Rapid detection of IDH mutations in gliomas by intraoperative mass spectrometry

Abstract

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.

Keywords: 2-hydroxyglutarate; intraoperative analysis; molecular diagnostics; oncometabolite; tandem mass spectrometry.

Fig. 1.

(A) IDH-mutant gliomas harbor significant metabolic aberrations. Specifically, α-ketoglutaric acid is reduced to 2HG which accumulates in IDH-mutant tumors. (B) Data on training (retrospective) and validation (prospective) cohorts in Mayo and Huashan studies. (C) Workflow of rapid metabolic analysis of gliomas by MS/MS measurement of 2HG (m/z 129) in IDH-mut gliomas using Glu (m/z 128) as reference. Typical results for (i) Mayo/Purdue study with DESI and benchtop ion trap MS and (ii) Huashan/Tsinghua study with direct capillary spray and miniature MS.

Fig. 2.

(A) Confusion matrix showing correlation between IHC and/or genetic testing and DESI-MS assessments of IDH genotype from 129 core biopsies from 31 patients. Intraoperative analysis of IDH mutation by 2HG/Glu achieved a sensitivity, specificity, and accuracy all of 100% by DESI-MS. (B) Box plots demonstrating separation of IDH-wt and IDH-mut tumors by DESI-MS measurements (***p < 0.0001). (C) ROC curves demonstrate perfect classification of IDH-wt and mut tumors by 2HG/Glu ratios irrespective of method. (D, E, and F) are parallel figures from intraoperative analysis of 156 core biopsies by miniature MS with sensitivity, specificity, and accuracy all at 100% and ***p < 0.0001.

Fig. 3.

(A and B) Comparison of the intraoperative and postoperative assessment of IDH genotype from tumor core and surgical margin biopsies with DESI-MS. (C) Intraoperative metabolic analysis by 2HG/Glu with miniature MS could separate IDH mutated gliomas and paratumor (margin) tissue significantly (***P < 0.001). (D) Multiple sampling with navigation from a left insular glioma shows the intratumoral heterogeneity of 2HG. A metabolic borderline was delineated with consecutive sampling in real-time by 2HG/Glu analysis with miniature MS (blue line).

Fig. 4.

(A) Left posterior temporal lesion with a high Cho/NAA ratio on MRS was diagnosed as lower grade glioma preoperatively. (B) Miniature MS analysis revealed a high log 2HG/Glu ratio of 0.31, indicating the presence of an IDH mutation. (C) Frozen section reported gliosis with a low cell population. (D) Second MS/MS analysis revealed a higher log 2HG/Glu ratio of 0.98, indicating the presence of an IDH mutation. (E) Second pathological examination confirmed a lower-grade glioma.