Navegando por Palavras-chave "RET"

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    Análise comparativa do perfil metabólico e funcional das variantes patogênicas G533c, C634y, M918v e M918t no gene Ret associadas à síndrome de neoplasia endócrina múltipla do tipo 2
    (Universidade Federal de São Paulo (UNIFESP), 2021) Hatanaka, Roxanne [UNIFESP]; Cerutti, Janete Maria [UNIFESP]; Universidade Federal de São Paulo
    Medullary thyroid carcinoma (MTC) is a tumor that originates from thyroid C cells, which can occur in sporadic or hereditary form. In the hereditary form, MTC is an integral part of multiple endocrine neoplasia type 2 (MEN 2) syndromes. MEN 2 is transmitted by an autosomal dominant inheritance pattern characterized by the presence of CMT (~100%), pheochromocytoma (~50%) and primary hyperparathyroidism (20-30%). The RET gene, located at 10q11.2 locus, is responsible for the predisposition to the development of the MEN 2 syndrome in approximately 98% of MEN 2 families. Due to the strong genotype-phenotype correlation in these syndromes, germline mutations in the RET gene have been used for the genetic screening of individuals at risk of developing MTC or family history of MEN 2. The American Society of Thyroid has developed a guideline (2015) for management of patients with hereditary MTC, being RET mutations classified according to the risk of aggressiveness of MTC, which is progressive. According to the risk (High, higher, or Highest), the age of prophylactic thyroidectomy is suggested. Despite the strong genotype-phenotype correlation in the MEN 2 syndrome, there has been observed an intra and interfamilial phenotypic heterogeneity in the individuals carrying of specific RET mutation. Considering that mutations in the RET gene constitutively activate the MAPK pathway and that different mutations can activate different substrates, generating alteration in cellular metabolism that affect the cellular processes associated with cancer (proliferation, migration, death, survival, stress responses and among others), our hypothesis is that different mutations in the RET gene can generate different metabolites, or even variable amounts of the same metabolite, resulting in the modification of the normal cellular metabolism and, consequently, of the biological behavior of the tumor. Therefore, the aim of this study was to evaluate the metabolic profile of the HEK293 cell line transfected with the RET gene Wild type and with RET G533C, C634Y, M918V and M918T mutations using the LC-MS/MS (liquid chromatography and tandem mass spectrometry) strategy. Additionally, we analyzed the biological and biochemical behavior of pathogenic variants in the RET gene and in control, by in vitro studies The analyzed data suggest that each RET mutation has different levels of metabolites, and its production of ATP and ROS (oxygen reactive species) can be influenced by molecules and indirectly or directly correlated pathways. In addition, differences observed among the RET mutations expand the knowledge about the cellular functioning of these specific cells, in which, when compared to the clinical heterogeneity presented in these carriers, it allows us to investigate other aspects that can assist and / or improve the conduct or treatment for these individuals.
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    Analysis of somatic mutations in BRAF, CDKN2A/p16 and PI3KCA in patients with medullary thyroid carcinoma
    (Spandidos Publ Ltd, 2016) Nascimento, Fabricio P. [UNIFESP]; Cardoso, Mirian G. [UNIFESP]; Lindsey, Susan C. [UNIFESP]; Kunii, Ilda S. [UNIFESP]; Valente, Flavia O. F. [UNIFESP]; Kizys, Marina M. L. [UNIFESP]; Delcelo, Rosana [UNIFESP]; Camacho, Cleber P. [UNIFESP]; Maciel, Rui M. B. [UNIFESP]; Dias-da-Silva, Magnus R. [UNIFESP]
    Medullary thyroid carcinoma (MTC), a neuroendocrine tumor originating from thyroid parafollicular cells, has been demonstrated to be associated with mutations in RET, HRAS, KRAS and NRAS. However, the role of other genes involved in the oncogenesis of neural crest tumors remains to be fully investigated in MTC. The current study aimed to investigate the presence of somatic mutations in BRAF, CDKN2A and PI3KCA in MTC, and to investigate the correlation with disease progression. DNA was isolated from paraffin-embedded tumors and blood samples from patients with MTC, and the hotspot somatic mutations were sequenced. A total of 2 novel HRAS mutations, p.Asp33Asn and p.His94Tyr, and polymorphisms within the 3' untranslated region (UTR) of CDKN2A (rs11515 and rs3088440) were identified, however, no mutations were observed in other genes. It was suggested that somatic point mutations in BRAF, CDKN2A and PI3KCA do not participate in the oncogenesis of MTC. Further studies are required in order to clarify the contribution of the polymorphisms identified in the 3' UTR of CDKN2A in MTC.
