Integrative analysis of DNA methylation suggests down-regulation of oncogenic pathways and reduced somatic mutation rates in survival outliers of glioblastoma
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.
KeywordsGlioblastoma Long-term survivor DNA methylation Genome-wide analyses
Glioma CpG Island Hypermethylator Phenotype
Long-term survivors of glioblastoma
O6-methylguanine DNA methyltransferase
Short-term survivors of glioblastoma
Transcription start sites
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 . 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, 36, 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)’ . 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 .
Although there have been some genome-wide studies for DNA methylation in survival outliers of brain cancer, most of them have largely focused on promoter regions and CpG islands (CGIs) in identifying aberrant methylation patterns or in classifying GBM due to its readiness of biological interpretation in terms of transcriptional regulation [16, 44]. However, the DNA methylation outside promotor-associated CGIs presents distinctive signatures in tumors and has significant effects on oncogenic pathways through multiple mechanisms. For example, DNA methylation of the CpG sites in gene body is known to be a major cause of cytosine to thymine transition mutations, as well as known to stimulate transcription elongation . Moreover, there is a genome-wide crosstalk, not limited to genic region, between DNA methylation and histone modifications [19, 32]. One good example is that trimethylation of histone H3 lysine 9 (H3K9me3) is required for DNMT3B dependent de novo DNA methylation . Therefore, unbiased analysis of DNA methylation across the whole genome is necessary to perform an integrative analysis of GBM of exceptional clinical course.
In the present study, we compared genome-wide DNA methylation profiles of IDH wild-type (IDH WT) GBM patients who lived longer than 3 years (n = 17, LTS-GBM) with the patients who lived less than 1 year (n = 12, STS-GBM). We found the striking differences in DNA methylation signatures between LTS- and STS- GBMs and performed integrative analyses for the differential patterns to understand their functional implications on epigenetic aspects of GBM. Also, we evaluated the classification potential of our identified patterns of DNA methylation with independent cohorts of mostly IDH WT GBM patients to test their possibility as prognostic marker.
Materials and methods
Survival data of patients with histologically-confirmed GBM at Seoul National University Hospital, Korea between 2000 and 2010 were obtained by retrospective review of patients’ charts, and from National Cancer Registry survival database of Korea. We identified 34 out of 429 newly diagnosed patients who lived longer than 3 years, and 17 patients were finally selected for discovery cohort of LTS-GBM group after central histological review. The central histological review included a complete agreement of histological diagnosis between two neuropathologists (S.H.P. and P.B.) on their independent slide review, and no evidence of IDH1 mutation on immuno-histochemical evaluation. For the comparison, 12 GBM patients who lived less than 1 year in spite of standard care were chosen for the STS-GBM group. For the validation cohort, 10 GBM samples were obtained from patients treated at Prince of Wales Clinical School, Australia between 2004 and 2009 who lived longer than 3 years. This cohort contained 5 wildtype IDH1, 1 mutated IDH1, and 4 patients with unknown IDH1 status. Patients’ clinical characteristics are summarized in the Additional file 1: Table S1. We also collected IDH WT samples that provide raw data of Illumina’s Infinium Human Methylation450K BeadChips from the cancer genome atlas (TCGA) dataset: 3 LTS and 39 STS samples.
DNA was extracted by standard methods for formalin-fixed paraffin-embedded tumor tissue. The QIAamp DNA FFPE Tissue Kit (Qiagen) was used to isolate and purify DNA from formalin fixed, paraffin embedded tissue. The protocol suggested by the manufacturer was used to isolate DNA. The quality and quantity of DNA was assessed both by Nanodrop and Bioanalyzer technology. The isolation of DNA from all clinical samples was performed by the Genetics Core Store of Johns Hopkins School of Medicine.
