Immunohistochemical analysis of H3K27me3 demonstrates global reduction in group-A childhood posterior fossa ependymoma and is a powerful predictor of outcome

Abstract

Posterior fossa ependymomas (EPN_PF) in children comprise two morphologically identical, but biologically distinct tumor entities. Group-A (EPN_PFA) tumors have a poor prognosis and require intensive therapy. In contrast, group-B tumors (EPN_PFB) exhibit excellent prognosis and the current consensus opinion recommends future clinical trials to test the possibility of treatment de-escalation in these patients. Therefore distinguishing these two tumor subtypes is critical. EPN_PFA and EPN_PFB can be distinguished based on DNA methylation signatures, but these assays are not routinely available. We have previously shown that a subset of poorly prognostic childhood EPN_PF exhibits global reduction in H3K27me3. Therefore, we set out to determine whether a simple immunohistochemical assay for H3K27me3 could be used to segregate EPN_PFA from EPN_PFB tumors. We assembled a cohort of 230 childhood ependymomas and H3K27me3 immunohistochemistry was assessed as positive or negative in a blinded manner. H3K27me3 staining results were compared with DNA methylation-based subgroup information available in 112 samples [EPN_PFA (n=72) and EPN_PFB tumors (n=40)]. H3K27me3 staining was globally reduced in EPN_PFA tumors and immunohistochemistry showed 99% sensitivity and 100% specificity in segregating EPN_PFA from EPN_PFB tumors. Moreover, H3K27me3 immunostaining was sufficient to delineate patients with worse prognosis in two independent, non-overlapping cohorts (n=133 and n=97). In conclusion, immunohistochemical evaluation of H3K27me3 global reduction is an economic, easily available and readily adaptable method for defining high-risk EPN_PFA from low-risk posterior fossa EPN_PFB tumors to inform prognosis and to enable the design of future clinical trials.

Case-based review: ependymomas in adults

“…The supratentorial ependymoma compartment includes the favorable prognostic category of ependymomas with YAP1 fusions. 32,34 By using an antibody against the trimethylated Histone H3 at Lysine 27 (H3K27me3), a recent study was able to distinguish 40 This is exciting as immunohistochemistry is cost effective compared to methylation arrays allowing for faster and easier clinical utilization.…”

Characterization of global 5-hydroxymethylcytosine in pediatric posterior fossa ependymoma

“…IHC analyses were performed as reported [9]. In brief, tissue sections were cut at 5 μm, followed by deparaffinization and rehydration using xylene and ethanol.…”

“…The Leica Bond Polymer Refine DAB detection kit was used according to the manufacturer's instructions. All IHC slides were evaluated by two independent neuropathologists; the scoring methods were performed as report described [9]. H3K27me3 positive staining was defined as scored positive when more than 80% cells had nuclear positivity and scored negative when they did not.…”

“…Group A ependymoma (EPN_PFA) is characterized by CpGi hypermethylation with 1q gain and occurs predominantly in infancy and young children. These subgroup tumors also exhibit global low H3K27me3 [7][8][9], global DNA hypomethylation [7], and high expression of EZHIP [10]. Conversely, group B ependymoma (EPN_PFB) presents with CpGi hypomethylation and primarily occurs in adolescences and young adults.…”

Put away your microscopes: the ependymoma molecular era has begun

“…Despite lacking a clear genetic driver mutation, PF-EPN-A tumors demonstrate widespread epigenomic alterations in the form of DNA CpG island hypermethylation and global DNA hypomethylation, a phenotype well described in many solid cancers[10,18–20]. Furthermore, PF-EPN-A tumors demonstrate global H3K27me3 loss, a histone modification important for regulating bivalent gene expression and maintaining cell identity[21,22]. Gene targets of DNA and H3K27 hypermethylation overlap significantly with patterns of DNA and histone H3K27 methylation seen in embryonic stem cells.…”