Altered methylation of imprinted genes in neuroblastoma: implications for prognostic refinement.

BACKGROUND: Neuroblastoma (NB) is a complex disease, and the current understanding of NB biology is limited. Deregulation in genomic imprinting is a common event in malignancy. Since imprinted genes play crucial roles in early fetal growth and development, their role in NB pathogenesis could be suggested. METHODS: We examined alterations in DNA methylation patterns of 369 NB tumours at 49 imprinted differentially methylated regions (DMRs) and assessed its association with overall survival probabilities and selected clinical and genomic features of the tumours. In addition, an integrated analysis of DNA methylation and allele-specific copy number alterations (CNAs) was performed, to understand the correlation between the two molecular events. RESULTS: Several imprinted regions with aberrant methylation patterns in NB were identified. Regions that underwent loss of methylation in > 30% of NB samples were DMRs annotated to the genes NDN, SNRPN, IGF2, MAGEL2 and HTR5A and regions with gain of methylation were NNAT, RB1 and GPR1. Methylation alterations at six of the 49 imprinted DMRs were statistically significantly associated with reduced overall survival: MIR886, RB1, NNAT/BLCAP, MAGEL2, MKRN3 and INPP5F. RB1, NNAT/BLCAP and MKRN3 were further able to stratify low-risk NB tumours i.e. tumours that lacked MYCN amplification and 11q deletion into risk groups. Methylation alterations at NNAT/BLCAP, MAGEL2 and MIR886 predicted risk independently of MYCN amplification or 11q deletion and age at diagnosis. Investigation of the allele-specific CNAs demonstrated that the imprinted regions that displayed most alterations in NB tumours harbor true epigenetic changes and are not result of the underlying CNAs. CONCLUSIONS: Aberrant methylation in imprinted regions is frequently occurring in NB tumours and several of these regions have independent prognostic value. Thus, these could serve as potentially important clinical epigenetic markers to identify individuals with adverse prognosis. Incorporation of methylation status of these regions together with the established risk predictors may further refine the prognostication of NB patients.

Supratentorial CNS-PNETs in children; a Swedish population-based study with molecular re-evaluation and long-term follow-up.

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.

DNA methylation alterations across time and space in paediatric brain tumours.

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.

DNA methylation profiling improves routine diagnosis of paediatric central nervous system tumours: A prospective population-based study.

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.

Patient injury-related alcohol use-underestimated in patients with facial fractures?

OBJECTIVE: The aim of this study was to highlight the current underestimation of the role of alcohol in facial fracture etiology and patients' daily life. STUDY DESIGN: A prospective cross-sectional study was conducted. Alcohol consumption habits were evaluated, and data were collected through a constructed questionnaire and interview. Case-related data collection was performed, with the primary predictor variables being mechanism of injury; fracture type; and associated injury (any). Outcome variables were alcohol involved in injury (yes/no) and heavy alcohol use (yes/no). The explanatory variables were gender and age. Descriptive and bivariate statistics were computed, and the P value was set at .05. RESULTS: In total, 166 patients were included in the study. Of these, 55% of patients reported being under the influence of alcohol when they sustained the injury. Alcohol was involved most often among male patients (P = .0006) and in the younger age groups (P < .0001). Of the study patients, 17% reported heavy alcohol use. The majority of the interpersonal violence events had taken place under the influence of alcohol (84%; P < .0001). CONCLUSIONS: The role of alcohol in facial fracture etiology is significant. A brief intervention for alcohol abuse should be included routinely in patient care to identify and, if necessary, address this problem.