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O melanoma é um tipo de câncer de pele geneticamente diverso, que surge diante das transformações em melanócitos. A mutação BRAFV600E está presente em mais de 90% de todas as mutações em BRAF, sendo assim ocorre em cerca de 50% dos casos registrados. As mutações em NRAS, ocupam o segundo lugar entre as mutações mais prevalentes, cerca de 20% dos casos. Informações sobre as assinaturas genéticas, permitiram o desenvolvimento de terapia alvo dirigida. O Vemurafenib, inibidor da quinase BRAFV600E, apresentou inicialmente resultados bastante satisfatórios, contudo existe registro de casos de recidiva e resistência. O receptor aril de hidrocarbonetos é expresso em vários componentes da pele, e assim está relacionado a homeostase e fisiopatologia da pele. Diante disso, a avaliação da expressão do receptor em um painel de linhagens mutadas para NRAS e BRAF, e BRAF resistentes, mostrou-se maior do que a encontrada em melanócitos. Também encontramos maior expressão de mRNA de AhR em linhagens de melanoma derivadas de sítio primário e metastático, mutadas para BRAFV600E, quando comparadas ao melanócito. Agregado a isto, a análise in silico no TCGA (The Cancer Genome Atlas) mostrou que há 18% de alteração genética em AhR, sendo em maior parte a alta regulação de mRNA. Também, a análise do banco público GSE12391, mostrou aumento de mRNA de AhR na fase de crescimento vertical do melanoma. Assim, concluímos que há maior expressão de mRNA e sua importância nas fases de desenvolvimento do melanoma, tanto nos processos iniciais quanto em processos de migração, invasão e metástase. Ainda, encontramos maior mRNA do receptor em linhagens resistentes ao Vemurafenib. Este resultado sustenta a hipótese de que AhR pode ser considerado um marcador de resistência em melanomas. O AhR, inicialmente no citoplasma, quando ativado pode atuar como fator de transcrição regulando vários genes que apresentam sequências definidas, participando de respostas carcinogênicas. Compostos halogenados e moléculas endógenas derivadas das vias de metabolização do triptofano são agonistas do receptor. Anteriormente, nosso grupo mostrou que linhagens de melanoma incubadas com triptamina e DMT exibiram menor clonogenicidade. Diante de uma literatura escassa sobre o papel do DMT no melanoma e com base nestes resultados, nosso objetivo foi avaliar o papel de AhR nesta interface DMT-melanoma. Para isto, nosso objetivo foi construir linhagem editada geneticamente para AhR através da ferramenta CRISPR-Cas9. Vários foram os esforços, sem sucesso, utilizados nas tentativas de comprovar a manutenção de células editadas na cultura. Atrelamos a este resultado a possibilidade de haver duas subpopulações editadas geneticamente pós CRISPR-Cas9, onde uma destas manteve o padrão de crescimento semelhante às células wild type. Devido a este crescimento diferencial, não obtivemos congruências nos ensaios e postulamos a perda do possível nocaute. A partir disso, realizamos ensaios de interactoma para avaliar a interação de DMT-AhR. Nosso resultado sugere a interação de DMT ao receptor sigma 1, e não ao receptor aril de hidrocarbonetos. Desta forma, o interactoma sustenta a hipótese de que DMT não é um ligante de AhR. Para certificar este resultado análises de docking associados a ensaios biológicos, avaliando o papel do receptor, devem ser realizados para averiguar a afinidade e seletividade de DMT como ligante do receptor na linhagem de melanoma
Melanoma is a genetically diverse type of skin cancer, which arises from changes in melanocytes. The BRAFV600E mutation is present in more than 90% of all BRAF mutations, so it occurs in about 50% of registered cases. Mutations in NRAS occupy the second place among the most prevalent mutations, about 20% of cases. Information on genetic signatures allowed the development of targeted therapy. vemurafenib, kinase inhibitor BRAFV600E, initially presented very satisfactory results, however there is a record of cases of relapse and resistance. The aryl hydrocarbon receptor is expressed in several components of the