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    Analysis of somatic mutations in BRAF, CDKN2A/p16 and PI3KCA in patients with medullary thyroid carcinoma
    (Spandidos Publ Ltd, 2016) Nascimento, Fabricio P. [UNIFESP]; Cardoso, Mirian G. [UNIFESP]; Lindsey, Susan C. [UNIFESP]; Kunii, Ilda S. [UNIFESP]; Valente, Flavia O. F. [UNIFESP]; Kizys, Marina M. L. [UNIFESP]; Delcelo, Rosana [UNIFESP]; Camacho, Cleber P. [UNIFESP]; Maciel, Rui M. B. [UNIFESP]; Dias-da-Silva, Magnus R. [UNIFESP]
    Medullary thyroid carcinoma (MTC), a neuroendocrine tumor originating from thyroid parafollicular cells, has been demonstrated to be associated with mutations in RET, HRAS, KRAS and NRAS. However, the role of other genes involved in the oncogenesis of neural crest tumors remains to be fully investigated in MTC. The current study aimed to investigate the presence of somatic mutations in BRAF, CDKN2A and PI3KCA in MTC, and to investigate the correlation with disease progression. DNA was isolated from paraffin-embedded tumors and blood samples from patients with MTC, and the hotspot somatic mutations were sequenced. A total of 2 novel HRAS mutations, p.Asp33Asn and p.His94Tyr, and polymorphisms within the 3' untranslated region (UTR) of CDKN2A (rs11515 and rs3088440) were identified, however, no mutations were observed in other genes. It was suggested that somatic point mutations in BRAF, CDKN2A and PI3KCA do not participate in the oncogenesis of MTC. Further studies are required in order to clarify the contribution of the polymorphisms identified in the 3' UTR of CDKN2A in MTC.
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    Comprehensive analysis of RET gene should be performed in patients with multiple endocrine neoplasia type 2 (MEN 2) syndrome and no apparent genotype- phenotype correlation: An appraisal of p.Y791F and p.C634Y RET mutations in five unrelated Brazilian families
    (Springer, 2013-12-01) Valente, F. O. F. [UNIFESP]; Dias-da-Silva, Magnus Régios [UNIFESP]; Camacho, C. P. [UNIFESP]; Kunii, I. S. [UNIFESP]; Bastos, A. U. [UNIFESP]; Fonseca, C. C. N. da [UNIFESP]; Simiao, H. P. C. [UNIFESP]; Tamanaha, R. [UNIFESP]; Maciel, R. M. B. [UNIFESP]; Cerutti, J. M. [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    Background: We previously identified a four-generation family with medullary thyroid cancer (MTC) and a germline p.Y791F RET mutation whose cancer lacked a strong genotype-phenotype correlation. the entire gene coding region of the RET gene should be sequenced when genotype-phenotype discrepancies are observed in patients with multiple endocrine neoplasia type 2 (MEN 2), even if a RET hotspot mutation has been identified. Methods: A new genetic test was performed in the index case of this family with the p.Y791F RET germline mutation. the entire coding region of the RET gene was investigated by direct sequencing of PCR products. Once a mutation was identified, the target exon was sequenced in all at-risk relatives. Results: An