RESUMEN
Adult-type diffuse gliomas and meningiomas are the most common primary intracranial tumors of the central nervous system. DNA methylation profiling is a novel diagnostic technique increasingly used also in the clinic. Although molecular heterogeneity is well described in these tumors, DNA methylation heterogeneity is less studied. We therefore investigated the intratumor genetic and epigenetic heterogeneity in diffuse gliomas and meningiomas, with focus on potential clinical implications. We further investigated tumor purity as a source for heterogeneity in the tumors. We analyzed genome-wide DNA methylation profiles generated from 126 spatially separated tumor biopsies from 39 diffuse gliomas and meningiomas. Moreover, we evaluated five methods for measurement of tumor purity and investigated intratumor heterogeneity by assessing DNA methylation-based classification, chromosomal copy number alterations and molecular markers. Our results demonstrated homogeneous methylation-based classification of IDH-mutant gliomas and further corroborates subtype heterogeneity in glioblastoma IDH-wildtype and high-grade meningioma patients after excluding samples with low tumor purity. We detected a large number of differentially methylated CpG sites within diffuse gliomas and meningiomas, particularly in tumors of higher grades. The presence of CDKN2A/B homozygous deletion differed in one out of two patients with IDH-mutant astrocytomas, CNS WHO grade 4. We conclude that diffuse gliomas and high-grade meningiomas are characterized by intratumor heterogeneity, which should be considered in clinical diagnostics and in the assessment of methylation-based and molecular markers.
Asunto(s)
Neoplasias Encefálicas , Glioma , Neoplasias Meníngeas , Meningioma , Adulto , Humanos , Metilación de ADN , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/metabolismo , Meningioma/genética , Homocigoto , Mutación , Eliminación de Secuencia , Glioma/genética , Glioma/patología , Neoplasias Encefálicas/patología , Aberraciones Cromosómicas , Neoplasias Meníngeas/genéticaRESUMEN
AIMS: Paediatric brain tumours are rare, and establishing a precise diagnosis can be challenging. Analysis of DNA methylation profiles has been shown to be a reliable method to classify central nervous system (CNS) tumours with high accuracy. We aimed to prospectively analyse CNS tumours diagnosed in Sweden, to assess the clinical impact of adding DNA methylation-based classification to standard paediatric brain tumour diagnostics in an unselected cohort. METHODS: All CNS tumours diagnosed in children (0-18 years) during 2017-2020 were eligible for inclusion provided sufficient tumour material was available. Tumours were analysed using genome-wide DNA methylation profiling and classified by the MNP brain tumour classifier. The initial histopathological diagnosis was compared with the DNA methylation-based classification. For incongruent results, a blinded re-evaluation was performed by an experienced neuropathologist. RESULTS: Two hundred forty tumours with a histopathology-based diagnosis were profiled. A high-confidence methylation score of 0.84 or more was reached in 78% of the cases. In 69%, the histopathological diagnosis was confirmed, and for some of these also refined, 6% were incongruent, and the re-evaluation favoured the methylation-based classification. In the remaining 3% of cases, the methylation class was non-contributory. The change in diagnosis would have had a direct impact on the clinical management in 5% of all patients. CONCLUSIONS: Integrating DNA methylation-based tumour classification into routine clinical analysis improves diagnostics and provides molecular information that is important for treatment decisions. The results from methylation profiling should be interpreted in the context of clinical and histopathological information.
Asunto(s)
Neoplasias Encefálicas , Neoplasias del Sistema Nervioso Central , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Neoplasias del Sistema Nervioso Central/diagnóstico , Neoplasias del Sistema Nervioso Central/genética , Neoplasias del Sistema Nervioso Central/patología , Niño , Estudios de Cohortes , Metilación de ADN , Humanos , Estudios ProspectivosRESUMEN
The leading cause of cancer-related mortality among children is brain tumour, and glioblastoma multiforme (GBM) has the worst prognosis. New treatments are urgently needed, but with few cases and clinical trials in children, pre-clinical models such as patient-derived tumour xenografts (PDTX) are important. To generate these, tumour tissue is transplanted into mice, but this yields highly variable results and requires serial passaging in mice, which is time-consuming and expensive. We therefore aimed to establish a cell line-based orthotopic mouse model representative of the patient tumour. Glioma stem cell (GSC) lines derived from paediatric GBM were orthotopically transplanted into immunodeficient mice. Overall survival data were collected and histological analysis of the resulting neoplasias was performed. Genome-wide DNA methylation arrays were used for methylation and copy-number alterations (CNA) profiling. All GSC lines initiated tumours on transplantation and the survival of the mice correlated well with the survival of the patients. Xenograft tumours presented histological hallmarks of GBM, and were also classified as GBM by methylation profiling. Each xenograft tumour clustered together with its respective injected GSC line and patient tumour based on the methylation data. We have established a robust and reproducible cell line-based xenograft paediatric GBM model. The xenograft tumours accurately reflected the patient tumours and mirrored the clinical course of the patient. This model can therefore be used to assess patient response in pre-clinical studies.
