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In 1979, Jeffrey Allen, MD and colleagues at Memorial Sloan-Kettering applied serum and CSF AFP/βHCG
radioimmunoassays for use in a group of 6 pediatric patients with biopsy proven intracranial germ cell
tumors. This study was not powered to allow for statistical discrimination of the results, but it demonstrated,
within individual cases, high correlations between serum AFP and CSF βHCG levels and the presence of
intracranial germ cell tumors. Furthermore, those patients with elevated AFP or βHCG assay levels had
complete normalization of their serum and CSF levels concurrent with successful tumor radiographic
[19]
response to therapy . This work has been expanded upon numerous times since, and the correlations
[19]
found in Allen et al. ’s paper have proven to be robust within larger cohorts. AFP and βHCG serum and
CSF assays have now become routine components of intracranial germ cell tumor diagnostics, in some cases
obviating the need for tissue sampling entirely [20-23] .

The story of AFP/βHCG serum and CSF assays is a rare success story in the realm of clinical oncologic
biomarkers for pediatric brain tumor patients, and one that has, frustratingly, not been matched since. No
other intracranial pediatric tumor has a widely accepted diagnostic peripheral biomarker capable - in select
[23]
circumstances - of replacing tissue biopsy in the clinical evaluation algorithm . Even for intracranial germ
cell tumors, AFP/βHCG serum and CSF assays can provide false negatives, and research continues as we
seek ever more reliable biofluid-based biomarkers.

CSF biomarkers
The most studied and fruitful biofluid target for pediatric oncologic biomarkers is CSF. A normally low-
[24]
protein, relatively acellular biofluid , CSF is a natural choice for mining biomarker data for central nervous
system (CNS) pathologies as it lies in direct contact with the whole of the CNS’s pial and ependymal surfaces.
Cells, both pathologic and normal, shed proteins and nucleic acids, be they naked or within extracellular
vesicles, into the extracellular spaces. These compounds and vesicles will accumulate within the local
biofluid compartments. In the case of the CNS, intravascular spaces, parenchymal interstitial spaces, and the
intraventricular/subarachnoid spaces represent the major extracellular compartments where biomarkers can
[25]
collect. With diffusion into the intravascular spaces being limited by the blood brain barrier (BBB) , the
CSF and interstitial spaces, theoretically, should be the most rich in cytologic, genetic, and protein markers
[16]
of tumor growth . Most CSF based biomarker studies have focused on detecting: (1) tumor cells; (2) cell-
free tumor DNA; (3) non-coding RNA; or (4) tumor-related metabolites. The authors have already discussed
CSF cytology (i.e., detecting tumor cells) in the previous section, so we will only focus on the last three
biomarker sources going forward.

Cell-free DNA
Circulating, cell-free tumor DNA (cfDNA) analysis employs several techniques to identify specific genetic
sequences or screen for arrays of genetic sequences within biofluids. Methods for detection of genomic
alterations in cfDNA are mainly PCR-based assays: real-time PCR, methylation-specific PCR, droplet
digital PCR, and next-generation sequencing (NGS). The latter two techniques have particularly improved
[26]
the sensitivity of cfDNA detection compared to older PCR-based assays . While not specifically confined
to use in the CSF, it has been most successful at correlating with brain tumor pathologies when examined
in the CSF, due to the fact that non-CSF biofluids typically contain a very small percentage (≤ 1%) of brain
[27]
tumor genetic material in a sea of normal tissue genetic material . Within CSF, cfDNA has been shown to
[28]
detect the presence of a CNS neoplasm at rates significantly higher than CSF cytologic analysis , as well as
identify clinically relevant mutations, even when present at strikingly low frequencies (1:1 × 10 ), through the
7
[29]
use of NGS techniques like duplex sequencing .
CSF cfDNA has been shown in several studies of adult intracranial tumor patients to exceed CSF cytologic
detection levels when specific mutations are screened for (EGFR for glioblastoma, KRAS for non-small
cell lung cancer, BRAF p. V600E for melanoma, etc.) [27,28,30,31] . Among pediatric brain tumor patients,