additional p.C634Y germline mutation in the RET gene was identified in the reported family. the double mutation occurred in cis and segregated with the phenotype. Through the Brazilian Genetic Screening Program developed at our institution, we additionally report the combination of these two mutations (p.C634Y/p. Y791F) in the RET gene in four other unrelated families. the overall penetrance of MTC and pheochromocytoma in patients with the p.C634Y/p.Y791F mutations was 79% and 13%, respectively. Conclusion: Our data emphasises that a comprehensive analysis of the RET gene may reveal multiple germline mutations in MEN 2 patients who exhibit an atypical clinical course of the disease. (C) 2013, Editrice Kurtis
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    DLK1 está associado ao fenótipo de células-tronco cancerígenas em linhagens celulares de carcinoma medular de tireoide
    (Universidade Federal de São Paulo, 2023-11-30) Silva, Danilo Dias da [UNIFESP]; Cerutti, Janete Maria [UNIFESP]; Araldi, Rodrigo Pinheiro; http://lattes.cnpq.br/1613522461963281; http://lattes.cnpq.br/1384038091754225; http://lattes.cnpq.br/3039373571526747
    O carcinoma medular de tireoide (CMT) é um tumor raro e altamente agressivo, comparado ao carcinoma diferenciado de tireoide. O CMT avançado ou metastático, progressivo e sintomático, pode exigir tratamento sistêmico adicional. No entanto, os pacientes podem desenvolver doença resistente. O mecanismo de resistência durante o tratamento ainda é pouco compreendido. Uma subpopulação de células cancerígenas denominadas células-tronco cancerígenas (CTCs) tem sido associada à resistência a medicamentos, metástase e recidiva do câncer. Procuramos caracterizar duas linhagens celulares de MTC (MZ-CRC-1 e TT) quanto à presença de CTCs. A análise de 15 marcadores associados ao fenótipo tronco, além de marcadores células-tronco (CT) previamente descritos em tireoide (CD133, CD44 e ALDH1) e proteínas associadas à resistência a múltiplas drogas (MRP1 e MRP3), demonstrou que essas células são caracterizadas por fenótipos específicos. MZ-CRC-1 (RET M918T) apresentou níveis mais elevados de marcadores tronco do que células TT (RET C634W) e foi mais eficiente na formação de esferóides, uma marca registrada dos CTCs. Como a via Notch estava associada à auto-renovação, sobrevivência de CTCs e metástase, investigamos a expressão do gene do ligante Notch 1 não canônico tipo Delta (DLK1) nessas duas linhagens celulares. DLK1, previamente encontrado com alteração funcional no número de cópias em amostras de CMT, foi expresso em ambas as linhagens celulares. A análise de populações enriquecidas (DLK1+ e DLK1-) revelou que as células DLK1+ apresentam marcadores tronco expressos em níveis distintos e níveis muito mais elevados do que as células DLK1-. Este estudo relatou pela primeira vez que ambas as linhagens de células MTC expressaram os principais marcadores de tronco e que o DLK1 pode desempenhar um papel no fenótipo tronco em células MTC.
