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this biomarker has been only sparsely examined and almost exclusively in CSF. Despite cfDNA’s limited
utilization, to date, in pediatric brain tumor biomarker development, the authors feel that it bears special
discussion, due to its potential as a low-risk diagnostic for midline gliomas.

Midline gliomas represent a one of the most difficult groups of pediatric primary brain tumors to treat.
[32]
Characterized by a H3K27M histone mutation , these tumors are generally aggressive and often not safely
[33]
surgically accessible , making it difficult for clinicians to acquire biopsies. This limits the amount of
molecular information available to direct therapy and inform prognosis, and it elevates the clinical need for
a safe, reliable method of extracting molecular information from these tumor patients.

Using cfDNA isolated from CSF samples from pediatric brain tumor patients, researchers have made
significant progress towards the detection of diffuse midline glioma histone mutations important for
[34]
prognostication. A study conducted by Huang et al. that reviewed CSF samples from 6 pediatric patients
with diffuse midline gliomas found that the H3F3A mutation could be isolated or detected in up to 83% of
patients, with a specificity of 100%. This methodology has, subsequently, been confirmed in a larger cohort
[35]
of 48 patients in a multi-institutional study. In that study by Panditharatna et al. , the authors found that
the H3 p. K27M mutations in CSF strongly correlated with the presence of a diffuse midline glioma with the
same H3K27M mutation. The amount of detectable genetic material also correlated with the response of that
tumor to radiation therapy. The accuracy of the H3 p. K27M cfDNA was 87% in this study. Interestingly,
Panditharatna’s group also looked at the presence of the H3 p. K27M mutant genetic material in matched
serum samples, as well. They reported comparable sensitivity and sensitivity results with serum as compared
[35]
to CSF, though with markedly lower quantities of detectable cfDNA .
CSF-based cfDNA does have important limitations that have yet to be overcome. While sensitivity for
intracranial tumor detection with CSF-based cfDNA is superior to CSF cytology in general, its sensitivity
for primary CNS tumors seems to be much less than for extra-CNS neoplasm. In a study of 53 adult patients
with intracranial tumors, 62.5% of patients with metastatic CNS tumors had detectable tumor-specific
mutant cfDNA in the CSF, while only 50% of patients with primary CNS neoplasms had detectable cfDNA
[27]
in the CSF . cfDNA detection in the CSF may be limited by anatomic factors, such as tumor location
relative to CSF cisterns. A study of 35 pediatric and adult patients with primary brain tumors found that
86% of tumors abutting a CSF cistern had detectable cfDNA in the CSF, but none of the tumors with entirely
[36]
parenchymal locations had detectable CSF cfDNA . Tumor biology may also limit detection as some
authors have noted low to absent rates of cfDNA detection even for some ependymomas with extensive
[37]
surface contact with CSF .

Non-coding RNA
Non-coding RNA, predominantly miRNA, are small (18-25 nucleotides) RNA that function to regulate
mRNA pre- and post-translationally. miRNA form from a precursor RNA (pri-miRNA) that is cleaved by
the RNase III nuclear enzyme Drosha into a 60-70 nucleotide intermediate known as pre-miRNA, which
is then exported from the nucleus via Exportin-5. Once in the cytoplasm, pre-miRNA is cleaved again by
Dicer, another RNase III enzyme, into miRNA, which then complexes with a transactivation-responsive
[38]
RNA binding protein and Argonaute to form an RNA-induced silencing complex . This miRNA-enzyme
complex is the active unit at the end of miRNA biogenesis, and it is capable of recognizing complementary
mRNA and (1) inhibiting translation initiation via cap-40S association; (2) inhibiting translation initiation
via 40S-AUG-60S association; (3) inhibiting elongation; (4) causing premature ribosome drop-off; (5) causing
cotranslational protein degradation; (6) causing sequestration of mRNA in P-bodies; (7) causing increased
mRNA degradation; (8) increasing mRNA cleavage; or (9) effecting transcriptional inhibition by interaction
[39]
with promoter sequences or increased methylation of promoters .
[40]
There are currently > 1,900 known human miRNA (miRbase.org) regulating at least 30% of human genes .
Table 1 lists some of the more prominent miRNA implicated in pediatric brain tumor biology [41-43] . These