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Supplementary Table 1 SampleSheet Supplementary Table 2 GEO_IDs Supplementary Table 3 EpiWNT-SHH_data Supplementary Table 4 EpiWNT-SHH_test Supplementary Table 5 EpiG3-G4_data Supplementary Table 6 EpiG3-G4_test

Supplementary Methods Supplementary Figure 1 Unsupervised DNA methylation analysis of the training cohort. Supplementary Figure 2 Comparison between the Illumina Infinium Human Methylation450K BeadChip and the Methylation EPIC BeadChip 850K array platforms. Supplementary Figure 3 Specificity of the epigenetic biomarkers for medulloblastoma subgroups. Supplementary Figure 4 Assay performance for low-input titration series. Supplementary Figure 5 Validation of the epigenetic classifier Panel EpiWNT-SHH using DNA methylation microarray data of medulloblastoma samples. Supplementary Figure 6 Validation of the methylation pattern of Panel EpiWNT-SHH using pyrosequencing and direct bisulfite sequencing. Supplementary Figure 7 Schematic overview of the experimental strategy applied for identification of the epigenetic biomarkers and the development of the classifier EpiG3-G4. Supplementary Figure 8 Validation of the epigenetic classifier Panel EpiG3-G4 using DNA methylation microarray data.

Purpose: The classification of medulloblastoma into WNT, SHH, group 3, and group 4 subgroups has become of critical importance for patient risk stratification and subgroup-tailored clinical trials. Here, we aimed to develop a simplified, clinically applicable classification approach that can be implemented in the majority of centers treating patients with medulloblastoma.Experimental Design: We analyzed 1,577 samples comprising previously published DNA methylation microarray data (913 medulloblastomas, 457 non-medulloblastoma tumors, 85 normal tissues), and 122 frozen and formalin-fixed paraffin-embedded medulloblastoma samples. Biomarkers were identified applying stringent selection filters and Linear Discriminant Analysis (LDA) method, and validated using DNA methylation microarray data, bisulfite pyrosequencing, and direct-bisulfite sequencing.Results: Using a LDA-based approach, we developed and validated a prediction method (EpiWNT-SHH classifier) based on six epigenetic biomarkers that allowed for rapid classification of medulloblastoma into the clinically relevant subgroups WNT, SHH, and non-WNT/non-SHH with excellent concordance (>99%) with current gold-standard methods, DNA methylation microarray, and gene signature profiling analysis. The EpiWNT-SHH classifier showed high prediction capacity using both frozen and formalin-fixed material, as well as diverse DNA methylation detection methods. Similarly, we developed a classifier specific for group 3 and group 4 tumors, based on five biomarkers (EpiG3-G4) with good discriminatory capacity, allowing for correct assignment of more than 92% of tumors. EpiWNT-SHH and EpiG3-G4 methylation profiles remained stable across tumor primary, metastasis, and relapse samples.Conclusions: The EpiWNT-SHH and EpiG3-G4 classifiers represent a new simplified approach for accurate, rapid, and cost-effective molecular classification of single medulloblastoma DNA samples, using clinically applicable DNA methylation detection methods. Clin Cancer Res; 24(6); 1355–63. ©2018 AACR.

Background The bone marrow (BM) is the most common site of dissemination in patients with aggressive, metastatic neuroblastoma (NB). However, the molecular mechanisms underlying the aggressive behavior of NB cells in the BM niche are still greatly unknown. In the present study, we explored biological mechanisms that play a critical role in NB cell survival and progression in the BM and investigated potential therapeutic targets. Methods Patient-derived bone marrow (BM) primary cultures were generated using fresh BM aspirates obtained from NB patients. NB cell lines were cultured in the presence of BM conditioned media containing cell-secreted factors, and under low oxygen levels (1% O2) to mimic specific features of the BM microenvironment of high-risk NB patients. The BM niche was explored using cytokine profiling assays, cell migration-invasion and viability assays, flow cytometry and analysis of RNA-sequencing data. Selective pharmacological inhibition of factors identified as potential mediators of NB progression within the BM niche was performed in vitro and in vivo. Results We identified macrophage migration inhibitory factor (MIF) as a key inflammatory cytokine involved in BM infiltration. Cytokine profiling and RNA-sequencing data analysis revealed NB cells as the main source of MIF in the BM, suggesting a potential role of MIF in tumor invasion. Exposure of NB cells to BM-conditions increased NB cell-surface expression of the MIF receptor CXCR4, which was associated with increased cell viability, enhanced migration-invasion, and activation of PI3K/AKT and MAPK/ERK signaling pathways. Moreover, subcutaneous co-injection of NB and BM cells enhanced tumor engraftment in mice. MIF inhibition with 4-IPP impaired in vitro NB aggressiveness, and improved drug response while delayed NB growth, improving survival of the NB xenograft model. Conclusions Our findings suggest that BM infiltration by NB cells may be mediated, in part, by MIF-CXCR4 signaling. We demonstrate the antitumor efficacy of MIF targeting in vitro and in vivo that could represent a novel therapeutic target for patients with disseminated high-risk NB.

