In Reply.

We appreciate the comments provided by Lombardi et al. [1] regarding our article [2] and acknowledge that they raise several important points. It is true that our patient population was heterogeneous, with glioma diagnoses at various grades and stages of treatment, and we noted these limitations in our report. In addition, our study had relatively few IDH wild-type patients and included heterogeneous IDH mutations, as Lombardi et al. point out. These factors may account for some of the discrepancy between our findings and those of Lombardi et al. Importantly, Lombardi et al. highlight that neither referenced study quantified the specific enantiomer of 2-hydroxyglutarate (2-HG) in their analyses. It is possible that the relative distribution of the d- or l-enantiomer of 2-HG is distinct in the urine, accounting for some of the discordance. We agree that analysis of the d-enantiomer, which is produced by the altered isocitrate dehydrogenase (IDH) enzyme, may be of utility in future validation studies. Ultimately, as detailed in our discussion, our objective was to explore in a pilot study whether 2HG in patient fluid samples could represent a potential biomarker. Given the suspected mechanism of altered IDH proteins, we would expect urine 2-HG to be comparatively elevated in IDH-mutant gliomas, as we found in our analysis. However, some of the discrepancies raised by Lombardi et al. highlight that preliminary findings to date require validation in larger samples with defined populations.