) have been found to be associated with cancer diagnosis and progression, and cf-DNA has become a potential candidate as biomarker for tumor detection. cf-DNA has been investigated in plasma or serum of many tumor patients affected by different malignancies, but not yet in thyroid cancer (TC). Furthermore, in TC cells the capability to metabolize iodine is frequently lost. SLC5A8 and SLC26A4 genes are both involved in the iodine metabolism, and SLC5A8 hypermethylation status is associated with the BRAF(V600E) mutation, which is the most frequent genetic event underlying the development of papillary TC. The aim of our study is the development of a new non-invasive tool for the diagnosis and prognosis of TC based on cf-DNA, SLC5A8 and SLC26A4 hypermethylation, and BRAF(V600E) analysis. METHODS: cf-DNA was measured by quantitative real-time PCR in nine cases of anaplastic thyroid cancer (ATC), 58 medullary thyroid cancers (MTC), five of synchronous medullary and follicular thyroid cancers (SMFC), 23 follicular adenomas (FA), 86 papillary thyroid cancers (PTC). A control group of 19 healthy subjects was taken. Moreover, in the PTC group we analyze the state of hypermethylation of SLC5A8 and SLC26A4, BRAF(V600E) mutation, and their involvement in the loss of function of the thyroid. RESULTS: cf-DNA showed a high ability to discriminate healthy individuals from cancer patients. cf-DNAALU83 and cf-DNAALU244 values were significantly correlated with the histological type of TC (P-value < 0.0001). A significant increase in the amount of cf-DNAALU83 and cf-DNAALU244 when methylation occurs was observed (P-value = 0.02). A correlation between BRAF(V600E) and cf-DNAALU244/ALU83 was also found (P-value = 0.02). CONCLUSIONS: According to our experimental results, the panel including cf-DNA, SLC5A8 and SLC26A4 hypermethylation, and BRAF(V600E) analysis appears easy, reproducible, and non-invasive for the diagnosis on TC. Its possible implication in clinical setting remains to be elucidated.

Differential gene expression of medullary thyroid carcinoma reveals specific markers associated with genetic conditions.

Medullary thyroid carcinoma accounts for 2% to 5% of thyroid malignancies, of which 75% are sporadic and the remaining 25% are hereditary and related to multiple endocrine neoplasia type 2 syndrome. Despite a genotype-phenotype correlation with specific germline RET mutations, knowledge of pathways specifically associated with each mutation and with non-RET-mutated sporadic MTC remains lacking. Gene expression patterns have provided a tool for identifying molecular events related to specific tumor types and to different clinical features that could help identify novel therapeutic targets. Using transcriptional profiling of 49 frozen MTC specimens classified as RET mutation, we identified PROM1, LOXL2, GFRA1, and DKK4 as related to RET(M918T) and GAL as related to RET(634) mutation. An independent series of 19 frozen and 23 formalin-fixed, paraffin-embedded (FFPE) MTCs was used for validation by RT-qPCR. Two tissue microarrays containing 69 MTCs were available for IHC assays. According to pathway enrichment analysis and gene ontology biological processes, genes associated with the MTC(M918T) group were involved mainly in proliferative, cell adhesion, and general malignant metastatic effects and with Wnt, Notch, NFκB, JAK/Stat, and MAPK signaling pathways. Assays based on silencing of PROM1 by siRNAs performed in the MZ-CRC-1 cell line, harboring RET(M918T), caused an increase in apoptotic nuclei, suggesting that PROM1 is necessary for survival of these cells. This is the first report of PROM1 overexpression among primary tumors.

RET codon 609 mutations: a contribution for better clinical managing.

Medullary thyroid carcinoma currently accounts for 5-8% of all thyroid cancers. The clinical course of this disease varies from extremely indolent tumors that can go unchanged for years to an extremely aggressive variant that is associated with a high mortality rate. As many as 75% of all medullary thyroid carcinomas are sporadic, with an average age at presentation reported as 60 years, and the remaining 25% are hereditary with an earlier age of presentation, ranging from 20 to 40 years. Germline RET proto-oncogene mutations are the genetic causes of multiple endocrine neoplasia type 2 and a strong genotype-phenotype correlation exists, particularly between a specific RET codon mutation and the (a) age-related onset and (b) thyroid tumor progression, from C-cell hyperplasia to medullary thyroid carcinoma and, ultimately, to nodal metastases. RET mutations predispose an individual to the development of medullary thyroid carcinomas and can also influence the individual response to RET protein receptor-targeted therapies. RET codon 609 point mutations are rare genetic events belonging to the intermediate risk category for the onset of medullary thyroid carcinoma. A large genealogy resulting in a less aggressive form of medullary thyroid carcinoma is associated with the high penetrance of pheochromocytoma and has been reported in the literature. In this short review article, we comment on our previous report of a large multiple endocrine neoplasia type 2A kindred with the same Cys609Ser germline RET mutation in which, conversely, the syndrome was characterized by a slightly aggressive, highly penetrant form of medullary thyroid carcinoma that was associated with low penetrance of pheochromocytoma and primary hyperparathyroidism.

RET codon 609 mutations: a contribution for better clinical managing

Medullary thyroid carcinoma currently accounts for 5-8% of all thyroid cancers. The clinical course of this disease varies from extremely indolent tumors that can go unchanged for years to an extremely aggressive variant that is associated with a high mortality rate. As many as 75% of all medullary thyroid carcinomas are sporadic, with an average age at presentation reported as 60 years, and the remaining 25% are hereditary with an earlier age of presentation, ranging from 20 to 40 years. Germline RET proto-oncogene mutations are the genetic causes of multiple endocrine neoplasia type 2 and a strong genotype-phenotype correlation exists, particularly between a specific RET codon mutation and the (a) age-related onset and (b) thyroid tumor progression, from C-cell hyperplasia to medullary thyroid carcinoma and, ultimately, to nodal metastases. RET mutations predispose an individual to the development of medullary thyroid carcinomas and can also influence the individual response to RET protein receptor-targeted therapies. RET codon 609point mutations are rare genetic events belonging to the intermediate risk category for the onset of medullary thyroid carcinoma. A large genealogy resulting in a less aggressive form of medullary thyroid carcinoma is associated with the high penetrance of pheochromocytoma and has been reported in the literature. In this short review article, we comment on our previous report of a large multiple endocrine neoplasia type 2A kindred with the same Cys609Ser germline RET mutation in which, conversely, the syndrome was characterized by a slightly aggressive, highly penetrant form of medullary thyroid carcinoma that was associated with low penetrance of pheochromocytoma and primary hyperparathyroidism.