In this report, we identified and characterized a distinct molecular subgroup in human gliomas. Analysis of epigenetic changes from TCGA samples identified the existence of a proportion of GBM tumors with highly concordant DNA methylation of a subset of loci, indicative of a CpG island methylator phenotype (G-CIMP). G-CIMP-positive samples were associated with secondary or recurrent (treated) tumors and tightly associated with IDH1
mutation. G-CIMP tumors also showed a relative lack of copy number variation commonly observed in GBM, including EGFR
amplification, chromosome 7 gain and chromosome 10 loss. Interestingly, G-CIMP tumors displayed copy-number alterations which were also shown in gliomas with IDH1
mutations in a recent report (Sanson et al., 2009
). Integration of the DNA methylation data with gene expression data showed that G-CIMP-positive tumors represent a subset of proneural tumors. G-CIMP-positive tumors showed a favorable prognosis within GBMs as a whole and also within the proneural subset, consistent with prior reports for IDH1
mutant tumors (Parsons et al., 2008
; Yan et al., 2009
). Interestingly, of the five discordant cases of G-CIMP-positive, IDH1
-wildtype tumors, two patients survived more than five years after diagnosis, suggesting that G-CIMP-positive status may confer favorable outcome independent of IDH1
mutation status. However, studies with many more discordant cases will be needed to carefully dissect the effects of G-CIMP status versus IDH1
mutation on survival. To a large extent, the improved prognosis conferred by proneural tumors (Phillips et al., 2006
) can be accounted for by the G-CIMP-positive subset. These findings indicate that G-CIMP could be use to further refine the expression-defined groups into an additional subtype with clinical implications.
G-CIMP is highly associated with IDH1 mutation across all glioma tumor grades, and the prevalence of both decreases with increasing tumor grade. Tumor grade is defined by morphology only, and therefore can be heterogeneous with respect to molecular subtypes. Within grade IV/glioblastoma tumors are a subset of patients who tend to be younger and have a relatively favorable prognosis. It is only through molecular characterization using markers such as IDH1 and G-CIMP status that one could prospectively identify such patients. Conversely, these markers could also be used to identify patients with low- and intermediate-grade gliomas who may exhibit unfavorable outcome relative to tumor grade.
In the non-TCGA independent validation set examined in this study, an IDH1
mutation was detected in 40/43 (93%) low- and intermediate-grade gliomas, but only 7/57 (13%) of primary GBMs. Similarly, we detected nearly 10-fold more G-CIMP-positive gliomas in grade II tumors as compared to grade IV GBMs. The improved survival of G-CIMP gliomas at all tumor grades suggests that there are molecular features within G-CIMP gliomas that encourage a less aggressive tumor phenotype. Consistent with this, we identified G-CIMP-specific DNA methylation changes within a broad panel of genes whose expression was significantly associated with patient outcome. We observed that this large subset of differentially silenced genes were involved in specific functional categories, including markers of mesenchyme, tumor invasion and extracellular matrix. This concept builds upon our prior finding of a mesenchymal subgroup of glioma which shows poor prognosis (Phillips et al., 2006
). According to this model, a lack of methylation of these genes in G-CIMP-negative tumors would result in a relative increase in expression of these genes, which in turn would promote tumor progression and/or lack of response to currently available treatment modalities. A comparison of the G-CIMP gene list with prior gene expression analyses (meta-analyses) suggests that G-CIMP positive tumors may be less aggressive due to silencing of key mesenchymal genes.
We found that a minority of genes with significant promoter hypermethylation showed a concomitant significant decrease in associated gene expression (293/1520, 19%). This is consistent with previous reports, in which we found similarly low frequencies of inversely correlated promoter hypermethylation and gene expression (Houshdaran et al., 2010
; Pike et al., 2008
). The lack of an inverse relationship between promoter hypermethylation and gene expression for most genes may be attributed to several scenarios, including the lack of appropriate transcription factors for some unmethylated genes and the use of alternative promoters for some genes with methylated promoters. Epigenetics controls expression potential, rather than expression state.
are the two genes showing the strongest evidence for epigenetic silencing in G-CIMP tumors. RBP1
has been previously reported to be epigenetically silenced in cancer cell lines and primary tumors, and the association of its encoded protein with retinoic acid receptors (RAR) has been well characterized (Esteller et al., 2002
gene expression is regulated by retinoic acid (RA), and encodes a protein that promotes apoptosis in primary cells suggesting a tumor suppressor role (Kitareewan et al., 2008
; Welch et al., 2009
). The vitamin A metabolite RA is important for both embryonic and adult growth. RA has a diverse roles involving neuronal development and differentiation mediated by RARs (reviewed in (Malik et al., 2000
)). Interestingly, studies in breast cancer cells have shown that silencing of RBP1
plays an important role in RA signaling by lowering all-trans
-retinoic acid production and loss of RAR levels and activity mediated by de-repression of PI3K/Akt signaling pathway, leading to loss of cell differentiation and tumor progression ((Farias et al., 2005a
),(Farias et al., 2005b
)). This mechanism may help describe the molecular features of tumorigenesis in G-CIMP tumors. Thus, dissecting the gene expression and DNA methylation alterations of G-CIMP tumors among lower grade gliomas will be helpful to better understand the roles of a mutant IDH1
and G-CIMP DNA methylation on tumor grade and patient survival.
The highly concerted nature of G-CIMP methylation suggests that this phenomenon may be caused by a defect in a trans-acting factor normally involved in the protection of a defined subset of CpG island promoters from encroaching DNA methylation. Loss of function of this factor would result in widespread concerted DNA methylation changes. We propose that transcriptional silencing of some CIMP genes may provide a favorable context for the acquisition of specific genetic lesions. Indeed, we have recently found that IGFBP7
is silenced by promoter hypermethylation in BRAF
-mutant CIMP+ colorectal tumors (Hinoue et al., 2009
). Oncogene-induced senescence by mutant BRAF
is known to be mediated by IGFBP7
(Wajapeyee et al., 2008
). Hence, CIMP-mediated inactivation of IGFBP7
provides a suitable environment for the acquisition of BRAF
mutation. The tight concordance of G-CIMP status with IDH1
mutation in GBM tumors is very reminiscent of colorectal CIMP, in which DNA hypermethylation is strongly associated with BRAF
mutation (Weisenberger et al., 2006
). We hypothesize that the transcriptional silencing of as yet unknown G-CIMP targets may provide an advantageous environment for the acquisition of IDH1
In our integrative analysis of G-CIMP tumors, we observed up-regulation of genes functionally related to cellular metabolic processes and positive regulation of macromolecules. This expression profile may reflect a metabolic adjustment to the proliferative state of the tumor, in conjunction with the gain-of-function IDH1
mutation (Dang et al., 2009
). Such a metabolic adjustment may be consistent with Warburg’s observation that proliferating normal and tumor cells require both biomass and energy production, and convert glucose primarily to lactate, regardless of oxygen levels, while non-proliferating differentiated cells emphasize efficient energy production (reviewed in (Vander Heiden et al., 2009
In summary, our data indicate that G-CIMP status stratify gliomas into two distinct subgroups with different molecular and clinical phenotypes. These molecular classifications have implications for differential therapeutic strategies for glioma patients. Further observation and characterization of molecular subsets of glioma will likely provide additional information enabling insights into the the development and progression of glioma, and may lead to targeted drug treatment for patients with these tumors.