A pioneering RET genetic screening study in the State of Ceará, Brazil, evaluating patients with medullary thyroid cancer and at-risk relatives: experience with 247 individuals

ABSTRACT Objective: Initial diagnosis of medullary thyroid carcinoma (MTC) is frequently associated with advanced stages and a poor prognosis. Thus, the need for earlier diagnoses and detection in relatives at risk for the disease has led to increased use of RET genetic screening. Subjects and methods: We performed RET screening in 247 subjects who were referred to the Brazilian Research Consortium for Multiple Endocrine Neoplasia (BRASMEN) Center in the State of Ceará. Direct genetic sequencing was used to analyze exons 8, 10, 11, and 13-16 in MTC index cases and specific exons in at risk relatives. Afterward, clinical follow-up was offered to all the patients with MTC and their affected relatives. Results: RET screening was performed in 60 MTC index patients and 187 at-risk family members. At the initial clinical assessment of the index patients, 54 (90%) were diagnosed with apparently sporadic disease and 6 (10%) diagnosed with hereditary disease. After RET screening, we found that 31 (52%) index patients had sporadic disease, and 29 (48%) had hereditary disease. Regarding at-risk relatives, 73/187 were mutation carriers. Mutations in RET codon 804 and the rare p.M918V mutation were the most prevalent. Conclusions: Performing RET screening in Ceará allowed us to identify a different mutation profile in this region compared with other areas. RET screening also enabled the diagnosis of a significant number of hereditary MTC patients who were initially classified as sporadic disease patients and benefited their relatives, who were unaware of the risks and the consequences of bearing a RET mutation.

A pioneering RET genetic screening study in the State of Ceará, Brazil, evaluating patients with medullary thyroid cancer and at-risk relatives: experience with 247 individuals.

OBJECTIVE: Initial diagnosis of medullary thyroid carcinoma (MTC) is frequently associated with advanced stages and a poor prognosis. Thus, the need for earlier diagnoses and detection in relatives at risk for the disease has led to increased use of RET genetic screening. SUBJECTS AND METHODS: We performed RET screening in 247 subjects who were referred to the Brazilian Research Consortium for Multiple Endocrine Neoplasia (BRASMEN) Center in the State of Ceará. Direct genetic sequencing was used to analyze exons 8, 10, 11, and 13-16 in MTC index cases and specific exons in at risk relatives. Afterward, clinical follow-up was offered to all the patients with MTC and their affected relatives. RESULTS: RET screening was performed in 60 MTC index patients and 187 at-risk family members. At the initial clinical assessment of the index patients, 54 (90%) were diagnosed with apparently sporadic disease and 6 (10%) diagnosed with hereditary disease. After RET screening, we found that 31 (52%) index patients had sporadic disease, and 29 (48%) had hereditary disease. Regarding at-risk relatives, 73/187 were mutation carriers. Mutations in RET codon 804 and the rare p.M918V mutation were the most prevalent. CONCLUSIONS: Performing RET screening in Ceará allowed us to identify a different mutation profile in this region compared with other areas. RET screening also enabled the diagnosis of a significant number of hereditary MTC patients who were initially classified as sporadic disease patients and benefited their relatives, who were unaware of the risks and the consequences of bearing a RET mutation.

Evidence for the founder effect of RET533 as the common Greek and Brazilian ancestor spreading multiple endocrine neoplasia 2A.

OBJECTIVES: About one-quarter of patients with medullary thyroid cancer (MTC) have inherited disease due to mutations in the RET gene. A rare mutation in exon 8 (G533C) of RET, previously described in a large Brazilian family with MEN2A, also appeared to be clustering in Greece, whereas it was rarely reported in other ethnic groups. The aim of this study was to identify a possible common ancestry between these carriers. PATIENTS AND METHODS: Twelve RET G533C mutation carriers, four randomly selected from the Brazilian cohort and eight from apparently unrelated Greek families, were studied for a possible common ancestral origin. RET flanking microsatellite markers at chromosome 10q (D10S197, D10S196, D10S1652 and D10S537) were used. RESULTS: Genomic DNA analysis using these markers showed that many of these apparently unrelated individuals shared a common haplotype indicating a common ancestral origin. CONCLUSION: Our data suggest that Brazilian and Greek patients with MTC carrying the G533C mutation in exon 8 of RET gene originate from a common ancestor. Due to historical reasons, we speculate that the more plausible explanation for the origin of this mutation is in Greece.

M918V RET mutation causes familial medullary thyroid carcinoma: study of 8 affected kindreds.

Germline mutations in codon 918 of exon 16 of the RET gene (M918T) are classically associated with multiple endocrine neoplasia type 2B (MEN 2B) with highly aggressive medullary thyroid cancer (MTC), pheochromocytoma and a unique phenotype. The objectives of this study are to describe the rare M918V RET mutation discovered in 8 MTC kindreds from Brazil lacking the MEN 2B phenotype classically observed in M918T patients and to investigate the presence of a founder effect for this germline mutation. Eight apparently sporadic MTC cases were diagnosed with the germline M918V RET mutation. Subsequently, their relatives underwent clinical and genetic assessment (n = 113), and M918V was found in 42 of them. Until today, 20/50 M918V carriers underwent thyroidectomy and all presented MTC/C-cell hyperplasia; the remainder carriers are on clinical follow-up. None of the M918V carriers presented clinical features of MEN 2B. Their clinical presentation was heterogeneous, and the age at tumor diagnosis ranged from 24 to 59 years. Lymph node metastases were present in 12/20 patients, and presumable distant metastases in 2/20; in contrast, we observed a carrier of up to 87 years of age without evidence of MTC. Ethnographic fieldwork and haplotype analyses suggested that the founder mutation first settled in that area fifteen generations ago and originated from Portugal. Our study is the first to demonstrate the RET M918V mutation co-segregating in 8 familial MTC kindreds with validated evidence of a founder effect. We suggest that M918V MTC should be clinically considered an American Thyroid Association (ATA) moderate-risk category.

