Intraoperative assessment of isocitrate dehydrogenase mutation status in human gliomas using desorption electrospray ionization–mass spectrometry

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The authors describe a rapid intraoperative ambient ionization mass spectrometry (MS) method for determining isocitrate dehydrogenase (IDH) mutation status from glioma tissue biopsies. This method offers new glioma management options and may impact extent of resection goals. Assessment of the IDH mutation is key for accurate glioma diagnosis, particularly for differentiating diffuse glioma from other neoplastic and reactive inflammatory conditions, a challenge for the standard intraoperative diagnostic consultation that relies solely on morphology.


Banked glioma specimens (n = 37) were analyzed by desorption electrospray ionization–MS (DESI-MS) to develop a diagnostic method to detect the known altered oncometabolite in IDH-mutant gliomas, 2-hydroxyglutarate (2HG). The method was used intraoperatively to analyze tissue smears obtained from glioma patients undergoing resection and to rapidly diagnose IDH mutation status (< 5 minutes). Fifty-one tumor core biopsies from 25 patients (14 wild type [WT] and 11 mutant) were examined and data were analyzed using analysis of variance and receiver operating characteristic curve analysis.


The optimized DESI-MS method discriminated between IDH-WT and IDH-mutant gliomas, with an average sensitivity and specificity of 100%. The average normalized DESI-MS 2HG signal was an order of magnitude higher in IDH-mutant glioma than in IDH-WT glioma. The DESI 2HG signal intensities correlated with independently measured 2HG concentrations (R2 = 0.98). In 1 case, an IDH1 R132H–mutant glioma was misdiagnosed as a demyelinating condition by frozen section histology during the intraoperative consultation, and no resection was performed pending the final pathology report. A second craniotomy and tumor resection was performed after the final pathology provided a diagnosis most consistent with an IDH-mutant glioblastoma. During the second craniotomy, high levels of 2HG in the tumor core biopsies were detected.


This study demonstrates the capability to differentiate rapidly between IDH-mutant gliomas and IDH-WT conditions by DESI-MS during tumor resection. DESI-MS analysis of tissue smears is simple and can be easily integrated into the standard intraoperative pathology consultation. This approach may aid in solving differential diagnosis problems associated with low-grade gliomas and could influence intraoperative decisions regarding extent of resection, ultimately improving patient outcome. Research is ongoing to expand the patient cohort, systematically validate the DESI-MS method, and investigate the relationships between 2HG and tumor heterogeneity.

ABBREVIATIONS ACN = acetonitrile; DESI = desorption electrospray ionization; DMF = dimethylformamide; GBM = glioblastoma; IDH = isocitrate dehydrogenase; LTQ = linear trap quadrupole; MRSI = MR spectroscopy imaging; MS = mass spectrometry; ROC = receiver operating characteristic; TCP = tumor cell percentage; WT = wild type; 2HG = 2-hydroxyglutarate.

Article Information

Correspondence Aaron Cohen-Gadol: Indiana University School of Medicine, Indianapolis, IN.

INCLUDE WHEN CITING Published online January 4, 2019; DOI: 10.3171/2018.8.JNS181207.

Disclosures The authors report no conflict of interest concerning the materials or methods used in this study or the findings specified in this paper.

© AANS, except where prohibited by US copyright law.



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    DESI-MS method overview. A: Image of the custom-made intraoperative DESI-MS system for intraoperative analysis of tissue biopsies. B: Diagram of the DESI process as described by Takáts et al. C: Workflow of intraoperative analysis protocol consisting of tissue collection (red spot) and smearing, DESI-MS analysis, and post hoc histopathological staining. Original magnification ×20. Figure is available in color online only.

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    Correlation of 2HG DESI-MS signal with 2HG concentration in the banked tissue specimens. A: Correlation between 2HG DESI-MS signal (normalized [norm.] relative to the full-scan total ion count), ion transition m/z 147→129→101, and 2HG concentration (ng/mg tissue). Adjacent tissue from the same specimens was used to perform both measurements. Only 28 of the 37 samples were used for the quantitative analysis. The remaining samples were excluded due to insufficient tissue quantity. Blue circles indicate IDH-WT samples (n = 21); red circles, IDH-mutant samples (n = 7). Quantitative data are described by Yannell et al. and also appear in the Supplemental Information. B: Box-and-whisker plot for 2HG concentrations (ng/mg tissue) in human glioma tissue specimens (n = 28) analyzed using direct infusion ESI-MS/MS as described by Yannell et al.27 The box represents the IQR with a median line and whiskers are at ± 1.5 standard deviation. Quantitative measurements were made using the MRM transitions m/z 147→129 for 2HG, and the signals were normalized by the MRM transition m/z 150→132 intensity of the added 2HG-d3 deuterium isotope labelled internal standard. Random numbers between 0 and the limit of detection were assigned to IDH-WT glioma specimens for which no 2HG was detected. The red line shows the cutoff of 45 ng/mg tissue determined from ROC curve analysis. Reproduced with permission from Yannell KE, Smith K, Alfaro CM, Jarmusch AK, Pirro V, Cooks RG: N-acetylaspartate and 2-hydroxyglutarate assessed in human brain tissue by mass spectrometry as neuronal markers of oncogenesis. Clin Chem 63:1766–1767, 2017 ©American Association for Clinical Chemistry, 2018. C: Intraoperative discrimination between IDH-WT and IDH-mutant gliomas via DESI-MS 2HG measurement. Box-and-whisker plot obtained from the patient cohort of the intraoperative study. IDH-WT, n = 14; IDH-mutant, n = 11. The average 2HG signal intensity, ion transition m/z 147→129→101, and normalized to the total ion count of the full scan, was plotted for each patient. The box represents the IQR with a median line and whiskers at ± 1.5 standard deviation. Mean values are shown as red squares. The outlier is shown as a black x. Signal intensities were significantly different (p < 0.0001). Zeros were assigned to the WT glioma specimens for which no 2HG was detected. Figure is available in color online only.

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    Patient 17, who had an IDH-mutant glioblastoma. A: H & E–stained tissue. Original magnification ×200. A diffusely infiltrative, moderately cellular glioma made up of abundant gemistocytic astrocytes with moderate atypia. Note the presence of vascular proliferation (arrow) indicating high-grade nature. B: Immunohistochemistry for IDH1 R132H (clone H09; Dianova) showing diffuse cytoplasmic immunoreactivity, indicating the presence of IDH1 R132H mutation. Original magnification ×200. C: Reconstruction of tumor volume (green) using the preoperative MRI scans (T2-weighted 3D FLAIR), showing the location of tumor core biopsies (dark objects) analyzed by DESI-MS. Sizes of the biopsies are not to scale. D: Average 2HG product ion scan m/z 147→129→○ from the 3 analyzed biopsy samples. Figure is available in color online only.



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