Diffuse gliomas cause significant morbidity across all age groups, despite decades of intensive research efforts. Here, we review the differences in diffuse gliomas in adults and children, as well as ...the World Health Organisation (WHO) 2021 classification of these tumours. We explain how DNA methylation-based classification works and list the methylation-based tumour types and subclasses for adult and paediatric diffuse gliomas. The benefits and utility of methylation-based classification in diffuse gliomas demonstrated to date are described. This entails the identification of novel tumour types/subclasses, patient stratification and targeted treatment/clinical management, and alterations in the clinical diagnosis in favour of the methylation-based over the histopathological diagnosis. Finally, we address several considerations regarding the use of DNA methylation profiling as a diagnostic tool, e.g., the threshold of the classifier, the calibrated score, tumour cell content and intratumour heterogeneity.
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.
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.
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).
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.
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.
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.
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.
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.
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 β 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 β 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 β 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.
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. Keywords: Childhood cancer, DNA methylation, Brain tumour, EPIC methylation array, Heterogeneity, Intratumour, Classification, Relapse
The results of this pilot randomized controlled trial suggest that Problem Management Plus (PM+), a low-intensity psychosocial intervention delivered by non-specialized 'helpers' is a feasible, ...well-accepted and safe treatment option for Syrian refugees in Switzerland.
Syrian refugees in Switzerland face several barriers in accessing mental health care. Cost-effective psychological interventions are urgently needed to meet the mental health needs of refugees. Problem Management Plus (PM+) is an evidence-based, psychological intervention delivered by trained non-specialist 'helpers'.
To assess the feasibility and acceptability of PM+ among Syrian refugees in Switzerland.
We conducted a single-blind pilot randomized controlled trial (RCT) with Syrian refugees impaired by psychological distress (K10 > 15 and WHODAS 2.0 > 16). Participants were randomized to PM+ or Enhanced Treatment As Usual (ETAU). Participants were assessed at baseline, and 1 week and 3 months after the intervention, and completed measures indexing mental health problems and health care usage. Semi-structured interviews were conducted with different stakeholders.
N = 59 individuals were randomized into PM+ (n = 31) or ETAU (n = 28). N = 18 stakeholders were interviewed about facilitators and barriers for the implementation of PM+. Retention rates in the trial (67.8%) and mean intervention attendance (M = 3.94 sessions, SD = 1.97) were high. No severe events related to the study were reported. These findings indicate that the trial procedures and PM+ were feasible, acceptable and safe.
The findings support the conduct of a definitive RCT and show that PM+ might have the potential to be scaled-up in Switzerland. The importance, as well as the challenges, of implementing and scaling-up PM+ in high-income countries, such as Switzerland, are discussed.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Abstract
Glioblastoma (GBM) is the most aggressive primary brain tumor with a median survival of less than 15 months, emphasizing the need for better treatments. Immunotherapy as a treatment for ...improving or aiding the patient’s own immune defense to target the tumor has been suggested for GBM. A randomized clinical trial of adoptive cell transfer using ALECSAT (Autologous Lymphoid Effector Cells Specific Against Tumor Cells) is currently ongoing in Sweden. Here we performed a paired pre-clinical study to investigate the composition and in vitro effect of ALECSAT and identify determinants for the effect using autologous GBM-derived cancer stem cells (CSC), immunocytochemistry and flow cytometry. We show a clear dose-response relationship of ALECSAT on CSC, suggesting that the number of infused cells is of importance. In addition, the in vitro effect of ALECSAT on CSC correlated significantly to the blood count of T helper (Th) cells in the patient indicating a potential benefit of collecting cells for ALECSAT preparation at an even earlier stage when patients generally have a better blood count. The factors identified in this study will be important to consider in the design of future immunotherapy trials to achieve prolonged survival.
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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