Population aging and the epidemiological transition have led to an increase in institutionalization as a lifestyle alternative for the elderly. It is necessary to discuss a model of LTCF quality, contextualized to the Brazilian reality to advance the process of evaluating their activities, in addition to the normative and regulatory aspects, seeking to maximize health and quality of life for residents. The objective of this qualitative study was to apprehend the meaning of quality of the LTCF from the point of view of the elderly residents, caregivers of the elderly and technical managers of some large and medium and small private and philanthropic ILPI located in Belo Horizonte, MG, Brazil. It is necessary to understand how this equipment works, looking for parameters that make it possible to offer solutions that are more favorable to life in institutional homes, transforming the current scenario where they are still compared to the old homes. The search for ambiences that make them desirable places may reduce the impact of the change in housing, characterizing them as worthy for the elderly at any level of dependency. El envejecimiento de la población y la transición epidemiológica han propiciado un aumento de la institucionalización como alternativa de estilo de vida para las personas mayores. Es necesario discutir un modelo de calidad LTCF, contextualizado a la realidad brasileña para avanzar en el proceso de evaluación de sus actividades, además de los aspectos normativos y regulatorios, buscando maximizar la salud y la calidad de vida de los residentes. El objetivo de este estudio cualitativo fue aprehender el significado de calidad del LTCF desde el punto de vista de los ancianos residentes, cuidadores de ancianos y gerentes técnicos de algunos ILPI privados y filantrópicos grandes y medianos y pequeños ubicados en Belo Horizonte, MG., Brasil. Es necesario entender cómo funciona este equipamiento, buscando parámetros que permitan ofrecer soluciones más favorables a la vida en los hogares institucionales, transformando el escenario actual donde aún se encuentran frente a los hogares antiguos. La búsqueda de ambientes que los conviertan en lugares deseables puede reducir el impacto del cambio de vivienda, caracterizándolos como dignos para las personas mayores en cualquier nivel de dependencia. O envelhecimento populacional e a transição epidemiológica levaram a um aumento da institucionalização como alternativa de vida para os idosos. Torna-se necessária a discussão de um modelo de qualidade das ILPI, contextualizado à realidade brasileira para o avanço do processo de avaliação de suas atividades, para além dos aspectos normativos e regulatórios, buscando-se maximizar saúde e qualidade de vida dos residentes. O objetivo deste estudo, de caráter qualitativo, foi apreender o significado de qualidade da ILPI do ponto de vista dos idosos residentes, cuidadores de idosos e responsáveis técnicos de algumas ILPI privadas e filantrópicas de grande, médio e pequeno porte situadas em Belo Horizonte, MG, Brasil. É preciso compreender como funcionam esses equipamentos, buscando parâmetros que possibilitem a oferta de soluções mais favoráveis à vida em moradias institucionais, transformando o cenário atual onde ainda são comparadas aos antigos asilos. A busca por ambiências que as tornem lugares desejáveis poderá diminuir o impacto da mudança de moradia, caracterizando-as como dignas para idosos em qualquer nível de dependência.

Purpose: The classification of medulloblastoma into WNT, SHH, group 3, and group 4 subgroups has become of critical importance for patient risk stratification and subgroup-tailored clinical trials. Here, we aimed to develop a simplified, clinically applicable classification approach that can be implemented in the majority of centers treating patients with medulloblastoma. Experimental Design: We analyzed 1,577 samples comprising previously published DNA methylation microarray data (913 medulloblastomas, 457 non-medulloblastoma tumors, 85 normal tissues), and 122 frozen and formalin-fixed paraffin-embedded medulloblastoma samples. Biomarkers were identified applying stringent selection filters and Linear Discriminant Analysis (LDA) method, and validated using DNA methylation microarray data, bisulfite pyrosequencing, and direct-bisulfite sequencing. Results: Using a LDA-based approach, we developed and validated a prediction method (EpiWNT-SHH classifier) based on six epigenetic biomarkers that allowed for rapid classification of medulloblastoma into the clinically relevant subgroups WNT, SHH, and non-WNT/non-SHH with excellent concordance (>99%) with current gold-standard methods, DNA methylation microarray, and gene signature profiling analysis. The EpiWNT-SHH classifier showed high prediction capacity using both frozen and formalin-fixed material, as well as diverse DNA methylation detection methods. Similarly, we developed a classifier specific for group 3 and group 4 tumors, based on five biomarkers (EpiG3-G4) with good discriminatory capacity, allowing for correct assignment of more than 92% of tumors. EpiWNT-SHH and EpiG3-G4 methylation profiles remained stable across tumor primary, metastasis, and relapse samples. Conclusions: The EpiWNT-SHH and EpiG3-G4 classifiers represent a new simplified approach for accurate, rapid, and cost-effective molecular classification of single medulloblastoma DNA samples, using clinically applicable DNA methylation detection methods. Clin Cancer Res; 24(6); 1355–63. ©2018 AACR.