Date Published: June 3, 2014
Publisher: BioMed Central
Author(s): Sama Ahsan, Eric H Raabe, Michael C Haffner, Ajay Vaghasia, Katherine E Warren, Martha Quezado, Leomar Y Ballester, Javad Nazarian, Charles G Eberhart, Fausto J Rodriguez.
Diffuse intrinsic pontine glioma (DIPG) is a malignant pediatric brain tumor associated with dismal outcome. Recent high-throughput molecular studies have shown a high frequency of mutations in histone-encoding genes (H3F3A and HIST1B) and distinctive epigenetic alterations in these tumors. Epigenetic alterations described in DIPG include global DNA hypomethylation. In addition to the generally repressive methylcytosine DNA alteration, 5-hydroxymethylation of cytosine (5hmC) is recognized as an epigenetic mark associated with active chromatin. We hypothesized that in addition to alterations in DNA methylation, that there would be changes in 5hmC. To test this hypothesis, we performed immunohistochemical studies to compare epigenetic alterations in DIPG to extrapontine adult and pediatric glioblastoma (GBM) and normal brain. A total of 124 tumors were scored for histone 3 lysine 27 trimethylation (H3K27me3) and histone 3 lysine 9 trimethylation (H3K9me3) and 104 for 5hmC and 5-methylcytosine (5mC). An H-score was derived by multiplying intensity (0–2) by percentage of positive tumor nuclei (0-100%).
We identified decreased H3K27me3 in the DIPG cohort compared to pediatric GBM (p < 0.01), adult GBM (p < 0.0001) and normal brain (p < 0.0001). H3K9me3 was not significantly different between tumor types. Global DNA methylation as measured by 5mC levels were significantly lower in DIPG compared to pediatric GBM (p < 0.001), adult GBM (p < 0.01), and normal brain (p < 0.01). Conversely, 5hmC levels were significantly higher in DIPG compared to pediatric GBM (p < 0.0001) and adult GBM (p < 0.0001). Additionally, in an independent set of DIPG tumor samples, TET1 and TET3 mRNAs were found to be overexpressed relative to matched normal brain. Our findings extend the immunohistochemical study of epigenetic alterations in archival tissue to DIPG specimens. Low H3K27me3, decreased 5mC and increased 5hmC are characteristic of DIPG in comparison with extrapontine GBM. In DIPG, the relative imbalance of 5mC compared to 5hmC may represent an opportunity for therapeutic intervention. The online version of this article (doi:10.1186/2051-5960-2-59) contains supplementary material, which is available to authorized users.
Tumors of the central nervous system are the second most common malignancy in pediatric patients. Diffuse intrinsic pontine gliomas (DIPGs) comprise approximately 10% of pediatric brain tumors and are universally fatal
. Due to their location, DIPGs have rarely been biopsied at diagnosis outside of recent clinical trials. No improvements in DIPG outcome have been noted in the past 20 years, perhaps due to the paucity of patient-derived samples and lack of molecular insights leading to novel treatments. Encouragingly, recent molecular and proteomic analyses of autopsy specimens have identified key genetic alterations in DIPG, including amplifications in genes encoding receptor tyrosine kinases (PDGFRA, MET)
, PDGFRA mutations
, as well as distinct DIPG subgroups based on Hedgehog (SHH) and MYCN pathway activation
. Mutations in the ACVR1 gene, encoding the transforming growth factor-beta (TGF-beta) superfamily member activin, have been reported in approximately 20% of DIPGs
We determined by IHC relative levels of H3K27me3, 5mC, and 5hmC in DIPG, pediatric GBM, and adult GBM tissue. Our data are consistent with previously reported low levels of H3K27me3 due to the H3K27M mutation in the majority of DIPG. H3K9me3 in DIPG was not significantly different from H3K9me3 immunoreactivity in extrapontine GBM and unlikely to be playing a role in the tumorigenicity of DIPG. Surprisingly, 5hmC immunoreactivity was significantly elevated in DIPG, in contrast to 5mC, suggesting that imbalance between 5hmC and 5mC plays a global role in the biology of DIPG. In general, high levels of 5hmC have been shown to be a feature of terminally differentiated cells
. In numerous solid tumors including carcinoma of the breast, prostate, colon, melanoma, and gliomas, 5hmC levels appear to be greatly reduced in neoplastic cells
. Our group has previously shown that in adult GBM and anaplastic astrocytoma high levels of 5hmC are associated with a less aggressive phenotype
. However, the global DNA hypomethylation that occurs in DIPG in conjunction with relatively increased 5hmC may represent signs of a novel global epigenetic dysregulation state distinct from that of adult high grade glioma. The elevation of 5hmC in DIPG must be considered in the context of global loss of 5mC in these tumors. The striking imbalance between 5hmC and 5mC in DIPG may be another sign of the marked epigenetic dysregulation which underlies these aggressive tumors.
The H3.3 Lysine 27 is a critical site for the deposition of an inhibitory epigenetic mark and is mutated in the majority of DIPG, suggesting that targeting epigenetics could be one therapeutic approach for this highly aggressive pediatric tumor. Understanding the epigenetic landscape of DIPG opens up the opportunity for epigenetic modifiers, which could potentially shift the active genome of this deadly tumor into a silent and regulated state. The role of epigenetic modifiers such as methylation inhibitors and histone deacetylase inhibitors
 in treating DIPG needs to be reevaluated as the epigenetic mechanisms of DIPG are better understood.