Terapias dirigidas con inhibidores del braf en gliomas pediátricos de alto y bajo grado con la mutación brafv600e. Experiencia del hospital Prof. Dr. Juan P. Garrahan / Targeted therapies with BRAF inhibitors for pediatric low- and high-grade gliomas with BRAF v600e mutation. Prof. Dr. Juan P. Garrahan Hospital experience

Resumen La mutación puntual V600E del gen BRAF juega un papel fundamental en la tumorigénesis de muchos gliomas. La inhibición de su producto forma parte de terapias innovadoras emergentes en los últimos años. Conocer el rol de estos tratamientos resulta imprescindible. El objetivo del trabajo fue describir la respuesta clínico-radiológica en niños con gliomas BRAF V600E mutado tratados con inhibidores BRAF. Para ello se realizó un estudio descriptivo y retrospectivo en pacientes menores de 16 años con gliomas BRAF V600E mu tado que recibieron vemurafenib o dabrafenib en el Hospital Garrahan. Trece pacientes tratados en los últimos 7 años fueron incluidos: 9 gliomas de bajo grado y 4 de alto grado. La mediana de edad al diagnóstico fue 8.6 años (0.89-14.04) y del comienzo del inhibidor 11.62 años (3.64-15.42). Inicialmente, todos habían realizado tratamiento quirúrgico, y 12/13 recibieron previamente otra terapia: 11 quimioterapia (eventualmente hasta 4 líneas distintas) y 4 radioterapia. Con la terapia dirigida, 10 pacientes tuvieron una disminución tumoral mayor o igual al 25%, quedando evidenciada en 7 niños la mejor respuesta dentro de los 6 meses del inicio. Hubo 4 progresados intratratamiento (todos alto grado), y 2 progresados prontamente luego de suspender el inhibidor (ambos bajo grado). Cinco presentaron efectos adversos grado 3-4, con recuperación ad-integrum. Se describe una buena y sostenida respuesta clínico-radiológica, con tolerancia aceptable, en pacientes con gliomas de bajo grado BRAF V600E mutado tratados con inhibidores BRAF V600E . En contraste, la respuesta en pacientes con gliomas de alto grado fue intermedia y de poca duración, con progresión tumoral precoz.

Abstract The BRAF V600E point mutation plays a key role in the tumorigenesis of many gliomas. Inhibiting its product is part of the innovative therapies emerging in recent years. Knowing the role of these treatments is essential. The aim of this experience was to describe the clinical-radiological response of pediatric BRAF V600E mutated gliomas treated with BRAF inhibitors. To this end, a descriptive and retrospective study was performed in patients under 16 years of age with BRAF V600E gliomas, who received vemurafenib or dabrafenib at Hospital Garrahan. Thirteen patients treated in the last 7 years were included: 9 were low-grade and 4 high-grade gliomas. The median age at diagnosis was 8.6 years (0.89-14.04) and at start of targeted therapy was 11.62 years (3.64-15.42). All patients had previously a surgical procedure, and 12/13 had received another therapy prior BRAF inhibition: 11 chemotherapy (in one case, up to 4 different protocols) and 4 radiotherapy. Under targeted therapy, tumour response was obtained in 10 patients (size reduction equal to or greater than 25%), and best response was observed in the first 6 months of treatment in 7 children. Four patients progressed under treatment (all high-grade gliomas) and 2 progressed shortly after stopping the inhibitor (both low-grade gliomas). Five patients had grade 3-4 toxicity, with subsequent full recovery. A good and sustained clinical-radiological response, with acceptable tolerance, is described in patients with BRAF V600E mutated low-grade gliomas treated with BRAF V600E inhibitors. In contrast, the response in patients with high-grade gliomas was intermediate and of short duration, with early tumour progression.

