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Necroptosis is a necrotic-like cell death pathway in which Receptor-interacting serine/threonine-protein kinase 3 (RIPK3) plays a central role and may induce inflammation and immunity. Lower RIPK3 levels have been correlated with a poor prognosis in breast and colorectal cancer patients. Instead, in gliomas, the most prevalent among central nervous system cancers, necrosis concurs with a more aggressive and lethal outcome, suggesting that, in these cases, necrotic-like pathways may be linked to worse prognoses. Lower-grade gliomas (LGG) exhibit highly diverse clinical behaviors, ranging from slow-paced growth to fast progression to glioblastoma yet patient outcomes cannot be fully predicted through the available markers. To date, IDH mutational status is the most broadly used prognostic marker, albeit several candidates have been proposed to refine LGG subgrouping. Here, we aimed to assess RIPK3 role as a prognostic marker for LGG patients, independently of or in combination with IDH. Using publicly available discovery (513 patients) and validation (134 patients) cohorts, we performed Kaplan Meier survival analysis and uni- and multivariate Cox regression models. RIPK3 is an independent prognostic marker in LGG patients, even when controlled by age and molecular or histological diagnostic criteria. Contrary to what was previously reported for other cancers, high RIPK3 expression levels correlates with an increased risk of death. Importantly, RIPK3 expression levels further split both the mutant and wild-type IDH patients into distinct risk groups. RIPK3 expression levels can be used in combination with IDH mutational status to better subgroup LGG patients regarding overall survival.

Medulloblastoma is the most frequent pediatric malignant brain tumor, and is divided into four main subgroups: WNT, SHH, group 3, and group 4. MYCN amplification is an important medulloblastoma prognostic biomarker. We aimed to molecular classify and predict MYCN amplification in a single assay.It was included 209 medulloblastomas from 205 patients (Brazil, Argentina, and Portugal), divided into training (n = 50) and validation (n = 159) sets. A nCounter assay was carried out using a custom panel for molecular classification, with additional genes, including MYCN. nSolver 4.0 software and the R environment were used for profiling and MYCN mRNA analysis. MYCN amplification by FISH was performed in 64 cases.The 205 medulloblastomas were classified in SHH (44.9%), WNT (15.6%), group 3 (18.1%) and group 4 (21.4%). In the training set, MYCN amplification was detected in three SHH medulloblastomas by FISH, which showed significantly higher MYCN mRNA counts than non-FISH amplified cases, and a cutoff for MYCN amplification was established ([Formula: see text] + 4σ = 11,124.3). Applying this threshold value in the validation set, we identified MYCN mRNA counts above the cutoff in three cases, which were FISH validated.We successfully stratified medulloblastoma molecular subgroups and predicted MYCN amplification using a single nCounter assay without the requirement of additional biological tissue, costs, or bench time.

As metástases cerebrais ocorrem comumente em pacientes com câncer. A freqüência dessa complicação está aumentando em decorrência do tratamento mais agressivo do câncer, bem como do uso de métodos de neuroimagem que permitem um diagnóstico mais preciso. Com o melhor controle dos tumores primários, permitido pelos tratamentos atuais, a importância do tratamento dessas metástases é óbvia. Os principais objetivos do tratamento são melhorar os déficits neurológicos e aumentar a expectativa e a qualidade de vida. Os autores revisam as manifestações clínicas, o diagnóstico e as principais opções para o tratamento das metástases cerebrais.