Genome-wide DNA methylation measurement
DNA methylation was measured by using Illumina’s Infinium Human Methylation450K BeadChips (“450K bead array”) according to the manufacturer’s manual. DNA bisulfite conversion was carried out using EZ DNA Methylation Kit (Zymo Research) by following manufacturer’s manual with modifications for Illumina Infinium Methylation Assay. Briefly, 400 ng of genomic DNA was first mixed with 5 ul of M-Dilution Buffer and incubated at 37 °C for 15 min and then mixed with 100 ul of CT Conversion Reagent prepared as instructed in the kit’s manual. Mixtures were incubated in a thermocycler with 16 thermal cycles at 95 °C for 30 s and 50C for 1 hour. Bisulfite-converted DNA samples were loaded onto 96-column plates provided in the kit for desulphonation and purification. Concentration of eluted DNA was measured using Nanodrop-1000 spectrometer.
Preprocessing of DNA methylation array data: filtering probes and intra-sample normalization
We filtered the following probes in the 450K bead array: i) probes whose detection p-values are greater than 0.01 in more than 5% of samples. According to Illumina, the detection p-value of a probe is calculated by comparing the intensity of target probes (the sum of methylated bead intensity and unmethylated bead intensity) with the intensities of negative control probes, ii) the probes whose bead counts are less than 3 in more than 5% of samples, iii) probes of non-CpG sites, iv) probes with SNP sites, v) probes whose sequences align to multiple locations in the reference genome, vi) probes associated with the sex chromosomes. The list of SNP-spanning probes and multiple-aligned probes were obtained from the R package “ChAMP” . The 407,351 probes among the total of 485,512 probes in the array were passed in the above filtering. The methylation level of a probe is represented by the “beta” value that is defined as a ratio of the intensity of methylation bead to the sum of total intensity of probe and the offset value of 100.
In the 450K bead array, a probe measures DNA methylation level with either ‘type I’ assay or ‘type II’ assay. While the probe with type I assay employs two different bead types for methylation and unmethylation with the same color channel, the probe with type II assay uses two different colors with one bead type to measure methylation and unmethylation status. Since the two types of probes measure DNA methylation with different chemistries, it is necessary to adjust technical variation between them. We adopted a beta-mixture quantile dilation (BMIQ) method proposed by Teschendorff et al. , to perform normalization of beta values within a sample.
Differentially methylated sites were identified between LTS- and STS- GBM groups by using R package, RUV-inverse for DNA methylation . Briefly, this package evaluates beta values for a probe with generalized least squares regression after controlling batch effects based on negative control probes in the array. The sites were marked as significant differential methylation if their corrected p-values for multiple testing by false discovery rate (FDR) were less than 0.01.
Pearson correlation coefficient was calculated between DNA methylation level (beta) for a given site and RNA expression level (log2 transformation of FPKM+ 1) of the corresponding refSeq mRNA using 32 GBM samples that are available for both of RNA-seq and 450K bead array in TCGA. When multiple sites were matched to a single gene, the maximum value of correlation coefficient was assigned for a gene. We only considered the sites whose standard deviation of beta values is greater than 0.1 and mean value of FPKM is greater than 0 to focus on the sites with enough variation of DNA methylation and detectable gene expression across the GBM samples.
The list of somatic mutation in GBM was downloaded from TCGA for 136 samples that have both of whole exome sequencing and 450K bead array. In each sample, a somatic mutation is associated with a probe site if the mutation is found between 5 K base-pairs upstream and downstream of a probe site.
Gene ontology (GO) analysis for a given set of sites on 450K bead array was performed adjusting for the selection bias of genes inherent from non-uniform distribution of probes across genome. We took a weighted resampling approach similar with the previous report . Specifically, 1000 random sets of as many genes as the test set were generated with selection of a gene weighted according to the number of probes assigned to a gene. Statistical significance of a GO term was determined by comparing the number of genes associated with the GO term between the test set and the randomly-selected gene sets. Significant GO terms were selected among the category of biological process based on FDR threshold of 0.05.
