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astrocytoma patients, with a combined sensitivity and specificity of 100% and 95%, respectively. Conditioned
media from cultured primary astrocytoma cells also showed relative enrichment of bFGF and TIMP3,
concordant with the findings in patient urine samples. Among 9 patient samples where pre- and post-
treatment imaging was available, bFGF and TIMP3 levels also significantly correlated with tumor volume
[93]
changes on imaging .

Another, earlier project from the same group looked at VEGF, MMP2, and MMP9 in urine and CSF samples
from a cohort of 28 brain tumor patients (n = 11 pediatric, n = 17 adult) and 23 control patients. Urinary
concentrations of VEGF, MMP2 and MMP9 were all significantly increased within the tumor patient cohort;
and urinary VEGF and MMP2, in particular, had excellent sensitivity and specificities for the presence of a
brain tumor. VEGF had 95.2% sensitivity and 89.5% specificity, while MMP2 demonstrated 82.1% sensitivity
and 95.7% specificity. Both of these proteins were undetectable in the urine of 5 patients for whom follow-up
[94]
imaging demonstrated complete resolution of their tumors .
Both of these studies are very encouraging for the prospect of developing a clinically deployable and
accurate laboratory assay for pediatric brain tumors. However, as with all biomarker sources, urine has
special limitations as a diagnostic media that have still not completely been addressed in either of the
[69]
aforementioned studies. Nolen et al. laid out in an extensive analysis of over 200 potential urinary
biomarkers the effects of normalization methods, population variability, and temporal variability on
measured biomarker concentrations. The authors of this study found that, while urine total protein had
the smallest impact on biomarker variability, none of the normalization methods tested (urine creatinine,
urine albumin, urine B2M) was clearly superior to the other. Additionally, the authors observed significant
intra- and inter-day variability in urine biomarker concentrations; in most cases, the coefficients of variation
[69]
exceeded 50% . These findings should prompt caution when interpreting urinary biomarker data, as robust
differences in biomarker concentrations between diseased and normal states will be needed to consistently
[93]
overcome such high levels of biomarker variability. Even so, the work by Pricola Fehnel et al. suggests that
such high levels discrimination may indeed exist for some pediatric brain tumor urinary biomarkers.

Challenges of developing biomarkers from pediatric populations
Compared to the adult neuro-oncology world, the development of circulating biomarker candidates for
pediatric brain tumor patients is still in its infancy. This is partially due to a significant statistical barrier
to research within pediatric brain tumor patient populations. There are an estimated 4,600 new pediatric
[1]
brain tumor diagnoses each year in the United States of America . This compares to an average of 22,172
[95]
new adult primary brain tumor diagnoses per year . As these statistics demonstrate, pediatric brain tumor
cases are relatively rare, and individual institutions often do not have enough volume to organize large scale
studies on their own. Multi-centered trials and novel screening assays are needed in order to create study
populations with enough power to generate meaningful conclusions.

Another issue is the lack of comparative data across biofluids and pediatric brain tumor clinical stages for
individual biomarker candidates. As mentioned in this review article, there exist significant theoretical and
practical limitations to each biofluid currently targeted for biomarker discovery. Additionally, there is mounting
evidence that tumor stage and location relative to the biofluid of interest can alter biomarker yields [36,41,96] . A
large study examining one or more biomarkers in multiple biofluids serially across early and late stages of brain
tumor therapy will be needed to answer the question of which biofluid is most suitable for diagnosing and
screening which pediatric brain tumors. This is a daunting ask given the difficulties researchers face accruing
[97]
and maintaining large population pediatric brain tumor studies. A study by Pages et al. is currently underway
examining the cfDNA in the CSF, serum, and urine of 192 pediatric brain tumor patients, but results are not
available as of the production of this manuscript. It is hoped that this study and others to come like it may help
to define the effect of biofluid choice on biomarker sensitivity and specificity for pediatric brain tumors.