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.