Cooks, R Graham

Gliomas are diffusely infiltrative, requiring accurate and sensitive diagnostic tools during surgical resection. Mutations in isocitrate dehydrogenase enzyme (IDH) alter its normal activity and result in accumulation of a unique oncometabolite, 2-hydroxyglutarate (2HG), in tumor cells. However, IDH-wild-type (IDH-wt) gliomas are more common and typically more aggressive. Here, 86 human brain samples were analyzed by syringe touch spray mass spectrometry to evaluate molecular alterations in gliomas. Tandem mass spectrometry (MS/MS) experiments were performed to minimize isobaric and isomeric interferences, and a ratiometric approach (using only signals for endogenous compounds) was employed to improve reliability of the measurements. Multiple ratios for pairs of metabolite signal intensities were found to differentiate tumor samples from nontumor tissue and they stratified glioma subtypes by IDH mutation status and by tumor grade, with several ratios showing 100% agreement with independent clinical assignments ( P < 0.0001, Mann–Whitney U test). Increased carnitine relative abundances were found across all glioma subtypes. Importantly, α-aminoadipate was significantly elevated in grade 4 glioma, which may correlate with increased tumor malignancy. Grade 3 gliomas showed markedly higher 2HG abundance than grade 4 samples. In addition, a selection of ratios showed a linear trend with varying tumor cell percentage. Overall, the multiple metabolic ratio alterations indicate a high degree of heterogeneity among glioma subtypes. Furthermore, there are metabolites that can differentiate glioma from nontumor tissue and this measurement can be made by a simple ratiometric method which has potential to be used intraoperatively for margin assessment without diverging from standard of care.

Abstract The mutation status of isocitrate dehydrogenase (IDH) in glioma patients has diagnostic and prognostic significance but is available only after surgery. Rapid and sensitive intraoperative molecular diagnosis is essential for distinguishing tumor from infiltrative and from normal brain tissue to maximize safe resection. IDH-mutant (mut)tumors produce high concentrations of 2-hydroxyglutarate (2HG), which serves as a surrogate marker of IDH mutation. Here, we use desorption electrospray ionization-multiple reaction monitoring (DESI-MRM), a form of mass spectrometry (MS), for rapid prediction of IDH mutation status. A set of 30 human brain smears was analyzed using DESI-MRM experiments to predict the mutation status of IDH and quantify 2HG in glioma samples within 2 min (endogenous reference molecule) or 7 min (external standard). Nanoelectrospray ionization (nESI) was employed as a validation method. An isotopically labeled surrogate internal standard allowed quantitative measurement of 2HG and clear separation between IDH-mut and IDH-wildtype (wt) samples, showing 100% agreement with clinical diagnosis. 2HG concentrations in IDH-mut samples ranged from 88 to 1445 ng mg–1, while IDH-wt samples exhibited 2HG concentrations of 9–15 ng mg–1. Calibration curves showed method accuracy within ≤15%. The quantitative results obtained using isotopically labeled standards showed a strong correlation between IDH mutation score and 2HG concentrations, as did an earlier semiquantitative (ratiometric, endogenous standard) method. These findings highlight the standalone capability of DESI-MRM for making measurements to justify assessments for intraoperative diagnosis and to support surgical decision-making to improve patient outcomes.

The development and performance of two mass spectrometry (MS) workflows for the intraoperative diagnosis of isocitrate dehydrogenase (IDH) mutations in glioma is implemented by independent teams at Mayo Clinic, Jacksonville, and Huashan Hospital, Shanghai. The infiltrative nature of gliomas makes rapid diagnosis necessary to guide the extent of surgical resection of central nervous system (CNS) tumors. The combination of tissue biopsy and MS analysis used here satisfies this requirement. The key feature of both described methods is the use of tandem MS to measure the oncometabolite 2-hydroxyglutarate (2HG) relative to endogenous glutamate (Glu) to characterize the presence of mutant tumor. The experiments i) provide IDH mutation status for individual patients and ii) demonstrate a strong correlation of 2HG signals with tumor infiltration. The measured ratio of 2HG to Glu correlates with IDH-mutant (IDH-mut) glioma ( P < 0.0001) in the tumor core data of both teams. Despite using different ionization methods and different mass spectrometers, comparable performance in determining IDH mutations from core tumor biopsies was achieved with sensitivities, specificities, and accuracies all at 100%. None of the 31 patients at Mayo Clinic or the 74 patients at Huashan Hospital were misclassified when analyzing tumor core biopsies. Robustness of the methodology was evaluated by postoperative re-examination of samples. Both teams noted the presence of high concentrations of 2HG at surgical margins, supporting future use of intraoperative MS to monitor for clean surgical margins. The power of MS diagnostics is shown in resolving contradictory clinical features, e.g., in distinguishing gliosis from IDH-mut glioma.