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    Estudo de alterações no número de cópias (CNA) associadas à gênese do carcinoma medular da tiroide
    (Universidade Federal de São Paulo (UNIFESP), 2017-11-14) Araujo, Aline Neves [UNIFESP]; Cerutti, Janete Maria [UNIFESP]; Mazzotti, Diego Robles [UNIFESP]; http://lattes.cnpq.br/1384038091754225; http://lattes.cnpq.br/9319492862937149; Universidade Federal de São Paulo (UNIFESP)
    O carcinoma medular da tiroide (CMT) é um tumor maligno originado das células C da tiroide, que pode ocorrer na forma hereditária (20-25%) como parte da síndrome de neoplasia endócrina múltipla do tipo 2 (NEM 2), ou na forma esporádica (75-80%). Embora o desenvolvimento do CMT hereditário esteja associado a mutações no gene RET nas células da linhagem germinativa, e em 50% dos CMT esporádico são identificadas mutações somáticas no gene RET; é sugerido que outras alterações ou mecanismos genéticos possam estar envolvidas na tumorigênese e progressão do CMT. Objetivo: Investigar a existência de alterações no número de cópias (CNAs) de DNA, relacionadas à gênese do CMT. Métodos: DNA de amostras de CMT (n=3) foram comparados aos seus respectivos sangues pareado (DNA constitutivo) por meio da plataforma Genome-Wide Human SNP Array 6.0. Os dados foram analisados através dos softwares PennCNV e Genotyping Console. As CNAs candidatas relacionadas à gênese do CMT foram validadas pela reação em cadeia da polimerase quantitativa (qPCR) nas amostras iniciais, além de terem sido testadas em um grupo expandido CMT (n=51). Os genes presentes nas CNAs validadas tiveram suas expressões avaliadas a nível de RNA mensageiro e proteína, para, possivelmente, identificar genes diferentemente expressos entre tumor e tecido normal. Resultados: A análise do genoma completo permitiu identificar sete alterações presentes em, pelo menos, 2 CMT (de 3). A validação das regiões permitiu confirmar o ganho de duas delas através da análise dos genes DLK1 (14q32.2) e AIFM3 (22q11.21) contidos nas mesmas, que apresentaram ganho no tumor em comparação ao DNA constitutivo (p<0,0001; ambos genes). A análise de expressão sugere que as CNAs sejam funcionais, uma vez que foi observada a superexpressão dos dois genes no tumor em relação ao DNA constitutivo (p<0,0001; ambos), além de ter sido observada expressão proteica de AIFM3 e DLK1 em 89% e 93% dos casos de CMT, respectivamente. Outra detecção do array foi a deleção de regiões sequenciais nos cromossomos 1p e 4q de um dos CMT, em comparação com o DNA constitutivo. A validação de uma dessas regiões por hibridização in situ fluorescente (FISH) confirmou a deleção. Conclusões: Esse estudo identificou duas CNAs de ganho envolvendo os genes AIFM3 e DLK1 como possivelmente envolvidos na tumorigênese do CMT. Além de detectar deleções em 1p e 4q, já descritas, em um dos CMT, reforçando a ideia dessas regiões abrigarem supressores tumorais.
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    Investigação da prevalência de mutações e fusões de genes que codificam efetores da via mapk e sua correlação com características clinico-patológicas e perfil de expressão em carcinomas papilíferos da tiroide
    (Universidade Federal de São Paulo (UNIFESP), 2016-10-25) Bastos, André Uchimura [UNIFESP]; Cerutti, Janete Maria [UNIFES]; http://lattes.cnpq.br/1384038091754225; http://lattes.cnpq.br/7991411234306363
    Papillary thyroid carcinoma (PTC) is the most prevalent subtype among thyroid carcinomas, representing about 80% of cases. Its prevalence has increased in recent years, mainly due to the increase of papillary thyroid microcarcinomas (PTC ?10 mm, PTMC). In recent decades, it became clear that genetic changes that lead to activation of the MAPK (Mitogen-Activated Protein Kinase pathway), a signaling cascade that regulates cell proliferation, differentiation and survival, are highly prevalent in PTC. Alterations in the genes encoding proteins of this pathway represent a total of approximately 70% of cases of PTC, and include chromosomal rearrangements like RET/PTC or NTRK, and mutations in RAS and BRAF genes. In addition to these changes, new fusions involving tyrosine kinases have been described recently by The Cancer Genome Atlas (TCGA). However, in a number of cases of PTCs, a genetic event associated with pathogenesis (driver changes) is still unknown ("dark-matter"). In order to