AGK-BRAF gene fusion is a recurrent event in sporadic pediatric thyroid carcinoma

Thyroid cancer is the fastest increasing cancer worldwide in all age groups. Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer in both adults and children. PTC genomic landscape has been extensively studied in adults, but information regarding sporadic pediatric patients is lacking. Although BRAF V600E mutation is highly prevalent in adults, this mutation is uncommon in pediatric cases. As adult and pediatric PTC is a mitogen-activated protein kinase-driven cancer, this altered pathway might be activated by different genetic events. The aim of this study was to investigate the occurrence of AGK-BRAF fusion gene, recently described in radiation-exposed pediatric PTC, in a cohort of exclusively sporadic pediatric PTC. The series consisted of 30 pediatric PTC younger than 18 years of age at the time of diagnosis and 15 matched lymph node metastases (LNM). Primary tumors and matched LNM were screened for the presence of the AGK-BRAF fusion transcript by RT-PCR. To confirm the identity of the amplified products, randomly selected samples positive for the presence of the fusion transcripts were sequenced. Moreover, BRAF dual-color, break-apart probes confirmed BRAF rearrangement. Overall, the AGK-BRAF fusion gene was detected in 10% (3/30) of primary tumors. For one of these cases, paired LNM was also available, which also shows the presence of AGK-BRAF fusion gene. This study described, for the first time, the presence of AGK-BRAF in sporadic pediatric PTC. Understanding the molecular events underlying pediatric PTC may improve preoperative diagnosis, allow molecular prognostication and define a therapeutic approach toward sporadic PTC patients.Sao Paulo State Research Foundation (FAPESP)CNPqFAPESP scholarUniv Fed Sao Paulo, Dept Morphol & Genet, Div Genet, Genet Bases Thyroid Tumors Lab, Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Dept Pathol, Sao Paulo, SP, BrazilIrmandade Santa Casa Misericordia Sao Paulo, Dept Pediat, Sao Paulo, SP, BrazilIrmandade Santa Casa Misericordia Sao Paulo, Dept Med, Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Dept Morphol & Genet, Div Genet, Genet Bases Thyroid Tumors Lab, Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Dept Pathol, Sao Paulo, SP, BrazilFAPESP: 2012/02902-9FAPESP: 2013/03867-5FAPESP: 2014/06570-6CNPq: 470441/2013-5Web of Scienc

Investigação De Rearranjos Cromossômicos No Gene Braf Em Carcinomas Pediatrícos Da Tiroide

The incidence of thyroid carcinoma, mainly of the papillary histological subtype (PTC), has increased in most populations, including pediatric patients. Genetic alterations leading to the MAPK pathway activation are highly prevalent in PTC. It has been demonstrated that BRAF V600E mutation is the most prevalent alteration found in sporadic PTC in adult (~40%). However, this event is rarely identified in pediatric PTC. In contrast, chromosomal rearrangements involving BRAF have been identified, mainly in radiation-exposed pediatric PTC. Our group described, for the first time, the presence of AGK-BRAF fusion in sporadic pediatric PTC. This suggests that it might be an alternative mechanismof aberrant BRAF activation in pediatric cases. Thus, the objective of this work was to investigate rearrangements involving BRAF. In the first part of the study, we performed the investigation on 22 pediatric PTC, by FISH break-apart (FISH BA), 5'RACE and sequencing. By FISH BA, it was revealed four positive cases for the BRAF gene breakage, with 10%, 11%, 17% and 36% of the primary tumor cells carrying the rearrangement. Using 5'RACE and sequencing techniques, it was possible to identify the AGK/BRAF rearrangement in one of them. In the second part of the study, we expanded our analysis to 52 pediatric PTC. AGK/BRAF fusion was investigated by RT-PCR and BRAF V600E mutation by PCR and sequencing methodologies. The AGK/BRAF fusion was identified in 27% of the cases and associated with younger ages while BRAF V600E mutation was found in 8% and associated with older ages. PTC with BRAF alterations tends to have a more aggressive biological behavior.A incidência de carcinoma da tiroide, principalmente do subtipo histológico papilífero (CPT), aumentou na maioria das populações, incluindo pacientes pediátricos. As alterações genéticas que conduzem à ativação da via MAPK são altamente prevalentes no CPT. Tem sido demonstrado que a mutação BRAF V600E é a mais frequente no CPT adulto (~40%). No entanto, essa alteração é raramente identificada no CPT pediátrico. Em contraste, rearranjos cromossômicos envolvendo BRAF têm sido identificados, principalmente,no CPT pediátrico exposto à radiação. Nosso grupo descreveu a presença da fusão AGK/BRAF pela primeira vez em CPT pediátrico esporádico, sugerindo que rearranjos neste gene podem ser um mecanismo alternativo de ativação alterada de BRAF em casos pediátricos. Desse modo, o objetivo deste trabalho foi investigar rearranjos envolvendo BRAF na faixa etária pediátrica. Na primeira parte do estudo, realizamos a investigação em 22 CPTs pediátricos, por FISH break-apart (FISH BA), 5’RACE e sequenciamento. A técnica de FISH BA revelou quatro casos positivos para a quebra no gene BRAF, nas proporções de 10%, 11%, 17% e 36% das células do tumor primário portando o rearranjo. A partir de 5’RACE e sequenciamento, foi possível identificar o rearranjo AGK/BRAF em uma delas. Na segunda parte do estudo, a casuística foi aumentada para 52 pacientes. Foi realizada uma triagem para a fusão AGK/BRAF por RT-PCR. A mutação BRAF V600E foi investigada por PCR e sequenciamento. A fusão AGK/BRAF foi identificada em 27% dos casos e associada com pacientes mais jovens, e a mutação BRAF V600E, em 8% e associada com pacientes mais velhos. CPTs com alterações em BRAF tendem a ter um comportamento biológico mais agressivo.Dados abertos - Sucupira - Teses e dissertações (2017