Probe annotation and open source datasets
The probes on the 450K bead array were annotated with R package, “IlluminaHumanMethylation450kanno.ilmn12.hg19”, except for their assignment to genes that were determined by the software ANNOVAR  using NCBI reference genes (Refseq). Note that the probes were grouped into four categories according to the distance from the closest CGI in the UCSC annotation (Additional file 1: Figure S1): i) “island” if a probe is inside CGI, ii) “shore” if a probe is within 2000 bases, iii) “shelf” if a probe is between 2000 and 4000 bases, iv) “open sea” otherwise. Further categories were made by distinguishing 5′ and 3′ direction from the closest CGI denoted by ‘N_’ and ‘S_’ respectively such as ‘N_shore’ and ‘S_shore’. The probes were also grouped according to the distance from transcription start sites (TSS) of the NCBI reference genes: i) “TSS1500” if a probe is within 200–1500 bases upstream TSS, ii) “TSS200” if a probe is between 0 and 200 bases upstream TSS, iii) “1st exon” if a probe is associated with first exon, iv) “Far” otherwise. If a probe has multiple categories, the following priority is applied to determine its single category: 1st Exon>TSS200 > TSS1500 > Far.
We obtained epigenetic annotation of human genomes from the ENCODE project [7, 38], specifically the regions of ChIP enrichment for 12 histone modifications (H2az, H3K27ac, H3K27me3, H3K36me3, H3K4me1, H3K4me2, H3K4me3, H3K79me2, H3K9ac, H3K9me1, H3K9me3, H4K20me1) across 66 cells (Additional file 1: Figure S2). The results from uniform processing by ENCODE Analysis Working Group were downloaded and manually parsed for further analyses. Also, we obtained H3K9me3 ChIP-seq and 450K bead array datasets of H1 from the ENCODE project.
TCGA datasets were downloaded for the level 3 data of RNA sequencing (RNA-seq), Illumina Infinium HumanMethylation450 BeadChip and exome sequencing of available GBM samples: FPKM, beta values and the compiled list of somatic mutation sites respectively.
For U87MG, a GBM cell line, DNA methylation profiled by 450K bead array was obtained from ENCODE project. ChIP-seq peak (q-value< 0.05) of H3K27ac was provided from  (GSE36354).
Differential DNA methylation patterns between LTS- and STS-GBM
Functional implications of LTS-GBM-specific DNA methylation patterns
Gene Ontology (GO) results of the hyper-methylated sites
Number of genes (intersection/ total)
Significance (p-value, FDR)
The multiplication or reproduction of cells, resulting in the expansion of a cell population.
50 / 263
The attachment of one cell to another cell via adhesion molecules that do not require the presence of calcium for the interaction.
6 / 13
Any process that results in a change in state or activity of a cell (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of an epidermal growth factor stimulus.
12 / 33
The hypomethylated sites in the open sea were slightly enriched with gene body region (Additional file 1: Figure S6b). Also, the they showed the enriched positive correlations between DNA methylation and gene expression, being consistent with the well-known association of DNA methylation with gene expression in gene body  (Additional file 1: Figure S7). However, their effect was not obvious as much as the hypermethylated sites. In addition, the hypomethylation-associated genes were not enriched with GO terms related to cancer (Additional file 1: Table S3). We concluded that hypomethylation in LTS-GBM is not involved with gene activity of oncogenic pathway, at least as much as hypermethylation.
Previous studies suggest that epigenetic modifications of the genome can affect the mutational rate in the human genome . We hypothesized that hypomethylation in the open sea might be related with local mutational rate. Using the exome sequencing data of GBM in TCGA, we looked at DNA methylation levels around the somatic mutation in the open sea (see materials and methods). Interestingly, we found that the distributions of DNA methylation levels around somatic mutations are different according to the sites in the open sea (Fig. 3b). In the LTS-GBM specific sites, somatic mutations were preferentially observed when they were highly methylated while the other sites in the open sea showed little bias of DNA methylation. We also looked into the relative location of the sample with somatic mutation in the distribution of DNA methylation levels (136 samples) by calculating Z-score of DNA methylation level. The hypo-methylated sites tend to have higher DNA methylation levels in the sample with somatic mutation relative to the mean DNA methylation levels across the 136 samples that were interrogated here (Additional file 1: Figure S8). This tendency is weaker in the insignificant sites (Kolmogorov-Smirnov test, number of hypo-methylated sites:1802, number of insignificant sites: 5032, p-value < 2.2e-16). These results imply that the hypomethylation in LTS-GBM occurs at the sites potentially contributing to lower somatic mutation rates.