Efeitos da 5-iodotubercidina em linhagens de melanoma humano parentais e resistentes ao inibidor de BRAF / Effects of 5-iodotubercidin on human melanoma lines of parental and resistant to the BRAF inhibitor

O melanoma é responsável por menos de 5% dos cânceres de pele, porém, 95% das mortes ocorrem devido a ocorrência de metástases. O melanoma metastático é refratário às terapias convencionais e rapidamente adquire resistência às terapias como as oncogene-dirigidas, como o inibidor de BRAF, da via de MAPK. Estudos prévios de screening in silico do nosso grupo, onde se utilizou as bases de dados TCGA e GEO, identificaram o gene adenosina quinase (ADK) como sendo diferencialmente expresso entre o melanoma invasivo e os nevus. A 5-iodotubercidina (5-ITu) é um potente inibidor farmacológico da ADK que dentre os diversos efeitos relatados na literatura destaca-se pelo potencial genotóxico. Os danos no DNA são os principais ativadores de checkpoint do ciclo celular, que levam a parada do ciclo celular transitória ou permanente, além de induzir morte celular, levando a hipótese de que ADK possa ser potencial agente anti-melanoma. Este trabalho objetivou avaliar a expressão do gene ADK em melanomas humanos e quimiorresistentes ao inibidor de BRAF (iBRAF), avaliou os impactos de 5-ITu sobre a proliferação, progressão do ciclo celular e morte celular e por fim avaliamos sua capacidade de aumentar a sensibilidade das células. Foi realizado PCR em tempo real para avaliar os níveis de expressão de mRNA de ADK em linhagens de melanoma e na cultura primária de melanócitos; a fim de avaliar a citotoxicidade de 5-ITu foram realizados os ensaios de exclusão por azul de tripan e de apoptose - Anexina V e PI e em modelo de esferoide, usando live/dead; também foi avaliada a influência de 5-ITu sobre a capacidade clonogênica e seus efeitos sobre a proliferação celular, a partir dos ensaios de ciclo celular e avaliação de marcadores de proliferação por imunofluorescência; as linhagens foram submetidas a diferentes regimes de tratamento com 5-ITu e o iBRAF, a fim de avaliar a curva de crescimento e a sensibilidade ao iBRAF por MTT níveis de expressão de mRNA de ADK maiores nas linhagens tumorais em relação aos melanócitos. 5-ITu mostrou-se capaz de inibir a proliferação (IC50) das linhagens de melanoma em concentrações de 1,9 a 3,5 µM. 5-ITu não foi capaz de induzir inviabilidade celular, apesar de reduzir a quantidade de células viáveis em todas as condições de tratamento, também não foi capaz de induzir aumento significativo de células apoptóticas, nem mesmo necróticas. No entanto, o tratamento com 5-ITu reduziu a capacidade clonogênica de linhagens de melanoma e promoveu parada de ciclo celular nas fases G1 e G2/M, levou ao aumento da população subG1. O tratamento com 5-ITu promoveu a redução da expressão de marcadores de proliferação, como ki67, e a combinação de tratamentos 5-ITu e iBraf foi capaz de aumentar o tempo de dobramento das linhagens de melanoma, embora tenha se mostrado incapaz de sensibilizar as células de melanoma ao tratamento com iBRAF. Desse modo, pode-se concluir que 5-ITu induz o efeito citostático e se mostra um potente agente antiproliferativo para melanoma parental e resistente

Melanoma accounts for less than 5% of skin cancers, but 95% of deaths occur due to metastases. Metastatic melanoma is refractory to conventional therapies and rapidly acquires resistance to therapies such as oncogene-directed, such as the BRAF inhibitor, of the MAPK pathway. Previous studies of screening in silico of our group, using the databases TCGA and GEO, identified the adenosine kinase gene (ADK) as differentially expressed between invasive melanoma and nevus. 5-iodotubercidin (5-ITu) is a potent pharmacological inhibitor of ADK that among the several effects reported in the literature stands out for the genotoxic potential. DNA damage is the main activator of the cell cycle checkpoint, which leads to transient or permanent cell cycle arrest, in addition to inducing cell death, leading to the hypothesis that ADK may be a potential anti-melanoma agent. This work aimed to evaluate the expression of the ADK gene in human melanomas and chemoresistants to the BRAF inhibitor (iBRAF), evaluated the impacts of 5-ITu on proliferation, cell cycle progression and cell death and finally we evaluated its ability to increase the sensitivity of cells. Real-time PCR was performed to assess the levels of ADK mRNA expression in melanoma lines and primary melanocyte culture; in order to evaluate the cytotoxicity of 5-ITu, the trypan blue and apoptosis - Annexin V and PI exclusion and blue spheroid models were performed using live / dead; the influence of 5-ITu on the clonogenic capacity and its effects on cell proliferation, from the cell cycle assays and the evaluation of proliferation markers by immunofluorescence; the cell lines were submitted to different treatment regimens with 5-ITu and iBRAF in order to evaluate the growth curve and the sensitivity to iBRAF by MTT levels of mRNA expression of ADK higher in the tumor lines in relation to the melanocytes. 5-ITu was able to inhibit the proliferation (IC 50) of melanoma lines at concentrations of 1.9 to 3.5 181;M. 5-ITu was not able to induce cell non-viability, although it reduced the amount of viable cells in all treatment conditions, nor was it able to induce a significant increase in apoptotic or even necrotic cells. However, treatment with 5-ITu reduced the clonogenic capacity of melanoma cells and promoted cell cycle arrest in the G1 and G2 / M phases, leading to an increase in the subG1 population. Treatment with 5-ITu promotes the reduction of expression of proliferation markers, such as ki67, and the combination of 5-ITu and iBRAF treatments was able to increase the doubling time of melanoma cells, although it has been shown to be unable to sensitize melanoma cells to treatment with iBRAF. Thus, it can be concluded that 5-ITu induces the cytostatic effect and shows a potent antiproliferative agent for parental and resistant melanoma

