DISCUSSION
This is one of the first pediatric studies comparing citrate levels in patients with midline glioma with or without the H3K27M mutation. Histone mutations have recently emerged as important molecular drivers and poor markers for pediatric brain tumors. The H3K27M midline gliomas have been included in the recent 2016 WHO classification of tumors of the central nervous system as a separate entity(16). Prior studies have also reported on elevated citrate levels in low-grade gliomas destined for aggressive behavior(4). Our study shows some interesting findings. Patients with the H3K27M mutation demonstrated higher citrate levels when compared to patients the mutation and the expression did not seem to be dependent on the histological grade of the tumor. Patients with H3K27M the mutation had a statistically significant worse median OS when compared those without. Patients with elevated citrate levels >1.2 mmol/kg tended to have a worse prognosis, but this was not statistically significant.
Similar findings have been reported in other studies. Bluml et al reported on elevated mean citrate levels in patients with aggressive when compared to indolent low-grade glioma (4.1±1.1 vs 0.6±0.8 mmol/kg; p<0.01)(4). Their citrate level measurements, however, were inconsistent in the high-grade glioma patients with some of these patients demonstrating elevated citrate levels and others not (4). A similar finding was noted in our study with the range varying from 0-7.5 nmol/g, but the median citrate level was significantly higher for the histologically WHO Grade III/IV when compared to the WHO Grade I/II patients. The majority of patients with the H3K27M mutation had grade III or IV tumors by histology (6/7), similar to other prior reports (10). One of the patients with a histologic low grade glioma also carried this mutation, supporting other published reports (11, 12). The prognosis of the patients with the H3K27M mutation in our study was also poor, consistent with other studies(6, 17-19). H3K27M mutant diffuse midline gliomas are now considered WHO Grade IV irrespective of the presence of anaplastic features in the 2016 WHO classification of tumors of the CNS because of this association with a poor prognosis (16).
The mechanism through which H3K27M and other histone mutations could alter citrate metabolism has not yet been studied. H3K27 is a substrate of the polycomb repressive complex 2 (PRC2), a major regulator of gene expression (20). PRC2 silences gene expression through trimethylation of K27 of the histone H3(H3K27me3) via its catalytic SET domain. H3K27M is a missense mutation in H3K27 and is associated with global decrease in H3K27me3, which is thought to be mediated through inhibition of PRC2 activity (20). These mutations are involved in oncogenesis of pediatric cancers but could in addition, be associated with alterations in the expression of other genes, leading to changes in downstream metabolic pathways, including that of citrate metabolism, thus leading to elevated citrate levels (5, 6) (13). More work obviously is required to fully understand these pathways. The finding that citrate is elevated in our study, if confirmed in larger studies could potentially suggest cellular pathways that could be explored to understand the metabolic alterations in these tumors.
Our study has a few limitations. The small sample size made it impossible to explore some of the relationships involving these variables in depth and suggest some of these findings could have occurred by chance. Although the histological grade, H3K27M mutations and age independently predicted a poor prognosis in univariate analysis, the finding that only the histological grading was statistically significantly associated with a poor outcome in multivariate analysis, suggests the sample size may have been too small to detect a change in the other variables. Although it would have been of interest to study how the H3K27M mutation affected the prognosis in histologically WHO Grade I/II and WHO Grade III/IV subgroups, this was not possible because of the small number of patients. Both a higher histological grading (WHO III/IV) and H3K27M mutation portended a worse prognosis in our report supporting the integrating of histologically and molecular data in diagnosing these tumors as in the 2016 WHO classification (21).
Our study, nonetheless, provides important hypotheses which could be tested in future studies, such as, exploring the relationship between the H3K27M mutation and citrate metabolism in a larger study to further understand the relationship between these two prognostic markers, and the utility of citrate levels preoperatively in consistently identifying a subgroup of more aggressive tumors histologically. In addition, with MR spectroscopy now more readily available in many different centers, this could improve the pre-operative characterization of tumors.