Validation of differential methylation in an independent LTS-GBM cohort
We also attempted to assign scores to each test sample in terms of hypermethylation and hypomethylation in order to predict LTS-GBM. The simple arithmetic average of beta values in each pattern is assigned to a sample. We predicted a sample as LTS-GBM when its mean beta values for both of hypermethylation and hypomethylation are greater than 0.2, otherwise it was called as STS-GBM. As a result, 9 out of 12 LTS-GBM were predicted correctly (sensitivity: 75%) and 38 out of 39 STS samples are correctly recalled (specificity: 97%) (Fig. 4b).
Epigenetic aberrations are increasingly regarded as a gateway to neoplastic transformation in gliomas . 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 . 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 . Zhang et al. analyzed methylation profiles of 13 LTS and 20 STS-GBM patients using Illumina Infinium Human Methylation 27 K Bead-Chips . 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.
In the present study, we pursued the genome-wide understanding of DNA methylation patterns specific to IDH WT LTS-GBM. We showed that LTS-GBM, compared with STS-GBM, is characterized by hypermethylation in the CGI and hypomethylation at open sea throughout the genome. It is well known that methylation of CGIs at promoter region is linked to silence of gene expression, and our results were consistent with this idea and previous findings of hypermethylation in LTS-GBM. However, hypomethylation of open sea in LTS-GBM is poorly appreciated so far. It is not compelling to understand this pattern in terms of gene activity since our GO analysis showed that the genes are not generally related with cancer. Also, as genome-wide hypomethylation in cancer is known to be ubiquitous feature of carcinogenesis , the hypomethylation pattern of LTS-GBM relative to STS-GBM seems to be enigmatic. However, a previous study showed that methylated cytosine bases are prone to mutation by spontaneous deamination to thymine . Additionally, there is evidence linking levels of regional mutation density of cancer cells with the heterochromatin-associated H3K9me3 that is a sole histone mark enriched with our hypomethylated sites . Our analysis of exome-sequencing of GBM showed that local somatic mutations tend to occur near the LTS-GBM-specific sites in open sea when these sites have higher DNA methylation levels. This suggests that significantly lower methylation around the identified sites in LTS-GBM can reduce the risk of de novo mutation contributable to oncogenesis possibly in the absence of proper DNA repair mechanisms in GBM. However, the mechanism of how the hypomethylation in the region distant from CGI in LTS-GBM affects the rates of somatic mutation and contributes to better survival relative to STS-GBM remains to be determined by further studies.
We also tested whether the two identified genome-wide patterns were able to serve as molecular markers of LTS-GBM by applying the identified sites to the independent cohorts of LTS samples. Although we confirmed the reasonable performance of the sites with the simple rule of prediction, the LTS samples from TCGA cohort did not show the predicted pattern. This might be due to small sample size (N = 3) or unknown batch effect. We anticipate a larger cohort as well as better prediction rules in the future study to validate applicability of the identified methylation patterns specific to LTS-GBM.
We like to thank Min Jung Park, R.N. for the support in summarization of clinical data and sample management. We also thank Waine Yu at the Oncology microarray core in Johns Hopkins Hospital for running microarray for our samples. We appreciate for ENCODE Consortium and the ENCODE production laboratories generating the GBM dataset. Also, we should note that the results here are in part based upon data generated by the TCGA Research Network: https://www.cancer.gov/about-nci/organization/ccg/research/structural-genomics/tcga.”
The study was designed by ML and CKP. TH performed all the computational analyses. Experimental work was undertaken by DM. Patient samples were collected and evaluated by KLM, ID, SK, YSD, PB, SHP, CKP. TH, DM, HC, CB, JHS, ML and CKP contributed the interpretation of results. TH, DM, ML and CKP wrote the manuscript and all the authors approved the manuscript.
This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Ministry of Science & ICT (NRF-2018M3A9H3021707). in Korea, and the Seoul National University Hospital Research Fund (3020180010).
Ethics approval and consent to participate
This study was approved by the institutional ethics committees of Seoul National University Hospital and Johns Hopkins University Hospital. All the patient samples were obtained after receiving informed consents for research purpose including genetic studies in accordance with its institutional review board.
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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