Asunto(s)
Neoplasias Encefálicas/patología , Glioblastoma/patología , Células Madre Neoplásicas/patología , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Animales , Neoplasias Encefálicas/genética , Línea Celular Tumoral , Niño , Preescolar , Metilación de ADN/genética , Femenino , Glioblastoma/genética , Humanos , Inmunohistoquímica , Masculino , Ratones , Células Madre Neoplásicas/metabolismo , Análisis de SupervivenciaRESUMEN
Statistical network modeling techniques are increasingly important tools to analyze cancer genomics data. However, current tools and resources are not designed to work across multiple diagnoses and technical platforms, thus limiting their applicability to comprehensive pan-cancer datasets such as The Cancer Genome Atlas (TCGA). To address this, we describe a new data driven modeling method, based on generalized Sparse Inverse Covariance Selection (SICS). The method integrates genetic, epigenetic and transcriptional data from multiple cancers, to define links that are present in multiple cancers, a subset of cancers, or a single cancer. It is shown to be statistically robust and effective at detecting direct pathway links in data from TCGA. To facilitate interpretation of the results, we introduce a publicly accessible tool (cancerlandscapes.org), in which the derived networks are explored as interactive web content, linked to several pathway and pharmacological databases. To evaluate the performance of the method, we constructed a model for eight TCGA cancers, using data from 3900 patients. The model rediscovered known mechanisms and contained interesting predictions. Possible applications include prediction of regulatory relationships, comparison of network modules across multiple forms of cancer and identification of drug targets.
Asunto(s)
Modelos Genéticos , Modelos Estadísticos , Neoplasias/genética , Antineoplásicos/farmacología , Deleción Cromosómica , Cromosomas Humanos Par 11 , Variaciones en el Número de Copia de ADN , Metilación de ADN , Genómica/métodos , Glioma/genética , Humanos , Internet , Isocitrato Deshidrogenasa/genética , Estimación de Kaplan-Meier , MicroARNs/metabolismo , Mutación , Neoplasias/mortalidad , ARN Mensajero/metabolismo , Programas InformáticosRESUMEN
The 2021 World Health Organization (WHO) grading system of isocitrate dehydrogenase (IDH)-mutant astrocytomas relies on histological features and the presence of homozygous deletion of the cyclin-dependent kinase inhibitor 2A and 2B (CDKN2A/B). DNA methylation profiling has become highly relevant in the diagnosis of central nervous system (CNS) tumors including gliomas, and it has been incorporated into routine clinical diagnostics in some countries. In this study, we, therefore, examined the value of DNA methylation-based classification for prognostication of patients with IDH-mutant astrocytomas. We analyzed histopathological diagnoses, genome-wide DNA methylation array data, and chromosomal copy number alteration profiles from a cohort of 385 adult-type IDH-mutant astrocytomas, including a local cohort of 127 cases and 258 cases from public repositories. Prognosis based on WHO 2021 CNS criteria (histological grade and CDKN2A/B homozygous deletion status), other relevant chromosomal/gene alterations in IDH-mutant astrocytomas and DNA methylation-based subclassification according to the molecular neuropathology classifier were assessed. We demonstrate that DNA methylation-based classification of IDH-mutant astrocytomas can be used to predict outcome of the patients equally well as WHO 2021 CNS criteria. In addition, methylation-based subclassification enabled the identification of IDH-mutant astrocytoma patients with poor survival among patients with grade 3 tumors and patients with grade 4 tumors with a more favorable outcome. In conclusion, DNA methylation-based subclassification adds prognostic information for IDH-mutant astrocytomas that can further refine the current WHO 2021 grading scheme for these patients.