AGK::BRAF no carcinoma papilífero da tiroide pediátrico: investigação do mecanismo associado à agressividade tumoral e análise funcional

Introdução: O carcinoma papilífero da tireoide (CPT), apesar de ser raro em crianças e adolescentes, geralmente têm uma apresentação clínica mais agressiva nesta população do que o CPT em adultos, apresentando multifocalidade, subtipos histológicos mais agressivos, presença de invasão tumoral, e maior prevalência de metástase loco-regional e à distância. Apesar disso, a mortalidade pelo câncer na faixa etária pediátrica é inferior àquela reportada em adultos. Tem sido sugerido que estas características clínico patológicas observadas em pacientes pediátricos (≤ 18 anos) deve-se, principalmente, às diferenças observadas quanto ao tipo de alteração observada nos genes “drivers” que levam à ativação constitutiva da via MAPK/ERK neste grupo de pacientes, bem como, maior responsividade ao tratamento convencional como iodo. Objetivos: Esta tese teve como objetivo analisar alterações genéticas associadas a ativação das vias RAS/MAPK/ERK e PI3K/AKT em 79 casos de CPT pediátricos e avaliar o efeito da presença da fusão AGK::BRAF na ativação da via MAPK, na expressão do gene NIS (do inglês, sodium/iodide symporter) e na atividade de NIS, bem como, em processos biológicos associados à progressão tumoral. Metodologia: As fusões gênicas (RET::PTC1, RET::PTC3, AGK::BRAF, ETV6::NTRK3 e STRN::ALK) foram avaliadas por RT-PCR e as variantes patogênicas do tipo SNVs (do inglês, Single Nucleotide Variants) (BRAF V600E, e TERT C228T/C250T) avaliadas por sequenciamento de Sanger. A análise funcional foi realizada na linhagem celular derivada de células foliculares de tiroide normal de rato (PCCL3) permanentemente e independentemente transfectadas com plasmídeo contendo cDNA de AGK::BRAF, ETV6::NTRK3, RET::PTC1, RET::PTC3, BRAF V600E, e BRAF WT (do inglês, wild type). Para examinar o efeito destes eventos genéticos nas vias de sinalização RAS/MAPK/ERK e PIK3/AKT e nas taxas da expressão de NIS, utilizamos a técnica de Western-blot. A atividade de NIS foi analisada por um ensaio de captação de iodeto não radioativo. A instabilidade genômica foi investigada por ensaio de micronúcleos com DAPI e microscopia fluorescente. A organização dos telômeros foi avaliada por hibridização in situ fluorescente quantitativa (Q-FISH) seguida por análise da organização tridimensional (3D) dos telômeros utilizando o software TeloView®. A organização da cromatina, por microscopia de super-resolução de iluminação estruturada tridimensional (3D-SIM), seguido por granulometria. Resultados: Na análise do perfil molecular, identificamos alterações genéticas em 53/79 (67%) dos casos de CPT, sendo que 59% (47/79) apresentavam fusões gênicas. RET::PTC1 foi a alteração genética mais frequente (22/79, 28%), seguida de AGK::BRAF (15/79, 19%), ETV6::NTRK3 (14/79, 18%), RET::PTC3 (12/79, 15%), BRAF V600E (11/79, 14%) e STRN::ALK (3/79, 4%). Entre as amostras analisadas para mutações do promotor de TERT (C228T e C250T) (n = 42), apenas uma (1/42, 2,4%) carregava uma variante patogênica (C250T). Além disso, identificamos a coexistência de mais de uma alteração genética associada à via MAPK em cerca de 1/3 desta coorte. Encontramos uma associação entre a presença de fusão RET::PTC3 e metástases linfonodais, multifocalidade e tamanho do tumor. A análise funcional demonstrou que a fusão AGK::BRAF leva a maior ativação da via MAPK e menor expressão e atividade de NIS em células PCCL3. Estes dados foram acompanhados por um maior número de micronúcleos e pontes anafásicas, alteração da organização 3D dos telômeros e da estrutura da cromatina. Demonstramos que os tumores de pacientes pediátricos têm diferentes assinaturas de organização telomérica e estrutura do DNA, em comparação com o tecido normal, sugerindo uma ruptura progressiva da organização da cromatina e dos telômeros, que pode estar relacionada à instabilidade genômica. Além disso, dados preliminares do 3D-SIM demonstram que pacientes com características clinicopatológicas de mau prognóstico (metástase à distância, multifocalidade e tamanho tumoral > 2 cm) apresentam mais espaços pobres em DNA nos núcleos dos tumores. Curiosamente, os tumores positivos para a fusão AGK::BRAF, mostraram uma tendência a ter mais espaços pobres em DNA em seus núcleos. De fato, demonstramos, em uma análise feita em um relato de caso, que a fusão AGK::BRAF resulta em uma organização diferente dos telômeros e do DNA, quando comparada a RET::PTC3. Conclusão: Aqui confirmamos que rearranjos são o evento genético mais comum no CPT pediátrico, e ainda demonstramos que co-ocorrência de mais de uma alteração genética que levam à ativação da via RAS/MAPK/ERK são prevalentes nos tumores desta população. Importante, observamos que o rearranjo RET::PTC3 está associado a características de mau prognóstico no CPT pediátrico. Os dados da análise funcional ajudaram a elucidar os mecanismos pelos quais a fusão AGK::BRAF causa a desdiferenciação das células tiroidianas e um comportamento mais agressivo das células tumorais em casos pediátricos de CPT com metástase à distância. Também demonstramos como o estudo da organização tridimensional nuclear e da