Treatment of advanced melanoma - A changing landscape / Tratamento de melanoma avançado - Um panorama em transformação

Summary Following decades of relative ostracism, advances in the treatment of melanoma have brought a new reality for patients, physicians and researchers. While antibodies targeting molecules involved in the modulation of the interaction between melanoma and immune cells changed the meaning of the term "cancer immunotherapy," a better characterization of the molecular aberrations involved in melanoma carcinogenesis prompted the development of inhibitors of the mitogen-activated protein kinase pathway (MAPK) that also led to significant improvements both in response rates and survival. As a result, new drugs have been approved for clinical use in the United States and Europe, including the immune-checkpoint blockers ipilmumab, pembrolizumab and nivolumab, the oncolytic herpesvirus talimogene laherparepvec, and the targeted-agents vemurafenib, dabrafenib, cobimetinib and trametinib. In this article, we review the results of studies that brought new approaches to the bedside and discuss how these developments are being incorporated into the care of patients in Brazil.

Resumo Após décadas de ostracismo, os recentes avanços no tratamento do melanoma trouxeram uma nova realidade para pacientes, médicos e pesquisadores. Enquanto anticorpos monoclonais voltados a moléculas envolvidas na modulação da interação entre células do melanoma e do sistema imune consolidaram o uso da "imunoterapia", um melhor conhecimento acerca das aberrações genômicas envolvidas na carcinogênese do melanoma viabilizaram o desenvolvimento de inibidores da via mitogen-activated protein kinase pathway (MAPK), o que também resultou em ganhos significativos em taxas de resposta e sobrevida. Consequentemente, novas modalidades de tratamento foram aprovadas para uso clínico nos Estados Unidos e na Europa, incluindo os bloqueadores de correceptores imunes ipilimumabe, nivolumabe e pembrolizumabe, o herpesvírus oncolítico talimogene laherparepvec (T-VEC), e os agentes-alvo vemurafenibe, dabrafenibe, cobimetinibe e trametinibe. Nesse artigo, revisamos os resultados que trouxeram novas alternativas para a prática clínica e discutimos a incorporação desses avanços ao cuidado de pacientes no Brasil.

An extremely rare case of concurrent BRAF V600E mutation driven hairy cell leukemia and melanoma: case report and review of literature

BRAF protein is a serine/threonine kinase with 766 amino acids. Approximately 15% of human cancers harbor BRAF mutations as well as other BRAF anomalies (amplifications, fusions). Somatic mutations mainly occur in the catalytic kinase domain (CR3), and the predominant mutation is p.V600E which is the substitution of glutamic acid (E) for valine (V) as result of a mutation at codon 600 of the kinase domain. To our knowledge, the vast majority of the cancers have non-germline BRAF mutations. Here we describe a case of a 60-year-old female with a history of hairy cell leukemia (HCL) who presented with aphasia and forgetfulness. A follow-up Brain CT scan showed three distinct brain lesions which were found to be diagnostic of melanoma (confirmed by immunohistochemistry) with no evidence of a concurrent brain involvement by a B-cell neoplasm. Molecular studies confirmed the same BRAF p.V600E mutation in both malignancies (hairy cell leukemia and melanoma). Thereafter the patient was started on BRAF inhibitor treatment and is now symptom-free after one year of follow up. Having two concurrent malignancies with a shared BRAF mutation is extremely rare and makes this an excellent example of a genomic marker-driven treatment in two histologically and immunophenotypically distinct tumors.