EPNs comprise a heterogeneous group of neuroepithelial tumors, accounting for about 10% of all intracranial tumors in children and up to 30% of brain tumors in those younger than 3 years. Actually, the pattern therapy for low-grade EPNs includes complete surgical resection followed by radiation therapy. Total surgical excision is often not possible due to tumor location. The aim of this study was to evaluate, for the first time, the anti-tumor activity of Amblyomin-X in 4 primary cultures derived from pediatric anaplastic posterior fossa EPN, Group A (anaplastic, WHO grade III) and one primary culture of a high grade neuroepithelial tumor with MN1 alteration, which was initially misdiagnosed as EPN: i) by in vitro assays: comparisons of temozolomide and cisplatin; ii) by intracranial xenograft model. Amblyomin-X was able to induce cell death in EPN cells in a more significant percentage compared to cisplatin. The cytotoxic effects of Amblyomin-X were not detected on hFSCs used as control, as opposed to cisplatin-treatment, which promoted a substantial effect in the hAFSCs viability. TEM analysis showed ultrastructural alterations related to the process of cell death: mitochondrial degeneration, autophagosomes and aggregate-like structures. MRI and histopathological analyzes demonstrated significant tumor mass regression. Our results suggest that Amblyomin-X has a selective effect on tumor cells by inducing apoptotic cell death and may be a therapeutic option for Group AEPNs.

OBJECTIVE: The objective was to establish a pattern of tumor growth of the C6 model of glioblastoma multiform in Wistar rats via magnetic resonance imaging (MRI) for the subsequent verification of tumor volume reduction due to magnetic hyperthermia therapy. METHODS: Young male Wistar rats weighing between 250 and 300 g were used for the C6 model. After the rates were anesthetized (55 mg/kg ketamine and 11 mg/kg xylazine), C6 lineage tumorigenic cells suspended in culture medium (10(5) cells in 10 µl) were stereotaxically injected into the right frontal cortex (bregma coordinates: 2.0 mm anteroposterior, 3.0 mm laterolateral, and 2.5 mm depth) of the rats using a Hamilton syringe. For the control group, the rats were injected with culture medium without cells. MRI scans were performed at 14, 21, and 28 d after the injection using a 2.0 T MRI scanner (Bruker BioSpec, Germany). The animals were anesthetized with 55 mg/kg ketamine and 11 mg/kg xylazine before being examined. Coronal multilayers were acquired using a standard spin echo sequence with the following parameters: repetition/echo time = 4.000 ms/67.1 ms, field of view = 3.50, matrix = 192, slice thickness = 0.4 mm, and slice separation = 0 mm. RESULTS: The MRI analysis enabled a clear visualization of the tumor mass, and it was possible to establish the tumor volume parameters on the various days that were examined. The volume at 14 d after induction was 13.7 ± 2.5 mm³. On days 21 and 28, the tumor volumes were 31.7 ± 6.5 mm³ and 122.1 ± 11.8 mm³, respectively. CONCLUSION: These results demonstrated that it is possible to evaluate the C6 model tumor volume in rats, which will allow for the future implementation and verification of magnetic hyperthermia therapy. OBJETIVO: Estabelecer um padrão de crescimento tumoral (volume) em ratos Wistar submetidos ao modelo C6 de glioblastoma multiforme por meio de imagens de ressonância magnética para posterior verificação de redução de volume tumoral com a terapia de magnetohipertermia. MÉTODOS: Para o modelo C6, utilizamos ratos Wistar, machos, jovens, pesando entre 250 e 300 g. Após anestesiados (cetamina 55 mg/kg e xilazina 11 mg/kg) foram injetadas estereotaxicamente células tumorigênicas linhagem C6 suspensas em meio de cultura (10(5) células em 10 µL) no córtex frontal direito (coordenadas a partir do bregma: anteroposterior = 2,0 mm; látero-lateral = 3,0 mm; profundidade = 2,5 mm) com uma seringa Hamilton. No Grupo Controle, houve a injeção do meio de cultura sem as células. Posteriormente, foram feitas imagens mediante a técnica de imagem por ressonância magnética em 14, 21 e 28 dias após a injeção em um escâner de imagem por ressonância magnética 2.0 T (Bruker BioSpec, Germany). Para o exame, os animais foram anestesiados com cetamina 55 mg/kg e xilazina 11 mg/kg. Multifatias coronais foram adquiridas utilizando uma sequência spin-echo padrão com os seguintes parâmetros: TR/TE = 4,000 ms/67,1 ms, FOV = 3,50, Matrix 192, slice thickness = 0,4 mm e slice separation = 0 mm. RESULTADOS: A análise das imagens de ressonância magnética do tumor possibilitou a clara visualização da massa tumoral, sendo possível ainda estabelecer parâmetros de volume tumoral nos diferentes dias analisados. O volume de 14 dias após a indução do foi de 13,7 ± 2,5 mm³. Aos 21 dias, o volume alcançado foi de 31,7 ± 6,5 mm³ e, aos 28 dias, a massa tumoral atingiu 122,1 ± 11,8 mm³. CONCLUSÃO: Estes resultados mostraram a possiblidade de avaliação do volume tumoral no modelo C6 em ratos, o que possibilitará, no futuro, a aplicação da terapia de magnetohipertermia bem como verificação de seus resultados.