Asunto(s)
Astrocitoma , Neoplasias Encefálicas , Metilación de ADN , Isocitrato Deshidrogenasa , Mutación , Humanos , Astrocitoma/genética , Astrocitoma/patología , Isocitrato Deshidrogenasa/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Masculino , Femenino , Pronóstico , Persona de Mediana Edad , Adulto , Anciano , Clasificación del Tumor/métodos , Estudios de Cohortes , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genéticaRESUMEN
Adenoid cystic carcinoma (ACC) of the head and neck is a malignant tumor with poor long-term prognosis. Besides the recently identified MYB-NFIB fusion oncogene generated by a t(6;9) translocation, little is known about other genetic alterations in ACC. Using high-resolution, array-based comparative genomic hybridization, and massively paired-end sequencing, we explored genomic alterations in 40 frozen ACCs. Eighty-six percent of the tumors expressed MYB-NFIB fusion transcripts and 97% overexpressed MYB mRNA, indicating that MYB activation is a hallmark of ACC. Thirty-five recurrent copy number alterations (CNAs) were detected, including losses involving 12q, 6q, 9p, 11q, 14q, 1p, and 5q and gains involving 1q, 9p, and 22q. Grade III tumors had on average a significantly higher number of CNAs/tumor compared to Grade I and II tumors (P = 0.007). Losses of 1p, 6q, and 15q were associated with high-grade tumors, whereas losses of 14q were exclusively seen in Grade I tumors. The t(6;9) rearrangements were associated with a complex pattern of breakpoints, deletions, insertions, inversions, and for 9p also gains. Analyses of fusion-negative ACCs using high-resolution arrays and massively paired-end sequencing revealed that MYB may also be deregulated by other mechanisms in addition to gene fusion. Our studies also identified several down-regulated candidate tumor suppressor genes (CTNNBIP1, CASP9, PRDM2, and SFN) in 1p36.33-p35.3 that may be of clinical significance in high-grade tumors. Further, studies of these and other potential target genes may lead to the identification of novel driver genes in ACC.
Asunto(s)
Carcinoma Adenoide Quístico/genética , Variaciones en el Número de Copia de ADN , Reordenamiento Génico , Genes myb , Neoplasias de Cabeza y Cuello/genética , Factores de Transcripción NFI/genética , Adulto , Anciano , Anciano de 80 o más Años , Hibridación Genómica Comparativa , Femenino , Genes Supresores de Tumor , Humanos , Masculino , Persona de Mediana Edad , Proteínas de Fusión Oncogénica/genética , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: Brain tumours are the leading cause of cancer-related death in children, and there is no effective treatment. A growing body of evidence points to deregulated epigenetics as a tumour driver, particularly in paediatric cancers as they have relatively few genomic alterations, and key driver mutations have been identified in histone 3 (H3). Cancer stem cells (CSC) are implicated in tumour development, relapse and therapy resistance and thus particularly important to target. We therefore aimed to identify novel epigenetic treatment targets in CSC derived from H3-mutated high-grade glioma (HGG) through a CRISPR-Cas9 knockout screen. RESULTS: The knockout screen identified more than 100 novel genes essential for the growth of CSC derived from paediatric HGG with H3K27M mutation. We successfully validated 12 of the 13 selected hits by individual knockout in the same two CSC lines, and for the top six hits we included two additional CSC lines derived from H3 wild-type paediatric HGG. Knockout of these genes led to a significant decrease in CSC growth, and altered stem cell and differentiation markers. CONCLUSIONS: The screen robustly identified essential genes known in the literature, but also many novel genes essential for CSC growth in paediatric HGG. Six of the novel genes (UBE2N, CHD4, LSM11, KANSL1, KANSL3 and EED) were validated individually thus demonstrating their importance for CSC growth in H3-mutated and wild-type HGG. These genes should be further studied and evaluated as novel treatment targets in paediatric HGG.