skin and is thus related to homeostasis and skin pathophysiology. Therefore, the evaluation of receptor expression in a panel of strains mutated to NRAS and BRAF, and resistant BRAF, proved to be greater than that found in melanocytes. We also found main expression of AhR mRNA in melanoma strains derived from primary and metastatic site, mutated to BRAFV600E, when compared to melanocyte. Added to this, the in silico analysis in TCGA (The Cancer Genome Atlas) showed that there is 18% of genetic alteration in AhR, being mostly the high regulation of mRNA. Also, an analysis by the public bank GSE12391, showed an increase in AhR mRNA in the vertical growth phase of melanoma. Thus, it is concluded that there is greater expression of mRNA and its importance in the stages of development of melanoma, both in recent processes and in the processes of migration, invasion and metastasis. In addition, we found higher receptor mRNA in strains resistant to vemurafenib. This result supports the hypothesis that AhR can be considered a marker of resistance in melanomas. AhR, initially in the cytoplasm, when activated can act as a transcription factor regulating several genes that have defined sequences, participating in carcinogenic responses. Along with this, we show that along the tumor progression, there is an increase in AhR in the radial growth phase of melanoma. Halogenated compounds and endogenous molecules derived from the tryptophan metabolism pathways are receptor agonists. Previously, our group showed that melanoma strains incubated with tryptamine and DMT exhibited less clonogenicity. In view of a scarce literature on the role of DMT in melanoma and based on these results, our objective was to evaluate the role of AhR in this DMT-melanoma interface. For this, our goal was to build genetically edited strain for AhR using the CRISPR-Cas9 tool. Several efforts were unsuccessful in attempts to prove the maintenance of cells edited in the culture. We linked to this result the possibility of having two subpopulations genetically edited after CRISPR-Cas9, where one of them maintained the growth pattern like wild type cells. Due to this differential growth, we did not obtain congruence in the tests and postulated the loss of the possible knockout. From that, we performed interactome assays to evaluate the DMT-AhR interaction. Our result suggests the interaction of DMT with the sigma 1 receptor, and not the aryl hydrocarbon receptor. Thus, the interactome supports the hypothesis that DMT is not an AhR ligand. To certify this result, docking analyses associated with biological assays, evaluating the role of the receptor, should be performed to ascertain the affinity and selectivity of DMT as a ligand of the receptor in the melanoma lineage
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Piel/lesiones , Genoma , Receptores de Hidrocarburo de Aril , Melanocitos/clasificación , Melanoma , Neoplasias/patología , Fosfotransferasas/antagonistas & inhibidores , Asociación , Factores de Transcripción/agonistas , Citoplasma/clasificación , Migración HumanaRESUMEN
Objective To investigate the role of BRAFV600E mutation in diagnosis of thyroid nodules when it is inconsonant with cytological results. Methods This study included 9837 patients who underwent US-FNA. We mainly analyzed 239 cases with benign or indeterminate cytology, but having a detection of BRAFV600E muta-tion. BRAFV600E mutation analysis was performed using a Amplification Refractory Mutation System Polymerase Chain Reaction. Results In 93 nodules with benign cytology results but positive BRAFV600E mutation, 84 nodules were malignant. Based on the results, US-FNA combined with BRAFV600E mutation analysis will improve sensitivity (Se=94.03％)and negative predictive value (NPV=2.69％) of the thyroid nodules diagnosis than using US-FNA alone(Se=71.03％, NPV=20.76％). Conclusion BRAFV600E mutation analysis is an important tool in the diagnosis of PTC with high sensitivity and NPV. When facing patients with benign or indeterminate cytology but positive BRAFV600E mutation, thyroidectomy should be considered.