Eficacia y seguridad de vemurafenib en el tratamiento de pacientes con melanoma maligno avanzado con mutación del BRAF/V600E que progresaron al uso de nivolumab / Efficacy and safety of vemurafenib in the treatment of patients with advanced malignant melanoma with BRAF / V600E mutation who progressed to the use of nivoluma

INTRODUCCIÓN: Antecedentes: El presente dictamen expone la evaluación de tecnología de la eficacia y seguridad del uso de vemurafenib como tratamiento de pacientes con diagnóstico de melanoma maligno, metastásico, irresecable, mutación BRAF V600 y que han progresado al tratamiento de primera línea con nivolumab. Aspectos Generales: El Melanoma es un tumor maligno que se origina en los melanocitos y afecta principalmente a la piel. Los melanomas pueden aparecer también en el ojo (úvea, conjuntiva y el cuerpo ciliar), las meninges o en varias superficies de mucosa. Mientras que los melanomas son causantes de cerca del 90% de las muertes asociadas a tumores cutáneos, incluso los tumores pequeños pueden tener tendencia a la metástasis y por lo tanto tener un pronóstico desfavorable. Tecnología Sanitaria de Interés: Vemurafenib (Zelboraf, Roche) es un inhibidor de la proteína quinasa oncogénica BRAF V600. Este medicamento está autorizado para el tratamiento de pacientes adultos con melanoma irresecable o metastásico con la mutación BRAF V600. METODOLOGÍA: Estrategia de Búsqueda: Se realizó una búsqueda de la literatura con respecto a la eficacia y seguridad de vemurafenib para el tratamiento de los pacientes con melanoma maligno, metastásico, irresecable, con mutación BRAF V600 que han progresado a primera línea con nivolumab. Esta búsqueda se realizó utilizando los meta-buscadores: Translating Research into Practice (TRIPDATABASE), National Library (Pubmed-Medline) y Health Systems Evidence. Adicionalmente, se a amplió la búsqueda revisando la evidencia generada por grupos internacionales que realizan revisiones sistemáticas (RS), evaluación de tecnologías sanitarias (ETS) y guías de práctica clínica (GPC), tales como la Cochrane Group, The National Institute for Health and Care Excellence (NICE), the Agency for Health care Research and Quality (AHRQ), The Canadian Agency for Drugs and Technologies in Health (CADTH) y The Scottish Medicine Consortium (SMC). ESta búsqueda se completó ingresando a la página web www,clinicaltrials.gov, para así pode identificar ensayos clínicos en elaboración o que no hayan sido publicados aún, y así disminuir el riesgo de sesgo de publicación. RESULTADOS: Sinopsis de la Evidencia: Se realizó la búsqueda bibliográfica y de eviencia científica para el sustento del uso de vemurafenib para el tratamiento de los pacientes con melanoma maligno, metastásico, irresecable, con mutación BRAF V600 y que han progresado a la primera línea con nivolumab. Se presenta la evidencia disponible según el tipo de publicación priorizada en los criterios de inclusión. Se incluyeron dos GPC, la guía realizada por la Sociedad Europea de Oncología Médica (ESMO) publicada en el 2015, y la guía del Instituto Nacional para Excelencia en el Cuidado de la Salud (NICE) también pulbicada en el 2015.Se incluyó el estudio BRIM3 que comparó vemurafenib con dacar bazina y en el que se basaron las GPC y la ETS. Aunque la población incluida en este estudio eran pacientes sin tratamiento sistémico previo, se trata del único ensayo clínico aleatorizado de fase III. No se encontraton estudios en progreso o sin publicar que respondan a la pregunta de interés de esta evaluación. Estudios excluidos: el estudio de Sosman et al., 2012 no ha sido incluido porque se trata de un ensayo de fase II sin grupo de comparación y la población no incluye a pacientes que han progresado con nivolumab. CONCLUSIONES: Es posible redefinir los tipos de melanomas en función a la presencia de ciertas mutaciones, las cuales conducen a la activación de proteínas señaladores mutantes que inducen la formación de tumores. Las mutaciones en el gen BRAF están presentes en el 40-70% de los melanomas, condiciendo a una activación de la proliferación celular incontrolada. En EsSalud está disponible el medicamento nivolumab para el tratamiento de primera línea de los pacientes con melanoma metastásico tengan o no la mutación BRAF V600. Frente a esta aprobación los clínicos plantearon la condición clínica de pacientes con melanoma metástasico positivos a la mutación BRAF V600, que progresan a pesar del tratamiento con nivolumab. Esta evaluación se centró en la búsqueda de toda la evidencia respecto a la eficacia de vemurafenib en pacientes que progresaron con nivolumab. Sin embargo, no se ha identificado evidencia respecto a la eficacia de vemurafenib en pacientes con melanoma maligno metastásico irresecable con la mutaciónBRAF V600 y que progresaron al uso de nivolumab. Después de considerar los riesgos de efectos adversos severos con el uso de vemurafenib, los probables costos asociados con el manejo de los mismos y la ausencia de evidencia directa, el balance riesgo beneficio resulta ser claramente de mayor riesgo, mayores inconvenientes para el paciente y probablemente mayores costos frente a un modesto benefício no observado directamente en los pacientes de interés de esta evaluación. En EsSalud está disponible del uso de nivolumad. La dacarbazina se viene usando por más de cuatro décadas y con un perfil de toxicidad conocido y manejable. El Instituto de Evaluación de Tecnologías en Salud e Investigación-IETSI, no aprueba el uso de vemurafenib para el tratamiento de pacientes con MA (irresecable o metastásico) con mutación BRAF V600 después de progresar con nivolumab.