Objetivo Avaliar o efeito do tempo de espera (TE) até radioterapia na sobrevida global de pacientes com glioblastoma como um fator prognóstico confiável. Método Estudo retrospectivo de 115 pacientes com glioblastoma, que foram submetidos à radioterapia pós-operatória, em dois serviços diferentes no Brasil (um público e outro privado). Resultados Mediana de TE para radioterapia foi de 6 semanas (variação, 1,3-17,6). A mediana de sobrevida para TE ≤ 6 semanas foi de 13,5 meses (IC95%, 9,1-17,9) e para TE > 6 semanas foi de 14,2 meses (IC95%, 11,2-17,2) (HR 1,165, 0,770-1,762; p = 0,470). Na análise multivariada, as variáveis associadas à sobrevida foram perfomance status (p < 0,001), extensão da ressecção (p = 0,009) e tratamento adjuvante (p = 0,001). Conclusão Não se observou impacto prognóstico para TE até a radioterapia na sobrevida. Diante de outros fatores prognósticos, é possível assegurar de que o espaço de tempo até a radioterapia não parece influenciar o controle da doença. Objective To evaluate the effect of waiting time (WT) to radiotherapy (RT) on overall survival (OS) of glioblastoma (GBM) patients as a reliable prognostic variable in Brazil, a scenario of medical disparities. Method Retrospective study of 115 GBM patients from two different health-care institutions (one public and one private) in Brazil who underwent post-operative RT. Results Median WT to RT was 6 weeks (range, 1.3-17.6). The median OS for WT ≤ 6 weeks was 13.5 months (95%CI , 9.1-17.9) and for WT > 6 weeks was 14.2 months (95%CI, 11.2-17.2) (HR 1.165, 95%CI 0.770-1.762; p = 0.470). In the multivariate analysis, the variables associated with survival were KPS (p < 0.001), extent of resection (p = 0.009) and the adjuvant treatment (p = 0.001). The KPS interacted with WT to RT (HR 0.128, 95%CI 0.034-0.476; p = 0.002), showing that the benefit of KPS on OS depends on the WT to RT. Conclusion No prognostic impact of WT to RT could be detected on the OS. Although there are no data to ensure that delays to RT are tolerable, we may reassure patients that the time-length to initiate treatment does not seem to influence the control of the disease, particularly in face of other prognostic factors. Hospital Israelita Albert Einstein Universidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Hospital do Coração Departamento de Oncologia Universidade de São Paulo Faculdade de Medicina UNIFESP, EPM SciELO

Background and purpose Previous studies have provided no clear conclusions regarding the effects of delaying radiotherapy (RT) in GBM patients. We present a systematic review and meta-analysis to address the effect of delayed RT on the overall survival (OS) of GBM patients. Methods A systematic search retrieved 19 retrospective studies published between 1975 and 2014 reporting on the waiting time (WT) to RT for GBM patients. The meta-analysis was performed by converting WT to RT studies intervals into a regression coefficient ( β ) and standard error expressing the effect size on OS per week of delay. Results Data required to calculate the effect size on OS per week of delay were available for 12 studies (5212 patients). A non-adjusted model and a meta-regression model based on well-recognized prognostic factors were performed. No association between WT to RT, per week of delay, and OS was found (HR=0.98; 95% CI 0.90–1.08; p =0.70). The meta-regression adjusted for prognostic factors weighted by the inverse-variance (1/SE 2 ) showed no clear evidence of the effect of WT to RT, per week of delay, on OS. Conclusions This meta-analysis, despite limitations, provided no evidence of a true effect on OS by delaying RT in GBM patients.