Asunto(s)
Sistemas CRISPR-Cas , Glioma , Humanos , Niño , Metilación de ADN , Glioma/genética , Genes Reguladores , Histonas/genéticaRESUMEN
Diffuse gliomas are the most prevalent malignant primary brain tumors in adults and remain incurable despite standard therapy. Tumor recurrence is currently inevitable, which contributes to a persistent high morbidity and mortality in these patients. In this study, we examined the genome-wide DNA methylation profiles of primary and recurrent adult-type IDH-mutant gliomas to elucidate DNA methylation changes associated with tumor progression (with or without malignant transformation). We analyzed DNA methylation profiles of 37 primary IDH-mutant gliomas and 42 paired recurrences using the DNA methylation EPIC beadChip array. DNA methylation-based classification reflected the tumor progression over time. We observed a methylation subtype switch in a proportion of IDH-mutant astrocytomas; the primary tumors were subclassified as low-grade astrocytomas, which progressed to high-grade astrocytomas in the recurrent tumors. The CNS WHO grade 4 IDH-mutant astrocytomas did not always resemble methylation subclasses of higher grades. The number of differentially methylated CpG sites increased over time, and astrocytomas accumulated more differentially methylated CpG sites than oligodendrogliomas during tumor progression. Few differentially methylated CpG sites were shared between patients. We demonstrated that DNA methylation profiles are mostly maintained during IDH-mutant glioma progression, but CpG site-specific methylation alterations can occur.
Asunto(s)
Astrocitoma , Neoplasias Encefálicas , Glioma , Adulto , Humanos , Metilación de ADN , Glioma/genética , Glioma/patología , Neoplasias Encefálicas/patología , Astrocitoma/genética , Isocitrato Deshidrogenasa/genética , Mutación/genéticaRESUMEN
BACKGROUND: Molecular analyses have shown that tumours diagnosed as supratentorial primitive neuro-ectodermal tumours of the central nervous system (CNS-PNETs) in the past represent a heterogenous group of rare childhood tumours including high-grade gliomas (HGG), ependymomas, atypical teratoid/rhabdoid tumours (AT/RT), CNS neuroblastoma with forkhead box R2 (FOXR2) activation and embryonal tumour with multi-layered rosettes (ETMR). All these tumour types are rare and long-term clinical follow-up data are sparse. We retrospectively re-evaluated all children (0-18 years old) diagnosed with a CNS-PNET in Sweden during 1984-2015 and collected clinical data. METHODS: In total, 88 supratentorial CNS-PNETs were identified in the Swedish Childhood Cancer Registry and from these formalin-fixed paraffin-embedded tumour material was available for 71 patients. These tumours were histopathologically re-evaluated and, in addition, analysed using genome-wide DNA methylation profiling and classified by the MNP brain tumour classifier. RESULTS: The most frequent tumour types, after histopathological re-evaluation, were HGG (35%) followed by AT/RT (11%), CNS NB-FOXR2 (10%) and ETMR (8%). DNA methylation profiling could further divide the tumours into specific subtypes and with a high accuracy classify these rare embryonal tumours. The 5 and 10-year overall survival (OS) for the whole CNS-PNET cohort was 45% ± 12% and 42% ± 12%, respectively. However, the different groups of tumour types identified after re-evaluation displayed very variable survival patterns, with a poor outcome for HGG and ETMR patients with 5-year OS 20% ± 16% and 33% ± 35%, respectively. On the contrary, high PFS and OS was observed for patients with CNS NB-FOXR2 (5-year 100% for both). Survival rates remained stable even after 15-years of follow-up. CONCLUSIONS: Our findings demonstrate, in a national based setting, the molecular heterogeneity of these tumours and show that DNA methylation profiling of these tumours provides an indispensable tool in distinguishing these rare tumours. Long-term follow-up data confirms previous findings with a favourable outcome for CNS NB-FOXR2 tumours and poor chances of survival for ETMR and HGG.