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Objective@#To investigate the role of BRAFV600E mutation in diagnosis of thyroid nodules when it is inconsonant with cytological results.@*Methods@#This study included 9837 patients who underwent US-FNA. We mainly analyzed 239 cases with benign or indeterminate cytology, but having a detection of BRAFV600E mutation. BRAFV600E mutation analysis was performed using a Amplification Refractory Mutation System Polymerase Chain Reaction.@*Results@#In 93 nodules with benign cytology results but positive BRAFV600E mutation, 84 nodules were malignant. Based on the results, US-FNA combined with BRAFV600E mutation analysis will improve sensitivity (Se=94.03%) and negative predictive value (NPV=2.69%) of the thyroid nodules diagnosis than using US-FNA alone (Se=71.03%, NPV=20.76%) .@*Conclusion@#BRAFV600E mutation analysis is an important tool in the diagnosis of PTC with high sensitivity and NPV. When facing patients with benign or indeterminate cytology but positive BRAFV600E mutation, thyroidectomy should be considered.
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Objective To observe and compare the different orthotopic models of papillary thyroid cancer ( PTC) cell lines of RET/PTC1 rearrangement and BRAFV600E mutation in nude mice. Methods Human PTC cell lines TPC-1, BHP5-16 and BHP2-7 were used. The genotypes of RET/PTC1 rearrangement and BRAFV600E mutation were determined by realtime-PCR and DNA sequencing analysis. The cells(2×105) were injected into the thyroid gland of nude mice. The nude mice were executed at 4th, 12th week, and then their thyroid tumors were removed and weighed. The levels of thyroid hormone were detected using chemiluminescent immunoassay. Results Both TPC-1 and BHP2-7 cells were identified as RET/PTC1 rearrangement by real time-PCR, and the expression of RET/PTC1 rearrangement in BHP2-7 cell was higher than that of TPC-1 cell. BRAFV600E mutation was found in BHP5-16 cell by DNA sequencing analysis, but was not found in TPC-1 and BHP2-7 cells. There were different characteristics in three orthotopic nude model groups. Tumorigenic rates of TPC-1 and BHP5-16 groups were 100%, but the growth of tumor was more rapid in BHP5-16 group than that in TPC-1 group, with more weight tumor. The changes of thyroid hormone levels in BHP5-16 group and TPC-1 group were the same, which were normal at 4th week and sharply decreased at 12 th week(P0. 05). Conclusions It showed difference in the orthotopic models of PTC cell lines of RET/PTC1 rearrangement and BRAFV600E mutation in nude mice. BRAFV600E mutation has obvious impacts on increasing tumorigenic rate and promotion of tumor growth in the orthotopic model. It should not be ignored that advanced thyroid tumor will lead to the destruction of thyroid function.
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Objective To compare the diagnostic efficiency of the thyroid imaging reporting and data system (TIRADS), the Bethesda system for reporting thyroid cytopathology(BSRTC) and BRAFV600E detection, and their combined use in the differentiation between benign and malignant thyroid nodules. Methods One hundred and twenty eight patients with 128 thyroid nodules who were scheduled for ultrasound-guided fine-needle aspiration biopsy (FNAB) were recruited for the study. All of them underwent ultrasound, fine-needle aspiration cytology(FNAC) examination, and BRAFV600E detection. TIRADS and BSRTC systems were adopted to judge the ultrasound and FNAC results. The receiver operating characteristic curve was established to assess the diagnostic value of each method. Results The sensitivity, specificity, and AUC of TIRADS were 74. 3%, 84. 5%, and 0. 794, respectively. BSRTC had higher specificity(98. 3%) and equal sensitivity compared to TIRADS. The sensitivity, specificity, and AUC of BRAFV600E detection were the highest ones among the three methods. Combinations of different methods could increase the diagnostic sensitivity and accuracy. The combination of FNAC and BRAFV600E detection significantly increased the diagnostic efficiency(AUC=0. 984), with sensitivity 98. 6% and specificity 98. 3%. Conclusions The diagnostic value of BRAFV600E detection in the differentiation of benign and malignant thyroid nodules is better than both TIRADS and BSRTC, and the combination of FNAC and BRAFV600E detection reaches the best diagnostic efficiency.
