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.

Keywords: Childhood ependymoma; Epigenetics; H3K27me3; Molecular subgrouping.

Conflict of interest statement

Declaration of interests: The authors declare no competing interests

Figures

Figure. 1. H3K27me3 immunohistochemistry segregates EPN_PFA from EPN_PF B tumor

a. Representative images of H3K27me3 immunostaining in grade II and grade III EPN_PFA and EPN_PFB. Arrowheads (black) indicate cells within the microenvironment of tumor including endothelial cells that stained positive for H3K27me3 and were used as internal positive control. Scale bars represents 200 µM b. Percent distribution of H3K27me3 negative (Red) and H3K27me3 positive (Blue) cases in predefined EPN_PFA and EPN_PFB tumors. Numbers in parentheses indicate the number of tumors studied. c. Quantification of H3K27me3 immunostaining in EPN_PFA and EPN_PFB. Each point represents a single tumor. Error bars represent mean +/− S.D. d. Percent sensitivity, specificity, positive predictive value, negative predictive value, false positive rate, false negative rate and false discovery rate for detection of childhood posterior fossa EPN_PFA vs. EPN_PFB molecular subgroups using H3K27me3 immunostaining.

Figure. 2. Global loss of H3K27me3 immunostaining relates with younger age and poor prognosis in two independent cohorts

a. Box and whisker plot of age distribution of H3K27me3 negative and H3K27me3 positive tumors in cohort 1. b–c. Progression free survival (PFS, B) or overall survival (OS, C) of EPN stratified by H3K27me3 staining in cohort 1. d. Box and whisker plot of age distribution of H3K27me3 negative and H3K27me3 positive tumors in cohort 2. e–f. Progression free survival (PFS, E) or overall survival (OS, F) of EPN stratified by H3K27me3 staining in cohort 2. The numbers below the X-axis indicates the number of persons at risk at each time point. g. Percentage of H3K27me3 negative and H3K27me3 positive tumors (Y-axis) plotted against corresponding patient age (X-axis). h–i. Progression free survival (PFS, H) or overall survival (OS, I) of EPN stratified by H3K27me3 staining in combined cohorts 1 and 2. For a and d, data are plotted as median with interquartile range with 95% CI and each point represents a single tumor. Data was analyzed using unpaired, two-tailed Mann Whitney test. For survival curves, the numbers below the X-axis indicates the number of persons at risk at each time point. Data was analyzed using the Log-Rank test.