estrutura do DNA do CPT pediátrico pode ser de extrema importância para o melhor entendimento desses tumores.Introduction: The papillary thyroid carcinoma (PTC), despite being rare in children and adolescents, generally has a more aggressive clinical presentation in this population than in adult PTC, with multifocality, more aggressive histological subtypes, presence of tumor invasion, and higher prevalence of locoregional and distant metastasis. Despite this, cancer mortality in the pediatric age group is lower than that reported in adults. It has been suggested that these clinical and pathological characteristics observed in pediatric patients (≤ 18 years old) are mainly due to the differences observed in the type of alteration observed in the ―driver‖ genes that lead to the constitutive activation of the MAPK/ERK pathway in this group of patients, as well as greater responsiveness to conventional treatment such as iodine. Objectives: This thesis aimed to analyze the genetic alterations associated with the activation of the RAS/MAPK/ERK and PI3K/AKT pathway in 79 cases of pediatric PTC and to evaluate the effect of the presence of the AGK::BRAF fusion on the activation of the MAPK pathway, on the expression of the NIS protein (sodium/iodide symporter) and NIS activity, as well as biological processes associated with tumor progression. Methodology: Gene fusions (RET::PTC1, RET::PTC3, AGK::BRAF, ETV6::NTRK3 and STRN::ALK) were evaluated by RT-PCR and point genetic alterations (BRAF V600E, and TERT C228T/C250T) were screened by sequencing from Sanger. Functional analysis was performed on a cell line derived from rat normal thyroid follicular cells (PCCL3) permanently and independently transfected with plasmid containing cDNA of AGK::BRAF, ETV6::NTRK3, RET::PTC1, RET::PTC3, BRAF V600E, and BRAF WT (wild type). To examine the effect of these genetic events on RAS/MAPK/ERK and PIK3/AKT signaling pathways and on NIS expression rates, we used the Western-blot technique. NIS activity was analyzed by a non-radioactive iodide uptake assay. Genomic instability was investigated by DAPI micronucleus assay and fluorescent microscopy. Telomere organization was assessed by xiv quantitative fluorescent in situ hybridization (Q-FISH) followed by three-dimensional (3D) telomere organization analysis using the TeloView® software. Chromatin organization was analyzed by super-resolution three-dimensional structured illumination microscopy (3D-SIM), followed by granulometry. Results: In the molecular profile analysis, we identified genetic alterations in 53/79 (67%) of PTC cases, with 59% (47/79) having gene fusions. RET::PTC1 was the most frequent genetic alteration (22/79, 28%), followed by AGK::BRAF (15/79, 19%), ETV6::NTRK3 (14/79, 18%), RET::PTC3 (12/79, 15%), BRAF V600E (11/79, 14%), and STRN::ALK (3/79, 4%). Among the samples analyzed for TERT promoter mutations (C228T and C250T) (n = 42), only one (1/42, 2.4%) carried a mutation (C250T). Furthermore, we identified the coexistence of more than one genetic alteration associated with the RAS/MAPK/ERK pathway in approximately 1/3 of our cohort. We found an association between the presence of RET::PTC3 fusion and lymph node metastases, multifocality and tumor size. Functional analysis demonstrated that the AGK::BRAF fusion leads to greater activation of the RAS/MAPK/ERK pathway and lower expression and activity of NIS in PCCL3 cells. These data were accompanied by a greater number of micronuclei and anaphase bridges, changes in the 3D organization of telomeres and chromatin structure. We demonstrated that tumors from pediatric patients have different signatures of telomere organization and DNA structure compared to normal tissue, suggesting a progressive disruption of chromatin and telomere organization, which may be related to genomic instability. Furthermore, preliminary data from 3D-SIM demonstrate that patients with clinicopathological features of poor prognosis (distant metastasis, multifocality, and tumor size > 2 cm) have more DNA-poor spaces in tumor nuclei. Interestingly, tumors positive for the AGK::BRAF fusion showed a tendency to have more DNA-poor spaces in their nuclei. In fact, we demonstrated, in an analysis made in a case report, that the AGK::BRAF fusion results in a different organization of telomeres and DNA, when compared to RET::PTC3. Conclusion: Here we confirm that rearrangements are the most common genetic event in pediatric PTC, and we also demonstrated that the co-occurrence of more xv than one genetic alteration that led to the activation of the RAS/MAPK/ERK pathway are prevalent in tumors in this population. Importantly, we observed that the RET::PTC3 rearrangement is associated with poor prognostic features in pediatric PTC. Functional analysis data helped to elucidate the mechanisms by which the AGK::BRAF fusion causes thyroid cell dedifferentiation and more aggressive tumor cell behavior in pediatric cases of PTC with distant metastasis. We also demonstrate how the study of the three-dimensional nuclear organization and DNA structure of pediatric PTC can be extremely important for a better understanding of these tumors.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)2018/26395-