Entendiendo las terapias actuales en melanoma metastásico / Understanding current therapies in metastatic melanoma

Cutaneous melanoma is a highly aggressive tumor developing from melanocytes, its incidence is increasing, and prognosis in advanced stages is daunting. New therapies have been approved during the recent years with unprecedented results, including inhibitors of MAPK/ERK pathway and immune checkpoint blockade (anti-cytotoxic T lymphocyte antigen-4 (CTLA-4) as ipilimumab, anti-programmed cell death protein 1 (PD-L1) as pembrolizumab and anti-programmed cell death protein 1 ligand (PD-L1), among many others). The aim of this paper is to review currently available metastatic melanoma therapies focusing mainly on new therapies that have demonstrated effectiveness, after several decades of little progress in the treatment of this disease.

Vemurafenib and cutaneous adverse events - report of five cases

Vemurafenib is a selective inhibitor of V600E-mutant BRAF protein used to treat metastatic and unresectable melanoma. Clinical trials have shown increased overall survival and progression-free survival in patients treated with Vemurafenib. However, cutaneous adverse events are common during treatment. We report fi ve cases of metastatic melanoma with BRAF V600E positivity, treated with Vemurafenib and its cutaneous adverse events. Dermatologists and oncologists need to be aware of possible skin changes caused by this medication, which is increasingly employed in melanoma treatment. Monitoring of patients during therapy is important for early treatment of adverse cutaneous cutaneous adverse events, improvement in quality of life and adherence to treatment.

Effects of Ischemic Postconditioning on the Hemodynamic Parameters and Heart Nitric Oxide Levels of Hypothyroid Rats / Efeito do Pós-Condicionamento Isquêmico na Hemodinâmica e nos Níveis Cardíacos de Óxido Nítrico em Ratos com Hipotireoidismo