Family members CREB and CREM control thyrotropin-releasing hormone (TRH) expression in the hypothalamus.

Thyrotropin-releasing hormone (TRH) in the paraventricular nucleus (PVN) of the hypothalamus is regulated by thyroid hormone (TH). cAMP response element binding protein (CREB) has also been postulated to regulate TRH expression but its interaction with TH signaling in vivo is not known. To evaluate the role of CREB in TRH regulation in vivo, we deleted CREB from PVN neurons to generate the CREB1(ΔSIM1) mouse. As previously shown, loss of CREB was compensated for by an up-regulation of CREM in euthyroid CREB1(ΔSIM1) mice but TSH, T4 and T3 levels were normal, even though TRH mRNA levels were elevated. Interestingly, TRH mRNA expression was also increased in the PVN of CREB1(ΔSIM1) mice in the hypothyroid state but became normal when made hyperthyroid. Importantly, CREM levels were similar in CREB1(ΔSIM1) mice regardless of thyroid status, demonstrating that the regulation of TRH by T3 in vivo likely occurs independently of the CREB/CREM family.

Infiltration of a mixture of immune cells may be related to good prognosis in patients with differentiated thyroid carcinoma.

OBJECTIVE: Immune responses against differentiated thyroid carcinomas (DTC) have long been recognized. We aimed to investigate the role of immune cell infiltration in the progression of DTC. DESIGN: We studied 398 patients - 253 with papillary and 13 with follicular thyroid cancers, as well as 132 with nonmalignant tissues. PATIENTS AND MEASUREMENTS: Immune cell infiltration was identified using CD3, CD4, CD8, CD20, CD68 and FoxP3 immunohistochemical markers. In addition, we assessed colocalization of CD4 and IL-17 to identify Th17 lymphocytic infiltration and colocalization of CD33 and CD11b to identify infiltration of myeloid-derived suppressor cells (MDSC). RESULTS: Immune cells infiltrated malignant tissues more often than benign lesions. The presence of chronic lymphocytic thyroiditis (CLT) concurrent to DTC, CD68+, CD4+, CD8+, CD20+, FoxP3+ and Th17 lymphocytes but not MDSCs was associated with clinical and pathological features of lower tumour aggressiveness and a more favourable patient outcome. A log-rank test confirmed an association between concurrent CLT, tumour-associated macrophage infiltration, and CD8+ lymphocytes and an increased in disease-free survival, suggesting that evidence of these immune reactions is associated with a favourable prognosis. CONCLUSION: Our data suggest that the tumour or peri-tumoural microenvironment may act to modify the observed pattern of immune response. Immune cell infiltration and the presence of concurrent CLT helped characterize specific tumour histotypes associated with favourable prognostic features.

Use of sodium iodide symporter expression in differentiated thyroid carcinomas.

OBJECTIVE: We aimed to investigate the use of NIS mRNA and protein expression as a diagnostic and/or prognostic marker in patients with differentiated thyroid cancer (DTC). DESIGN: This is a case-control study. PATIENTS: We studied 397 thyroid nodules tissue samples, including 224 papillary thyroid carcinomas (PTCs), 41 follicular carcinomas, 58 nodular goiters, 56 follicular adenomas and 18 normal tissues assembled in a tissue microarray. MEASUREMENTS: NIS protein was identified using a monoclonal antibody that labelled only the follicular cell basolateral membrane of all 397 tissue samples. In addition, NIS mRNA was quantified in 145 DTC patients and 85 PTC cases were screened for BRAF(V600E) mutation. RESULTS: We found low NIS mRNA expression and low or negative NIS protein expression in most DTC. NIS expression was lower in DTC patients over 45 years old and in tumours larger than 2 cm. There was a tendency for lower NIS expression in advanced stages and patients presenting recurrences. All 13 DTC patients who succumbed to the disease were NIS negative at immunohistochemistry and had very low mRNA expression. NIS expression was lower in PTC presenting BRAF(V600E) mutation. However, neither NIS immunohistochemical analysis nor NIS mRNA quantified expression could identify individuals with poor prognosis. CONCLUSIONS: Our data suggest that NIS expression may help characterize patients' risk and individuals with a poor response to therapy, but is not useful as a diagnostic or prognostic marker, reinforcing the current concept that an appropriate management of DTC patient is the most important and modifiable prognostic factor.

Lack of cAMP-response element-binding protein 1 in the hypothalamus causes obesity.

The melanocortin system in the hypothalamus controls food intake and energy expenditure. Its disruption causes severe obesity in mice and humans. cAMP-response element-binding protein 1 (CREB1) has been postulated to play an important role downstream of the melanocortin-4 receptor (MC4R), but this hypothesis has never been confirmed in vivo. To test this, we generated mice that lack CREB1 in SIM1-expressing neurons, of the paraventricular nucleus (PVN), which are known to be MC4R-positive. Interestingly, CREB1(ΔSIM1) mice developed obesity as a result of decreased energy expenditure and impairment in maintaining their core body temperature and not because of hyperphagia, defining a new role for CREB1 in the PVN. In addition, the lack of CREB1 in the PVN caused a reduction in vasopressin expression but did not affect adrenal or thyroid function. Surprisingly, MC4R function tested pharmacologically was normal in CREB1(ΔSIM1) mice, suggesting that CREB1 is not required for intact MC4R signaling. Thus CREB1 may affect other pathways that are implicated in the regulation of body weight.