Asunto(s)
Neoplasias Encefálicas , Neoplasias del Sistema Nervioso Central , Glioma , Neoplasias de Células Germinales y Embrionarias , Tumores Neuroectodérmicos Primitivos , Humanos , Niño , Recién Nacido , Lactante , Preescolar , Adolescente , Suecia/epidemiología , Estudios de Seguimiento , Estudios Retrospectivos , Metilación de ADN , Neoplasias Encefálicas/genética , Tumores Neuroectodérmicos Primitivos/genética , Tumores Neuroectodérmicos Primitivos/patología , Neoplasias del Sistema Nervioso Central/genética , Neoplasias del Sistema Nervioso Central/patología , Glioma/genética , Neoplasias de Células Germinales y Embrionarias/genética , Factores de Transcripción Forkhead/genéticaRESUMEN
DNA copy number aberrations (CNAs) are a hallmark of cancer genomes. However, little is known about how such changes affect global gene expression. We develop a modeling framework, EPoC (Endogenous Perturbation analysis of Cancer), to (1) detect disease-driving CNAs and their effect on target mRNA expression, and to (2) stratify cancer patients into long- and short-term survivors. Our method constructs causal network models of gene expression by combining genome-wide DNA- and RNA-level data. Prognostic scores are obtained from a singular value decomposition of the networks. By applying EPoC to glioblastoma data from The Cancer Genome Atlas consortium, we demonstrate that the resulting network models contain known disease-relevant hub genes, reveal interesting candidate hubs, and uncover predictors of patient survival. Targeted validations in four glioblastoma cell lines support selected predictions, and implicate the p53-interacting protein Necdin in suppressing glioblastoma cell growth. We conclude that large-scale network modeling of the effects of CNAs on gene expression may provide insights into the biology of human cancer. Free software in MATLAB and R is provided.
Asunto(s)
Dosificación de Gen , Glioblastoma/genética , Proteínas del Tejido Nervioso/metabolismo , Neoplasias del Sistema Nervioso/genética , Proteínas Nucleares/metabolismo , Activación Transcripcional/genética , Proteína p53 Supresora de Tumor/metabolismo , Línea Celular Tumoral , Aberraciones Cromosómicas , Bases de Datos Factuales , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Genoma Humano , Estudio de Asociación del Genoma Completo , Glioblastoma/metabolismo , Glioblastoma/mortalidad , Glioblastoma/patología , Humanos , Modelos Genéticos , Proteínas del Tejido Nervioso/genética , Neoplasias del Sistema Nervioso/metabolismo , Neoplasias del Sistema Nervioso/mortalidad , Neoplasias del Sistema Nervioso/patología , Proteínas Nucleares/genética , Pronóstico , Programas Informáticos , Proteína p53 Supresora de Tumor/genéticaRESUMEN
One of the central problems of cancer systems biology is to understand the complex molecular changes of cancerous cells and tissues, and use this understanding to support the development of new targeted therapies. EPoC (Endogenous Perturbation analysis of Cancer) is a network modeling technique for tumor molecular profiles. EPoC models are constructed from combined copy number aberration (CNA) and mRNA data and aim to (1) identify genes whose copy number aberrations significantly affect target mRNA expression and (2) generate markers for long- and short-term survival of cancer patients. Models are constructed by a combination of regression and bootstrapping methods. Prognostic scores are obtained from a singular value decomposition of the networks. We have previously analyzed the performance of EPoC using glioblastoma data from The Cancer Genome Atlas (TCGA) consortium, and have shown that resulting network models contain both known and candidate disease-relevant genes as network hubs, as well as uncover predictors of patient survival. Here, we give a practical guide how to perform EPoC modeling in practice using R, and present a set of alternative modeling frameworks.