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Radioiodine ablation (RIA) therapy is one of the most important treatments for papillary thyroid carcinoma (PTC), but some patients who received (131)I have radioiodine-refractory disease caused by the decreased expression of the Na(+)/I(-) symporter (NIS). BRAF(V600E) mutation is one possible risk factor that can disturb the NIS expression, but the roles are unclear in clinical practice. This research discussed the association of BRAF(V600E) mutation and NIS expression in PTC tissue and the clinical implications in RIA therapy. 134 PTC samples were collected between June 2013 and June 2014 from Tongji Hospital affiliated to Tongji Medical College, and their clinical characteristics were analyzed. RT-PCR was used to detect the BRAF(V600E) mutation from formalin-fixed paraffin-embedded samples, and immunohistochemistry was applied to detect the NIS expression. IPP software was used to calculate the relative expression quantity of NIS. We found that there was no significant correlation between the absorbance (A) values of NIS and clinicopathologic features in these cases, even thyroid stimulating hormone. BRAF(V600E) mutation showed inhibitory effect on the NIS expression without statistically significant difference in all PTC cases (β=-0.0195, P=0.085), but in the subgroup without hashimoto's thyroiditis (HT), BRAF(V600E) mutation could significantly inhibit the NIS expression (β=-0.0257, P=0.046). The results indicate that BRAF(V600E) mutation is correlated with a lower expression of NIS in PTCs without HT, suggesting the radioiodine-refractory effects during RIA therapy in these patients.
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Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Carcinoma , Genética , Metabolismo , Carcinoma Papilar , Estudios de Casos y Controles , Mutación Missense , Proteínas Proto-Oncogénicas B-raf , Genética , Simportadores , Genética , Metabolismo , Neoplasias de la Tiroides , Genética , MetabolismoRESUMEN
Objective BRAFV600E mutation, RET/PTC rearrangement, and the concomitant of Hashimoto's thyroiditis(HT) could influence clinicopathological features of papillary thyroid carcinoma (PTC).This study is to investigate the distribution of three factors in PTC and to analyze their associations with clinicopathological characteristics.Methods Fine-needle aspiration samples were collected in a total of 122 conventional PTC patients, who were confirmed by surgery.The clinicopathological features were collected to analyze its association with different factors.BRAFV600E mutation and RET/PTC rearrangement were detected by pyrosequencing and Taqman-qPCR, respectively.Results BRAFV600E mutation was significantly correlated with an older age and a less coexistence with HT(P＜0.05).In contrast, RET/PTC rearrangement was more prevalent in young patients and was associated with the concomitant of HT(P＜0.05).In the age of ≥20 year and＜45 year groups, BRAFV600E mutation was significantly associated with extrathyroidal invasiveness.RET/PTC rearrangement was significantly associated with bilateral lymph node metastasis and the number of metastatic lymph node.Conclusions The distribution of three factors were different in PTC patients.In addition to the age at diagnosis, all of three factors should also be considered together to analyze the association of clinicopathological features of PTC.
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reference index of lymph node in central region.
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PURPOSE: This study evaluated the association of the BRAF(V600E) mutation with known prognostic factors and ultrasonographic characteristics in cases of papillary thyroid carcinoma. METHODS: Subjects included 169 patients who received thyroidectomy at Wonju Christian Hospital under the diagnosis of papillary thyroid cancer from February 2010 to October 2011. RESULTS: Of the total patients who received thyroidectomy, there were 128 cases (75,7%) of BRAF(V600E) mutation. Neither age nor sex were associated with the BRAF(V600E) mutation. Tumor size, shape, margin, extrathyroidal extension, central node metastasis and lateral node metastasis were found not to be associated with the BRAF(V600E) mutation. Tumor calcification, echogenicity and vascularity were also not associated with the mutation. CONCLUSION: As debate remains about the association between the BRAF(V600E) mutation and clincopathologic factors and ultrasonographic characteristics in cases of papillary thyroid carcinoma, further study is needed.