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Hosp Israelita Albert Einstein, Inst Israelita Ensino & Pesquisa Albert Einstein, Sao Paulo, Brazil

In Regard to Lassman et al. Neuro-Oncol. 2011;13:649–659. The treatment of patients with anaplastic oligodendroglial tumors remains controversial. Initial treatment with radiation therapy associated or not with concomitant and/or sequential procarbazine, lomustine, and vincristine (PCV) chemotherapy is historically the most frequent approach to treat this disease.1 Radiation therapy has been reported for the treatment of the oligodendrogliomas since the 1960s2 and has been established as an effective treatment for increasing these patients’ probability of surviving 5 years after subtotal tumor resection since the 1980s.3 From the RTOG 94024 and EORTC 269515 well-designed phase III trials, we have the information that patients presenting with anaplastic oligodendroglial tumors with 1p19q codeletion have better prognosis and response to treatment, either radiation or chemotherapy. The early addition of chemotherapy in these trials improved the progression-free survival but not overall survival. In addition, the neurologic function was not assessed; thus, the real impact of this approach was not fully evaluated. The strength of this article is based on an impressive number of patients, with the retrospective analysis of >1000 patients with anaplastic olidendroglial tumors. However, the 3 initial treatment groups (chemotherapy, radio-chemotherapy, and radiation therapy) were too heterogeneous. Most received combined radio-chemotherapy as the first treatment, demonstrating the preference of the clinicians for this approach. Seventy-three percent of the patients who received chemotherapy as the first treatment and had a known 1p19q status had a codeleted status, compared with 85% in the chemotherapy and radiation therapy arm, compared with

Treating elderly cancer patients is a challenge for oncologists, especially considering the several therapeutic modalities in glioblastoma. Extensive tumor resection offers the best chance of local control. Adequate radiotherapy should always be given to elderly patients if they have undergone gross total resection and have maintained a good performance status. Rather than being ruled out, chemotherapy should be considered, and temozolomide is the chosen drug. A comprehensive geriatric assessment is a valuable tool to help guiding treatment decisions in elderly patients with glioblastoma. O tratamento de idosos com câncer é um desafio para a prática oncológica, especialmente no que se refere à terapêutica multimodal do glioblastoma. Nessa população, a ressecção ampla do tumor oferece a melhor chance de controle local e, naqueles pacientes que mantenham um bom performance status, a radioterapia complementar deve sempre ser levada em consideração. A quimioterapia também tem um papel no tratamento, sendo a temozolomida a droga de eleição. Frente à heterogeneidade desses pacientes, uma avaliação geriátrica ampla é um instrumento valioso no auxílio da decisão terapêutica em idosos com glioblastoma.