Incorporation of 5-ethynyl-2'-deoxyuridine (EdU) as a novel strategy for identification of the skewed X inactivation pattern in balanced and unbalanced X-rearrangements

X-chromosome inactivation occurs randomly in normal female cells. However, the inactivation can be skewed in patients with alterations in X-chromosome. In balanced X-autosome translocations, normal X is preferentially inactivated, while in unbalanced X alterations, the aberrant X is usually inactivated. Here, we present a novel strategy to verify the skewed X inactivation pattern through the incorporation of 5-ethynyl-2'-deoxyuridine (EdU) into cells, in 11 patients: five carriers of balanced X-autosome translocations and six of unbalanced X-chromosome alterations. Since EdU is a labeled nucleoside analog of thymidine, its incorporation during DNA synthesis can reveal late replication regions and the inactive X-chromosome. All EdU findings were validated by the human androgen receptor gene (HUMARA) assay. The late replication regions were easily and quickly visualized in all cells, where inactive Xs are marked with strong green fluorescence. It was observed that the normal X-chromosome was preferentially inactivated in patients with balanced X-autosome translocations; while the aberrant X-chromosome was inactivated in most cells from patients with unbalanced alterations. By performing the fluorescence-based EdU assay, the differences between the active and inactive X-chromosomes are more easily recognizable than by classic cytogenetic methods. Furthermore, EdU incorporation allows the observation of the late replication regions in autosomal segments present in X derivatives from X-autosome translocations. Therefore, EdU assay permits an accurate and efficient cytogenetic evaluation of the X inactivation pattern with a low-cost, easy to perform and highly reproducible technique.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Univ Fed Sao Paulo, Dept Morphol & Genet, BR-04023900 Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Biochem, Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Med, Sao Paulo, BrazilSao Paulo State Univ, Inst Biociencias Botucatu, Dept Genet, BR-18618970 Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Morphol & Genet, BR-04023900 Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Biochem, Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Med, Sao Paulo, BrazilWeb of Scienc