Background: Ischemic postconditioning (IPost) is a method of protecting the heart against ischemia-reperfusion (IR) injury. However, the effectiveness of IPost in cases of ischemic heart disease accompanied by co-morbidities such as hypothyroidism remains unclear. Objective: The aim of this study was to determine the effect of IPost on myocardial IR injury in hypothyroid male rats. Methods: Propylthiouracil in drinking water (500 mg/L) was administered to male rats for 21 days to induce hypothyroidism. The hearts from control and hypothyroid rats were perfused in a Langendorff apparatus and exposed to 30 min of global ischemia, followed by 120 min of reperfusion. IPost was induced immediately following ischemia. Results: Hypothyroidism and IPost significantly improved the left ventricular developed pressure (LVDP) and peak rates of positive and negative changes in left ventricular pressure (±dp/dt) during reperfusion in control rats (p < 0.05). However, IPost had no add-on effect on the recovery of LVDP and ±dp/dt in hypothyroid rats. Furthermore, hypothyroidism significantly decreased the basal NO metabolite (NOx) levels of the serum (72.5 ± 4.2 vs. 102.8 ± 3.7 μmol/L; p < 0.05) and heart (7.9 ± 1.6 vs. 18.8 ± 3.2 μmol/L; p < 0.05). Heart NOx concentration in the hypothyroid groups did not change after IR and IPost, whereas these were significantly (p < 0.05) higher and lower after IR and IPost, respectively, in the control groups. Conclusion: Hypothyroidism protects the heart from IR injury, which may be due to a decrease in basal nitric oxide (NO) levels in the serum and heart and a decrease in NO after IR. IPost did not decrease the NO level and did not provide further cardioprotection in the hypothyroid group. .

Fundamento: O pós-condicionamento isquêmico (PCI) é um método potente utilizado para proteger o coração contra a lesão de isquemia-reperfusão (I/R). Não está claro se o PCI é eficaz quando a doença cardíaca isquêmica é acompanhada de comorbidades, tais como hipotireoidismo. Objetivo: O objetivo deste estudo foi determinar o efeito do PCI sobre a lesão de I/R do miocárdio em ratos machos com hipotireoidismo. Métodos: O hipotireoidismo foi induzido pela administração de propiltiouracila em água potável na concentração de 500 mg/L durante 21 dias. Os corações de ratos controle e com hipotireoidismo foram perfundidos utilizando o aparelho de Langendorff e expostos a isquemia global por 30 minutos, seguido de reperfusão por 120 minutos. O PCI foi iniciado imediatamente após a isquemia. Resultados: O hipotireoidismo e PCI aumentaram significativamente a pressão ventricular esquerda desenvolvida (PVED) e as taxas máximas de variação positiva (+dp/dt) e negativa (–dp/dt) da pressão ventricular esquerda durante a reperfusão em ratos controle (p < 0,05). No entanto, o PCI não teve efeito aditivo no restabelecimento da PVED e das ±dp/dt em ratos com hipotireoidismo. Além disso, o hipotireoidismo diminuiu significativamente os níveis basais séricos (72,5 ± 4,2 vs. 102,8 ± 3,7 μmol/L; p < 0,05) e cardíacos (7,9 ± 1,6 vs. 18,8 ± 3,2 μmol/L; p < 0,05) de NOx. Os níveis cardíacos de NOx não se alteraram no grupo com hipotireoidismo após I/R e PCI mas foram significativamente maiores e menores (p < 0,05) nos grupos controle após I/R e PCI, respectivamente. Conclusão: O hipotireoidismo protegeu o coração da lesão de I/R, o que pode ser devido à diminuição dos níveis séricos e cardíacos basais de óxido nítrico (NO) e à diminuição dos níveis de NO após I/R. No entanto, o PCI não diminuiu os níveis de NO e não conferiu proteção adicional ao grupo com hipotireoidismo. .

Molecular targeting for treatment of advanced hepatocellular carcinoma / 대한간학회지

Hepatocellular carcinoma (HCC) is a major global health problem, which has a grave morbidity and mortality. Over the past few decades, no effective systemic therapeutic modalities have been established for patients with the unresectable HCC in advanced stage. Sorafenib is a small molecule that blocks cancer cell proliferation by targeting the intracellular signaling pathway at the level of Raf-1 and B-Raf serine-threonine kinases, and exerts an anti-angiogenic effect by targeting the vascular endothelial growth factor receptor-1, 2 and 3, and platelet-derived growth factor receptor-beta tyrosine kinases. Recently, two clinical successful applications, SHARP and Asia-Pacific trial, of multikinase inhibitor sorafenib represent a significant advance in the treatment of advanced HCC patients without a curative chance. However, because the results of clinical trials show diverse responses in a subset of HCC patients, a molecular classification of HCC through the excavation of specific biomarkers related to its biological behavior is necessary for sorting HCC patients to each group with a biological homogeneity, ultimately leading to the most suitable individualization of molecular targeted therapy in HCC.