Uma mulher de 45 anos apresentou quadro de perda da iniciativa, dificuldade de memória, choro imotivado e distúrbio de comportamento, rapidamente progressivos ao longo de 6 meses, quando tornou-se não responsiva a estímulos verbais e totalmente apática. Um EEG mostrou atividade periódica bilateral, sugerindo doença de Creutzfeldt-Jacob (DCJ), mas a investigação para outras causas de demência mostrou hypercalcemia e hipofoefatemia que, exames posteriores, indicaram dever-se a adenoma de células oxífilas de paratireóide. Com a correção do distúrbio eletrolítico e posterior cirurgia houve normalização tanto do EEG como do exame neurológico. Não temos conhecimento de outros relatos de hiperparatireoidismo associado a síndrome demencial e atividade periodica no EEG. Este deveria ser am diagnóstico diferencial em todos os casos de DCJ recente. A 45 years-old woman presented with loss of iniciative and memory, imotivated ciy and behaviour disturbance with childish traits, quickly progressive along 6 months until total apathy. An EEG showed periodic activity with bilateral triphasic waves against a flattened background activity suggesting Creutzfeldt-Jacob disease (CJD), but investigation for treatable causes of dementia disclosed hypercalcemia and hypophosphatemia. Further investigation showed a mass at the thyroid region that at surgery was identified as an oxyphilic ceils adenoma. With electrolyte disturbance correction and posterior surgery there was normalization of both EEG and clinical status. Though it was previously reported mental confusion and EEG alteration associated with hyperparathyroidism we do not know of any previous case o>: confusion associated with periodic activity in EEG as in this disease. Hyperparathyroidism should be a differential diagnosis in every «de novo» case of CJD. Escola Paulista de Medicina UNIFESP, EPM SciELO

A 20-year-old man with a germ cell tumor who experienced an ischemic stroke as a complication of cisplatin/etoposide/bleomycin based chemotherapy is reported. The previously reported cases are reviewed as well as the different physiopathologic mechanisms associated with vascular toxicity of this regimen.

