Research Article: Integrative analysis of DNA methylation suggests down-regulation of oncogenic pathways and reduced somatic mutation rates in survival outliers of glioblastoma

Date Published: June 3, 2019

Publisher: BioMed Central

Author(s): Taeyoung Hwang, Dimitrios Mathios, Kerrie L. McDonald, Irene Daris, Sung-Hye Park, Peter C. Burger, Sojin Kim, Yun-Sik Dho, Hruban Carolyn, Chetan Bettegowda, Joo Heon Shin, Michael Lim, Chul-Kee Park.


The study of survival outliers of glioblastoma can provide important clues on gliomagenesis as well as on the ways to alter clinical course of this almost uniformly lethal cancer type. However, there has been little consensus on genetic and epigenetic signatures of the long-term survival outliers of glioblastoma. Although the two classical molecular markers of glioblastoma including isocitrate dehydrogenase 1 or 2 (IDH1/2) mutation and O6-methylguanine DNA methyltransferase (MGMT) promoter methylation are associated with overall survival rate of glioblastoma patients, they are not specific to the survival outliers. In this study, we compared the two groups of survival outliers of glioblastoma with IDH wild-type, consisting of the glioblastoma patients who lived longer than 3 years (n = 17) and the patients who lived less than 1 year (n = 12) in terms of genome-wide DNA methylation profile. Statistical analyses were performed to identify differentially methylated sites between the two groups. Functional implication of DNA methylation patterns specific to long-term survivors of glioblastoma were investigated by comprehensive enrichment analyses with genomic and epigenomic features. We found that the genome of long-term survivors of glioblastoma is differentially methylated relative to short-term survivor patients depending on CpG density: hypermethylation near CpG islands (CGIs) and hypomethylation far from CGIs. Interestingly, these two patterns are associated with distinct oncogenic aspects in gliomagenesis. In the long-term survival glioblastoma-specific sites distant from CGI, somatic mutations of glioblastoma are enriched with higher DNA methylation, suggesting that the hypomethylation in long-term survival glioblastoma can contribute to reduce the rate of somatic mutation. On the other hand, the hypermethylation near CGIs associates with transcriptional downregulation of genes involved in cancer progression pathways. Using independent cohorts of IDH1/2- wild type glioblastoma, we also showed that these two patterns of DNA methylation can be used as molecular markers of long-term survival glioblastoma. Our results provide extended understanding of DNA methylation, especially of DNA hypomethylation, in cancer genome and reveal clinical importance of DNA methylation pattern as prognostic markers of glioblastoma.

Partial Text

Despite advances in modern neuro-oncology, glioblastoma (GBM) continues to have a poor prognosis. Survival rates of adult GBM patients in the United States are quite low with 1-year, 2-year, 3-year, and 5-year relative survival rates estimated at 39.3, 16.9, 9.9, and 5.5%, respectively [26]. While the majority of GBM patients live no longer than 2 years, there is a subset of patients who live longer than 3 years and are classified as long-term survivors (LTS). This group of patients remains a puzzle to researchers in the field, as studies on clinical, radiological, histological, and molecular characteristics have yet to yield consensus regarding determinants of durable response to the current treatment [2, 3, 12, 14, 15, 20, 22, 24, 28, 29, 35–37]. For example, efforts to identify specific gene expression profiling patterns for LTS-GBM failed to uncover consistent features [8, 9, 30]. The classic genetic markers of favorable prognosis of GBM such as O-6-methylguanine-DNA methyltransferase (MGMT) promoter methylation or isocitrate dehydrogenase (IDH) mutation do not fully account for long term survivors of glioblastoma (LTS-GBM) [1, 9, 10, 21, 33, 39]. In particular, there are few studies for identification of molecular features associated with glioblastoma independent from IDH mutation or the IDH mutation-related signatures such as DNA methylation pattern called ‘Glioma CpG Island Hypermethylator Phenotype (G-CIMP)’ [8]. Although there is a report of concurrent gain of chromosomes 19 and 20 as a favorable prognostic factor for a subset of LTS-GBM that did not show G-CIMP, multiple other studies revealed no distinctive DNA copy number changes in LTS-GBM [9, 10, 30]. These results suggest that there is little chance to define LTS-GBM with a single genetic or epigenetic mechanism, emphasizing the importance of integrative understanding of molecular signatures in LTS-GBM. In fact, a recent integrated genomic analysis comparing LTS and short-term survivors (STS) GBM showed that multiple genetic and epigenetic factors are involved in divergent molecular features between the two extremes of the survival spectrum [28].

Epigenetic aberrations are increasingly regarded as a gateway to neoplastic transformation in gliomas [16]. In particular, a recent study showed that DNA methylation was the strongest predictor of prolonged survival in GBM compared to any of clinical variables, RNA expressions for mRNA/miRNA, and the available genomic data including germline/somatic point mutation and copy number variation [14]. They found the importance of DNA methylation based on the statistical analyses for clinical data and multimodal molecular profiles of 44 patients (7.4%) who lived longer than 3 years among 591 GBM patients from TCGA dataset. However, the effect of DNA methylation is often confounded with genetic perturbation. For example, although G-CIMP signatures were found to be a favorable prognostic marker of GBM [4, 25], the majority of them overlap with IDH mutation [27, 41]. Therefore, it is important to evaluate the effect of DNA methylation in LTS-GBM after controlling genetic background such as IDH mutation. There have been several studies identifying DNA methylation signatures specific for IDH WT LTS-GBMs. Mock et al. compared global DNA methylation profiling using Methyl-CpG-Immunoprecipitation in 14 LTS and 15 STS-GBM patient samples with IDH1 wild-type, and found that hypermethylation of multiple CpGs mapping to the promoter region of LOC283731 correlated with improved patient outcome [22]. Zhang et al. analyzed methylation profiles of 13 LTS and 20 STS-GBM patients using Illumina Infinium Human Methylation 27 K Bead-Chips [44]. They identified the promoter methylation in ALDH1A3 is a prognostic biomarker in a IDH1 wild-type and unmethylated MGMT promoter GBM sample. However, these studies only focused on DNA methylation in promoter regions and did not provide comprehensive understanding of landscape of DNA methylation signatures in LTS-GBM.

Our finding provides a clue on functional implications of global DNA methylation in survival outliers of glioblastoma, which are related to oncogenic pathways through the two distinct mechanisms of transcriptional suppression and somatic mutation depending on their genomic location (Fig. 5). The implication of DNA hypomethylation specific to long term survivors of glioblastoma call more attention to its dual aspects on both oncogenic contribution and survival benefits of patients.Fig. 5Genome-wide DNA methylation pattern of glioblastoma. The genomes of long-term survivors in glioblastoma are differentially methylated relative to short-term survival patients depending on CpG density: hypermethylation near CpG islands (CGIs) and hypomethylation far from CGIs (open sea). The hypermethylation at CGIs frequently occurs around regions with histone marks of active transcription such as H3K27ac, correlating with downregulation of gene expression in cancer progression pathways. The hypomethylated region at open sea are enriched with a histone mark of heterochromatin, H3K9me3. The rate of de novo mutation is high in this region when it is methylated, implying survival advantage of hypomethylation of the region in glioblastoma. In the figure, we highlighted genic regions such as first exon and gene body to emphasize potential effect of perturbed DNA methylation in glioblastoma