Asunto(s)
Biología Computacional/métodos , Redes Reguladoras de Genes/genética , Modelos Genéticos , Neoplasias/genética , Biología de Sistemas/métodos , Algoritmos , Biología Computacional/clasificación , Dosificación de Gen , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes/efectos de los fármacos , Predisposición Genética a la Enfermedad/genética , Glioblastoma/tratamiento farmacológico , Glioblastoma/genética , Humanos , Neoplasias/tratamiento farmacológico , Pronóstico , Reproducibilidad de los Resultados , Análisis de SupervivenciaRESUMEN
DNA methylation is increasingly used for tumour classification and has expanded upon the > 100 currently known brain tumour entities. A correct diagnosis is the basis for suitable treatment for patients with brain tumours, which is the leading cause of cancer-related death in children. DNA methylation profiling is required for diagnosis of certain tumours, and used clinically for paediatric brain tumours in several countries. We therefore evaluated if the methylation-based classification is robust in different locations of the same tumour, and determined how the methylation pattern changed over time to relapse. We sampled 3-7 spatially separated biopsies per patient, and collected samples from paired primary and relapse brain tumours from children. Altogether, 121 samples from 46 paediatric patients with brain tumours were profiled with EPIC methylation arrays. The methylation-based classification was mainly homogeneous for all included tumour types that were successfully classified, which is promising for clinical diagnostics. There were indications of multiple subclasses within tumours and switches in the relapse setting, but not confirmed as the classification scores were below the threshold. Site-specific methylation alterations did occur within the tumours and varied significantly between tumour types for the temporal samples, and as a trend in spatial samples. More alterations were present in high-grade tumours compared to low-grade, and significantly more alterations with longer relapse times. The alterations in the spatial and temporal samples were significantly depleted in CpG islands, exons and transcription start sites, while enriched in OpenSea and regions not affiliated with a gene, suggesting a random location of the alterations in less conserved regions. In conclusion, more DNA methylation changes accumulated over time and more alterations occurred in high-grade tumours. The alterations mainly occurred in regions without gene affiliation, and did not affect the methylation-based classification, which largely remained homogeneous in paediatric brain tumours.
Asunto(s)
Neoplasias Encefálicas , Metilación de ADN , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Niño , Islas de CpG , Humanos , Mutación , Recurrencia Local de Neoplasia/genéticaRESUMEN
Several DNA methylation clocks have been developed to reflect chronological age of human tissues, but most clocks have been trained on adult samples. The rapid methylome changes in children and the role of epigenetics in pediatric tumors calls for tools accurately estimating methylation age in children. We aimed to evaluate seven methylation clocks in multiple tissues from healthy children to inform future studies on the optimal clock for pediatric cohorts, and analyzed the methylation age in brain tumors. We found that clocks trained on pediatric samples were the best in all tested tissues, highlighting the need for dedicated clocks. For blood samples, the Skin and blood clock had the best correlation with chronological age, while PedBE was the most accurate for saliva and buccal samples, and Horvath for brain tissue. Horvath methylation age was accelerated in pediatric brain tumors and the acceleration was subtype-specific for atypical teratoid rhabdoid tumor (ATRT), ependymoma, medulloblastoma and glioma. The subtypes with the highest acceleration corresponded to the worst prognostic categories in ATRT, ependymoma and glioma, whereas the relationship was reversed in medulloblastoma. This suggests that methylation age has potential as a prognostic biomarker in pediatric brain tumors and should be further explored.
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Envejecimiento/genética , Neoplasias Encefálicas/genética , Metilación de ADN , Epigénesis Genética , Relojes Biológicos , Niño , HumanosRESUMEN
BACKGROUND: Radiation is an important therapeutic tool. However, radiotherapy has the potential to promote co-evolution of genetic and epigenetic changes that can drive tumour heterogeneity, formation of radioresistant cells and tumour relapse. There is a clinical need for a better understanding of DNA methylation alterations that may follow radiotherapy to be able to prevent the development of radiation-resistant cells. METHODS: We examined radiation-induced changes in DNA methylation profiles of paediatric glioma stem cells (GSCs) in vitro. Five GSC cultures were irradiated in vitro with repeated doses of 2 or 4 Gy. Radiation was given in 3 or 15 fractions. DNA methylation profiling using Illumina DNA methylation arrays was performed at 14 days post-radiation. The cellular characteristics were studied in parallel. RESULTS: Few fractions of radiation did not result in significant accumulation of DNA methylation alterations. However, extended dose fractionations changed DNA methylation profiles and induced thousands of differentially methylated positions, specifically in enhancer regions, sites involved in alternative splicing and in repetitive regions. Radiation induced dose-dependent morphological and proliferative alterations of the cells as a consequence of the radiation exposure. CONCLUSIONS: DNA methylation alterations of sites with regulatory functions in proliferation and differentiation were identified, which may reflect cellular response to radiation stress through epigenetic reprogramming and differentiation cues.