A Multifocal Pediatric Papillary Thyroid Carcinoma (PTC) Harboring the <i>AGK-BRAF</i> and <i>RET/PTC3</i> Fusion in a Mutually Exclusive Pattern Reveals Distinct Levels of Genomic Instability and Nuclear Organization

The spectrum and incidence of gene fusions in papillary thyroid carcinoma (PTC) can differ significantly depending on the age of onset, histological subtype or radiation exposure history. In sporadic pediatric PTC, RET/PTC1-3 and AGK-BRAF fusions are common genetic alterations. The role of RET/PTC as a prognostic marker in pediatric PTC is still under investigation. We recently showed that AGK-BRAF fusion is prevalent in young patients (mean 10 years) and associated with specific and aggressive pathological features such as multifocality and lung metastasis. In this pilot study, we report a unique patient harboring three different foci: the first was positive for AGK-BRAF fusion, the second was positive for just RET/PTC3 fusion and the third was negative for both rearrangements. To investigate whether AGK-BRAF and RET/PTC3 are associated with genomic instability and chromatin modifications, we performed quantitative fluorescence in situ hybridization (Q-FISH) of telomere repeats followed by 3D imaging analysis and 3D super-resolution Structured Illumination Microscopy (3D-SIM) to analyze the DNA structure from the foci. We demonstrated in this preliminary study that AGK-BRAF is likely associated with higher levels of telomere-related genomic instability and chromatin remodeling in comparison with RET/PTC3 foci. Our results suggest a progressive disruption in chromatin structure in AGK-BRAF-positive cells, which might explain a more aggressive disease outcome in patients harboring this rearrangement

AGK

Thyroid cancer is the fastest increasing cancer worldwide in all age groups. Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer in both adults and children. PTC genomic landscape has been extensively studied in adults, but information regarding sporadic pediatric patients is lacking. Although BRAF V600E mutation is highly prevalent in adults, this mutation is uncommon in pediatric cases. As adult and pediatric PTC is a mitogen‐activated protein kinase‐driven cancer, this altered pathway might be activated by different genetic events. The aim of this study was to investigate the occurrence of AGK‐BRAF fusion gene, recently described in radiation‐exposed pediatric PTC, in a cohort of exclusively sporadic pediatric PTC. The series consisted of 30 pediatric PTC younger than 18 years of age at the time of diagnosis and 15 matched lymph node metastases (LNM). Primary tumors and matched LNM were screened for the presence of the AGK‐BRAF fusion transcript by RT‐PCR. To confirm the identity of the amplified products, randomly selected samples positive for the presence of the fusion transcripts were sequenced. Moreover, BRAF dual‐color, break‐apart probes confirmed BRAF rearrangement. Overall, the AGK‐BRAF fusion gene was detected in 10% (3/30) of primary tumors. For one of these cases, paired LNM was also available, which also shows the presence of AGK‐BRAF fusion gene. This study described, for the first time, the presence of AGK‐BRAF in sporadic pediatric PTC. Understanding the molecular events underlying pediatric PTC may improve preoperative diagnosis, allow molecular prognostication and define a therapeutic approach toward sporadic PTC patients

Telomere Architecture Correlates with Aggressiveness in Multiple Myeloma

The prognosis of multiple myeloma (MM), an incurable B-cell malignancy, has significantly improved through the introduction of novel therapeutic modalities. Myeloma prognosis is essentially determined by cytogenetics, both at diagnosis and at disease progression. However, for a large cohort of patients, cytogenetic analysis is not always available. In addition, myeloma patients with favorable cytogenetics can display an aggressive clinical course. Therefore, it is necessary to develop additional prognostic and predictive markers for this disease to allow for patient risk stratification and personalized clinical decision-making. Genomic instability is a prominent characteristic in MM, and we have previously shown that the three-dimensional (3D) nuclear organization of telomeres is a marker of both genomic instability and genetic heterogeneity in myeloma. In this study, we compared in a longitudinal prospective study blindly the 3D telomeric profiles from bone marrow samples of 214 initially treatment-naïve patients with either monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), or MM, with a minimum follow-up of 5 years. Here, we report distinctive 3D telomeric profiles correlating with disease aggressiveness and patient response to treatment in MM patients, and also distinctive 3D telomeric profiles for disease progression in smoldering multiple myeloma patients. In particular, lower average intensity (telomere length, below 13,500 arbitrary units) and increased number of telomere aggregates are associated with shorter survival and could be used as a prognostic factor to identify high-risk SMM and MM patients.Medicine, Faculty ofNon UBCMedicine, Department ofReviewedFacult