O Receptor do Fator de Crescimento Epidérmico (do inglês, EGFR) é uma proteína de membrana celular que consiste em um domínio extracelular para o acoplamento do ligante e em um domínio intracelular apresentando sítio catalítico de tirosinoquinase. Em ~40% dos GBMs primários é observada a amplificação de EGFR resultando na sua hiperexpressão, o que raramente ocorre em GBM secundário. Mais da metade dos casos de GBM com amplificação do receptor está associado com rearranjo do gene, uma forma deletada de EGFR (EGFRvIII). Adicionalmente, o receptor de interleucina 13 alfa-2 (IL-13R 2), apresenta-se abundante e especificamente hiperexpresso em gliomas de alto grau, em particular, GBM. O objetivo do presente estudo é analisar a expressão, amplificação, e deleção de EGFR em astrocitomas, bem como a coexpressão entre esse gene e o da IL-13R 2. Foram analisadas 145 astrocitomas (22 astrocitomas pilocíticos (AP); 22 astrocitomas grau II (AGII); 17 astrocitomas anaplásico (AA); e 84 GBM) e 17 tecidos cerebrais não tumorais provenientes de cirurgia de epilepsia. A deleção EGFRvIII foi analisada por RT-PCR, e confirmada por PCR em tempo real (RQ-PCR). A expressão relativa de EGFR e IL-13R 2 foi estudada por RQ-PCR utilizando-se o método SYBR Green, comparado ao tecido não tumoral, e normalizado para os genes de referência endógena, HPRT e Gus- . A amplificação de EGFR foi também determinada por RQ-PCR com relação ao gene da beta-hemoglobina, descrito como um gene de cópia única. Foi realizada imunohistoquímica para analisar a expressão da proteína EGFR. A deleção EGFRvIII foi somente encontrada em GBM (19/84, 23%), demonstrando a exclusividade dessa alteração num grau tumoral de maior malignidade e uma diminuição da sobrevida desses pacientes (p = 0,030). A hiperexpressão de EGFR foi encontrada em 88 casos (61%), correspondendo a 50% de GBM, 88% de AA, e interessantemente em 77% de AGII e 64% de AP. Da mesma maneira, a hiperexpressão de IL-13R 2 foi encontrada em 62 casos (43%), correspondendo a 48% de GBM, 29% de AA, 18% de AGII e, surpreendentemente em 59% de AP. Embora tenha havido um aumento de expressão de ambos os genes em todos os graus de astrocitomas, não houve coexpressão dos mesmos. A amplificação de EGFR foi observada em 29 casos (20%) correspondendo a 31% de GBM e ainda um caso para cada um dos demais graus de astrocitomas, sendo que dos 29 casos amplificados, 21 pacientes eram mais velhos que 45 anos (p < 0,001) e 50% dos casos de GBM com amplificação de EGFR, apresentaram simultaneamente EGFRvIII. Adicionalmente, o acúmulo citoplasmático da proteína foi detectado em 74 casos (51%), correspondendo a 55% de GBM, 47% de AA, 54,5% de AGII e 37% de AP, além de um acúmulo nuclear detectado em 15% dos casos de astrocitomas difusamente infiltrativos. Assim, a alta freqüência de hiperexpressão dos genes estudados em todos os graus de astrocitomas, principalmente a amplificação de EGFR e a presença da deleção EGFRvIII foram observados entre os astrocitomas de alto grau, especialmente em GBM. Os resultados apresentados contribuem para um melhor direcionamento no futuro em métodos terapêuticos específicos, salientando a importância da análise de expressão molecular, protéica e das alterações mutacionais que envolvem genes candidatos, em particular, EGFR e IL-13R 2 Epidermal Growth Factor Receptor, EGFR is a transmembrane protein consisting of an extracellular EGF-binding domain and an intracellular domain with ligand-activated tyrosine kinase activity. In ~40% of primary GBM is observed amplification leading to overexpression of EGFR, but rarely in secondary GBM. Over the half of primary GBM with EGFR amplification is associated to gene rearrangement, a deleted form of EGFR (EGFRvIII). Additionally, the interleukin-13 alpha 2 receptor (IL13R 2) is abundant and specifically overexpressed in high-grade gliomas, particularly in GBM. The aim of the present study is to analyze the EGFR expression, amplification, and deletion in astrocytomas as well as its coexpression with IL13R 2. We have analyzed 145 surgical astrocytoma samples (22 pilocytic astrocytomas (PA); 22 low-grade astrocytomas (LGA); 17 anaplastic astrocytomas (AA); and 84 GBM) and 17 non-neoplastic brain tissue from epilepsy surgery. EGFRvIII deletion was analyzed by RT-PCR, and also confirmed by real time PCR (RQ-PCR). The relative EGFR and IL-13R 2 expression was studied by RQ-PCR using SYBR Green method, compared to non-neoplastic tissue, normalized for HPRT and Gus- genes. The EGFR amplification was also determined by RQ-PCR relative to the hemoglobin beta gene, described as a single copy gene. Immunohistochemistry was performed to analyze the protein expression in tumor samples. The EGFRvIII deletion was found only in GBM cases (19/84, 23%) demonstrating the exclusivity of this alteration in higher tumor grade and survival was decreased in these patients (p = 0.030). EGFR overexpression was found in 88 cases (61%), corresponding to 50% of GBM, 88% of AA, and interestingly in 77% of LGA and 64% of PA. In the same way, the overexpression of IL-13R 2 was found in 62 cases (43%), corresponding to 48% of GBM, 29% of AA, 18% of LGA and, surprising in 59% of PA. Although increased expression of both genes was demonstrated in all astrocytoma grades, it was no coexpression of the genes. The amplification was observed in 29 cases (20%) corresponding to 31% of GBM and only one case each of PA, LGA and AA. Among 29 cases with EGFR amplification, 21 patients were older than 45 years (p < 0.001) and 50% of GBM with EGFR amplification presented simultaneously the EGFRvIII. Moreover, the EGFR cytoplasmic accumulation was detected in 74 cases (51%), corresponding to 55% of GBM, 47% of AA, 54.5% of LGA and 37% of PA, and nuclear accumulation was detected in 15% of diffusely infiltrative astrocytomas. Thus, the high overexpression frequency of the genes studied in all grades of astrocytomas, mainly the EGFR amplification and presence of EGFRvIII deletion were observed among high-grade astrocytomas, mainly in GBM. The present results contribute to better tailoring specific future therapeutical approach in patients with astrocytomas, pointing out the importance of the molecular, protein expressions and mutational analyses of candidates genes, in particular, EGFR and IL-13R 2