Asunto(s)
Neoplasias del Sistema Nervioso Central/patología , Metilación de ADN/genética , Epigénesis Genética/genética , Glioma/genética , Células Madre Neoplásicas/metabolismo , Diferenciación Celular , Niño , Preescolar , Daño del ADN/efectos de la radiación , Metilación de ADN/efectos de la radiación , Fraccionamiento de la Dosis de Radiación , Epigénesis Genética/efectos de la radiación , Femenino , Glioma/radioterapia , Humanos , Masculino , Recurrencia Local de Neoplasia/genética , Células Madre Neoplásicas/efectos de la radiaciónRESUMEN
This chapter discusses analysis and interpretation of large-scale Illumina DNA methylation microarray data, used in the context of cancer studies. We outline commonly used normalization procedures and list issues to consider regarding data preprocessing. Focusing on software packages for R, we describe methods for finding features in the methylation data that are of importance for generating and testing hypotheses in cancer research, like differentially methylated positions or regions and global methylation trends.
Asunto(s)
Metilación de ADN , Epigenómica/métodos , Neoplasias/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Programas Informáticos , Genoma Humano , Humanos , Neoplasias/metabolismoRESUMEN
BACKGROUND: A feature of glioblastoma (GBM) is cellular and molecular heterogeneity, both within and between tumors. This variability causes a risk for sampling bias and potential tumor escape from future targeted therapy. Heterogeneous intratumor gene expression in GBM is well documented, but little is known regarding the epigenetic heterogeneity. Variability in DNA methylation within tumors would have implications for diagnostics, as methylation can be used for tumor classification, subtyping, and determination of the clinically used biomarker O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation. We therefore aimed to profile the intratumor DNA methylation heterogeneity in GBM and its effect on diagnostic properties. METHODS: Three to 4 spatially separated biopsies per tumor were collected from 12 GBM patients. We performed genome-wide DNA methylation analysis and investigated intratumor variation. RESULTS: All samples were classified as GBM isocitrate dehydrogenase (IDH) wild type (wt)/mutated by methylation profiling, but the subclass differed within 5 tumors. Some GBM samples exhibited higher DNA methylation differences within tumors than between, and many cytosine-phosphate-guanine (CpG) sites (mean: 17 000) had different methylation levels within the tumors. MGMT methylation status differed in IDH mutated patients (1/1). CONCLUSIONS: We demonstrated that intratumor DNA methylation heterogeneity is a feature of GBM. Although all biopsies were classified as GBM IDH wt/mutated by methylation analysis, the assigned subclass differed in samples from the same patient. The observed heterogeneity within tumors is important to consider for methylation-based biomarkers and future improvements in stratification of GBM patients.
Asunto(s)
Biomarcadores de Tumor/genética , Neoplasias Encefálicas/patología , Metilación de ADN , Regulación Neoplásica de la Expresión Génica , Glioblastoma/clasificación , Glioblastoma/patología , Regiones Promotoras Genéticas , Anciano , Anciano de 80 o más Años , Neoplasias Encefálicas/clasificación , Neoplasias Encefálicas/genética , Femenino , Estudios de Seguimiento , Glioblastoma/genética , Humanos , Isocitrato Deshidrogenasa/genética , Masculino , Persona de Mediana Edad , Mutación , PronósticoRESUMEN
DNA methylation is the most studied epigenetic modification due to its role in regulating gene expression, and its involvement in the pathogenesis of cancer and several diseases upon aberrations in methylation. The method of choice to evaluate genome-wide methylation has been the Illumina HumanMethylation450 BeadChip (450K), but it was recently replaced with the MethylationEPIC BeadChip (EPIC). We therefore sought to validate the EPIC array in comparison to the 450K array for both fresh-frozen (FF) and formalin-fixed paraffin-embedded (FFPE) tumours. We also performed analysis on the EPIC array with paired FF and FFPE samples to adapt to a clinical setting where FFPE is routinely used. Further, we compared two restoration methods, REPLI-g and Infinium, for FFPE-derived DNA on the EPIC array. The Pearson correlation of ß values for common probes on the 450K and EPIC array was high for both FF (mean: 0.992) and FFPE (mean: 0.984) samples. The ß values generated from the EPIC array for FFPE samples correlated well with the paired FF tumours, but varied between 0.901 and 0.987. We did note that sample pairs with lower correlation had less bimodal density distributions of ß values and displayed higher noise in the copy number alteration plots (generated from the methylation array data) in the FFPE sample. Both REPLI-g and the Infinium restoration for FFPE samples performed well on the EPIC array and generated equivalent correlation scores to the paired FF sample.
