A novel GC-MS/MS assay for the measurement of 2-hg enantiomers and the utility of 2-hg enantiomer levels as a biomarker for IDH mutant gliomas

The isocitrate dehydrogenase (IDH) gene has recently been identified to be mutated in gliomas, a malignant brain tumor. Mutant IDH (IDHmut) produces the oncometabolite, (R)-2-hydroxyglutarate (R-2-hg), resulting in a significant increase of intracellular concentrations above physiological levels. However, a lack of correlation between circulating 2-hg levels and IDH status has been observed. This is likely due to the lack of discrimination from the enantiomer of R-2-hg and (S)-2-hydroxyglutarate (S-2-hg). S-2-hg is also normally made in the body but can increase to levels comparable to R-2-hg during hypoxic and acidic conditions. Thus, it is important to differentiate between R-2-hg and S-2-hg to determine the utility of R-2-hg as a biomarker. Furthermore, the current mass spectrometry (MS) methods available for the separation and detection are lacking. Current assays use laborious methods that can result in interconversion of enantiomers during sample preparation, and since low-resolution mass spectrometry instruments have been utilized, incorrect characterization of 2-hg MS data has occurred. A novel chiral gas chromatography-tandem mass spectrometry (GC-MS/MS) assay was developed which improves upon current methods. The assay utilizes a simplified ethyl acetate extraction, separates 2-hg enantiomers using a chiral column which avoids racemization, and quantifies 2-hg enantiomers using stable-isotope dilution MS. Using 2-hg isotopologues, unique EI fragmentation pathways for both 2-hg and the 2-hg lactone have also been described resulting in the ability to simultaneously detect both 2-hg and 2-hg lactone enantiomers. The assay was then validated and serum 2-hg levels from healthy patients were measured, establishing a new, comprehensive reference range for normal levels of each enantiomer. Differences in basal levels of 2-hg enantiomers were observed between races but not sex. Finally, serum levels of 2hg enantiomers were measured in patients with and without IDHmut gliomas. An increase in R-2hg levels was observed for a number of patients with growing IDHmut gliomas, however, not all patients with IDHmut gliomas had an increase in R-2-hg levels. Increased S-2-hg levels were also observed in patients who received prior chemotherapy/radiotherapy. Further work is needed to fully understand circulating 2-hg enantiomer biology, but the work presented herein takes a significant first step in providing the tools and framework for understanding the clinical utility of R-2-hg as a biomarker for IDHmut gliomas.