describe the genetic events associated with pathogenesis of PTC in the Brazilian population, our group investigated the prevalence of BRAF V600E mutation in a sample set of 120 PTCs. In addition to determining the prevalence of this mutation (48%; n=58), we observed the association with histological subtype, clinical and pathological features associated with a poor prognosis, and loss of expression of genes involved in iodine uptake and metabolism (NIS and TSHR). These data associated with the reported in the literature suggest that BRAF V600E is a marker of poor prognosis. In the articles that originate this thesis, we initially investigated the prevalence of RET/PTC, which is the second most frequent genetic alteration in the PTC, and we evaluated the expression of TPO, PDS and TG. Fusions involving the RET gene (RET/PTC1, RET/PTC2 and RET/PTC3) were identified in 27% (32/118) of PTCs. There was no association between RET/PTC, expression of genes associated with iodine uptake and metabolism and clinic-pathologic characteristics associated with aggressiveness. In fact, the role of RET/PTC as a prognostic marker is still controversial. Besides NIS, previously reported, in this study we demonstrate that BRAF V600E mutation is also associated with the loss of TPO expression. Additionally, we identified the mutation in codon 61 of NRAS in 9% (11/118) of PTCs. When we evaluate the genetic changes in PTMC (n=40), we found no association with clinic-pathologic characteristics associated with aggressiveness, but we found that the BRAF V600E mutation is associated with a decreased expression of NIS and TPO. When PTMCs were divided into two subgroups (<7 mm vs. ?7 mm) we observed that MCPTs ?7 mm have higher extrathyroidal extension, metastasis to lymph nodes and recurrence rates, and that the decreased expression of NIS and TPO is much more evident in this group. This is probably due to the accumulation of PTMCs with BRAFV600E in the ?7 mm subgroup. These data are similar to those found in tumors larger than 10 mm, more aggressive, which suggests that despite PTMCs being, most often, clinically indolent, the subgroup of 7-10 mm with BRAF V600E could be treated in a similar manner to conventional PTC with BRAF V600E. In the second phase of this study, we investigated the presence of ETV6-NTRK3, STRN-ALK and AGK-BRAF fusion genes, recurring events in PTCs that are able to activate the MAPK pathway. We also investigated the presence of mutations in the KRAS and HRAS genes (codons 12, 13 and 61), and only one of the samples was positive for HRAS Q61R. ETV6-NTRK3 was identified in about 5% (6/116) of PTCs. ETV6- NTRK3 was exclusively found in the follicular variant of PTC (FVPTC), leading to a prevalence of 16% (6/45) in this variant. This fact is interesting, since most of the "dark-matter" is composed of PTCs of this variant. In addition, we investigate whether such mutation would have some association with the presence of the tumor capsule. ETV6-NTRK3 was found in both variants, infiltrative and encapsulated, in 50% each. As a recently new nomenclature was suggested for encapsulated FVPTC without any invasion, called noninvasive follicular thyroid neoplasm with papillary-like nuclear characteristics (NIFTP), the relationship between ETV6-NTRK3 and NIFTP variant should be further investigated. STRN-ALK was identified in 3% (4/116) of PTC and was found in both classic and FVPTC. There was no association with clinic-pathological features associated with aggressiveness. We did not find any positive sample for AGK-BRAF in this series. This finding confirms the proposition that AGK-BRAF is a likely event associated with PTCs of pediatric patients. Finally, in this initial screening, we identified that 27% (n=30) of the samples were negative for BRAF V600E (and adjacent codons), NRAS Q61, HRAS (codons 12, 13 and 61), KRAS (codons 12, 13 and 61) RET/PTC1, RET/PTC2, RET/PTC3, ETV6-NTRK3, STRN-ALK and AGK-BRAF. Our data contribute to a better understanding of the pathogenesis of PTC and collects important data regarding the use of various alterations as prognostic markers. Along with other previously published articles, and articles that will be published in future in several other populations, we hope this information can help to establish effective guidelines for the management and treatment of patients with papillary carcinoma of the thyroid.