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Neoplasias Encefálicas/genética , Metilación de ADN , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Neoplasias Encefálicas/patología , Islas de CpG , Epigénesis Genética , Humanos , Adhesión en Parafina , Fijación del TejidoRESUMEN
High-grade gliomas (HGG) are the most common brain tumors, with an average survival time of 14 months. A glioma-CpG island methylator phenotype (G-CIMP), associated with better clinical outcome, has been described in low and high-grade gliomas. Mutation of IDH1 is known to drive the G-CIMP status. In some cases, however, the hypermethylation phenotype is independent of IDH1 mutation, suggesting the involvement of other mechanisms. Here, we demonstrate that DNMT1 expression is higher in low-grade gliomas compared to glioblastomas and correlates with phosphorylated c-Jun. We show that phospho-c-Jun binds to the DNMT1 promoter and causes DNA hypermethylation. Phospho-c-Jun activation by Anisomycin treatment in primary glioblastoma-derived cells attenuates the aggressive features of mesenchymal glioblastomas and leads to promoter methylation and downregulation of key mesenchymal genes (CD44, MMP9 and CHI3L1). Our findings suggest that phospho-c-Jun activates an important regulatory mechanism to control DNMT1 expression and regulate global DNA methylation in Glioblastoma.
Asunto(s)
Neoplasias Encefálicas/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1/genética , Metilación de ADN , Glioma/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Anisomicina/farmacología , Neoplasias Encefálicas/genética , Línea Celular Tumoral , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Metilación de ADN/efectos de los fármacos , Epigénesis Genética/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genoma Humano , Glioma/genética , Humanos , Fosforilación , Pronóstico , Regiones Promotoras Genéticas/efectos de los fármacos , Análisis de Supervivencia , Regulación hacia Arriba/efectos de los fármacosRESUMEN
High-grade gliomas (HGGs) are very aggressive brain tumors with a cancer stem cell component. Cells, including cancer stem cells, release vesicles called exosomes which contain small non-coding RNAs such as microRNAs (miRNAs). These are thought to play an important role in cell-cell communication. However, we have limited knowledge of the types of exosomal miRNAs released by pediatric HGG stem cells; a prerequisite for exploring their potential roles in HGG biology. Here we isolated exosomes released by pediatric glioma stem cells (GSCs) and compared their repertoire of miRNAs to genetically normal neural stem cells (NSCs) exosomes, as well as their respective cellular miRNA content. Whereas cellular miRNAs are similar, we find that the exosomal miRNA profiles differ between normal and tumor cells, and identify several differentially expressed miRNAs. Of particular interest is miR-1290 and miR-1246, which have previously been linked to 'stemness' and invasion in other cancers. We demonstrate that GSC-secreted exosomes influence the gene expression of receiving NSCs, particularly targeting genes with a role in cell fate and tumorigenesis. Thus, our study shows that GSCs and NSCs have similar cellular miRNA profiles, yet differ significantly in the repertoire of exosomal miRNAs and these could influence malignant features of HGG.
RESUMEN
Glioblastomas are characterized by transcriptionally distinct subtypes, but despite possible clinical relevance, their regulation remains poorly understood. The commonly used molecular classification systems for GBM all identify a subtype with high expression of mesenchymal marker transcripts, strongly associated with invasive growth. We used a comprehensive data-driven network modeling technique (augmented sparse inverse covariance selection, aSICS) to define separate genomic, epigenetic, and transcriptional regulators of glioblastoma subtypes. Our model identified Annexin A2 (ANXA2) as a novel methylation-controlled positive regulator of the mesenchymal subtype. Subsequent evaluation in two independent cohorts established ANXA2 expression as a prognostic factor that is dependent on ANXA2 promoter methylation. ANXA2 knockdown in primary glioblastoma stem cell-like cultures suppressed known mesenchymal master regulators, and abrogated cell proliferation and invasion. Our results place ANXA2 at the apex of a regulatory cascade that determines glioblastoma mesenchymal transformation and validate aSICS as a general methodology to uncover regulators of cancer subtypes.