Cooks, R. Graham

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.

2071 Background: Despite advances in our understanding of brain cancer, the ability to discern between infiltrated and non-infiltrated tissue with current adjuncts, remains elusive. Isocitrate dehydrogenase (IDH) mutation is unique to IDH-mut glioma cells and can be used as a surrogate for tumor infiltration. Further, emerging evidence has shown unique signatures in brain cancer lipidomic profiling, with lipid metabolism playing a key role in brain cancer biology. Here, we aimed to evaluate the impact of desorption electrospray ionization-mass spectrometry (DESI-MS) as a near real-time method to determine infiltration status and the use of DESI-MS to perform intra-operative histological diagnosis. Methods: This is a prospective multi-institution cohort study using intra-operative DESI-MS on freshly obtained tumor samples to evaluate 2-hydroxyglutarate (2HG), with post-hoc validation by review by a board-certified neuropathologist. For tumor cell estimation and tissue diagnosis, high-throughput DESI-MS was used for rapid analysis of high-density arrays using lipidomic profiling. Analysis of the data was performed using a pre-written algorithm employing a combination of Python- and MATLAB-based custom software allowing results in less than a second per sample. Results: A total of 496 tumor biopsies from 72 patients have been analyzed. Earlier iterations focused on absolute quantitation of 2HG in 247 biopsies from 49 patients to determine IDH genotype yielding sensitivity, specificity, and accuracy values of 89, 100, and 94% for core biopsies (71). Improvement in accuracy was obtained with relative measurement of 2HG in comparison to glutamate allowing for detection of IDH-mut infiltration at the margin, resulting in 93% sensitivity, 100% specificity, and 98% accuracy with turnover times averaging 3 minutes in 183 biopsies from 23 patients. Margin assessment beyond the limits of neuro-navigation and surgeon assessment showed tumor infiltration in 88% of margin biopsies from IDH mutant tumors (30 of 34 biopsies) with 100% accuracy. Intra-operative tissue diagnosis was pursued in 66 unique tumor samples using DESI-MS lipidomic profiling, including samples of normal brain parenchyma, gliomas, meningiomas and pituitary adenomas, split into two microarray sets (36 and 40 samples). Using estimated principal components as input features, high accuracy ( > 90%; ROC AUCs ≥ 0.89) was obtained for supervised tissue classification using simple machine learning algorithms. Conclusions: We present a novel system for intra-operative evaluation of tumor infiltration at the margin in gliomas and for intra-operative tissue diagnosis of brain cancer. Our method identified tumor infiltration beyond the surgical margins and serves as a foundation for future uses employing DESI-MS to allow for molecularly guided surgery in IDH-mut gliomas.

Abstract BACKGROUND Despite advances in cancer biology, surgery and its extent of resection remains the most important prognostic modulator in gliomas, with the distinction between infiltrated and non-infiltrated tissues being an issue at the penumbra. Isocitrate dehydrogenase (IDH) mutation is unique to IDH-mutant glioma cells and can be used as a potential surrogate for tumor infiltration. Here we evaluate desorption electrospray ionization-mass spectrometry (DESI-MS) as a near real-time method for detecting infiltration at the margin of IDH-mutant gliomas. METHODS We conducted a prospective study in which DESI-MS analysis was performed on freshly obtained tissue biopsies to detect the presence of 2-Hydroxyglutarate (2HG), with subsequent review of each sample by a board-certified neuropathologist. RESULTS A total of 247 biopsies from a 49-patient study were previously collected and analyzed at Indiana University. Assessment of IDH mutation status of 71 core biopsies yielded sensitivity, specificity, and accuracy values of 89, 100, and 94%, respectively. Subsequent improvement of the method, including the measurement of 2HG relative to an internal control (Glutamate, Glu), allowed for detection of IDH-mutant infiltration at surgical margins. We have collected 183 biopsies (116 core and 67 margin) from 23 patients. Preliminary results of IDH mutation assessments of core biopsies indicate 93% sensitivity, 100% specificity, and 98% accuracy and margin biopsies indicate 88% sensitivity, 100% specificity, and 94% accuracy. Tumor infiltration at the margin of the surgical resection by detection of IDH-mut infiltration was detected in 88% of margin biopsies from IDH mutant tumors (30 of 34 biopsies). CONCLUSION We present a novel method to allow intraoperative evaluation of IDH mutation presence in glioma margin biopsies. Our method and its ongoing validation identified tumor infiltration beyond surgical margins and serves as a foundation for molecularly guided surgery in IDH-mut gliomas via DESI-MS.

Abstract BACKGROUND Intraoperative detection of residual tumor and isocitrate dehydrogenase (IDH) mutations can assist in maximizing surgical resection beyond contrast enhancing margins and guide intraoperative surgical decision making for glioma patients. We aimed to evaluate the use of desorption electrospray ionization-mass spectrometry (DESI-MS) for intraoperative assessment of IDH mutations and estimation of tumor cell percentage (TCP). METHODS This is a prospective study using intraoperative DESI-MS analysis of freshly obtained tissue samples to evaluate IDH mutations via 2-hydroxyglutarate (2-HG) intensity and TCP via measurement of N-acetylaspartic acid (NAA) intensity and characteristic lipid profiles. These IDH mutation and TCP estimates were subsequently validated by a senior neuropathologist. RESULTS A total of 247 biopsies from a 49-patient study were previously collected and analyzed at Indiana University. Assessment of TCP in 203 margin and core biopsies based on NAA intensity yielded sensitivity, specificity, and accuracy values of 91, 76, and 83%, whereas TCP assessment based on characteristic lipid profiles yielded 76, 85, and 81%, respectively. Assessment of IDH mutation status of 71 core biopsies yielded sensitivity, specificity, and accuracy values of 89, 100, and 94%. Further validation of the methodology is being performed in an ongoing collaboration with Mayo Clinic-Jacksonville, where we have collected 178 biopsies from 24 patients. Preliminary results of IDH mutation assessments indicate 100% sensitivity, specificity, and accuracy. DISCUSSION/CONCLUSION We present a novel system to allow intraoperative evaluation of IDH status and to guide surgical resection by TCP measurement from tissue biopsies. Prospectively, we propose to modify our DESI-MS system by placing a surgical material (e.g. cottonoid) along the surgical margin and transferring material from the blot to a microscope slide prior to DESI-MS analysis. This will allow the retention of the spatial distribution of diagnostic molecules while analyzing a wall of the surgical cavity without the need for biopsy.

Abstract Introduction/Objective Maximizing surgical resection in gliomas, while avoiding compromising non-infiltrated tissue, is associated with survival benefit. Current methodologies are suboptimal in providing rapid, intraoperative molecular characterization of tissue. We address this unmet need by using desorption electrospray ionization mass spectrometry (DESI-MS) for the intraoperative molecular assessment of gliomas. Methods/Case Report This prospective study uses intraoperative DESI-MS analysis of fresh tissue to evaluate IDH mutations via 2-hydroxyglutarate intensity and TCP via measurement of N-acetylaspartic acid (NAA) intensity and characteristic lipid profiles in less than three minutes. Blinded review of the tissue smears by a neuropathologist is used to validate IDH mutation status and TCP estimates. Results (if a Case Study enter NA) Presently, 529 biopsies from 85 enrolled patients have been collected and analyzed at two institutions. TCP assessment based on NAA intensity in 203 biopsies at the first institution yielded sensitivity, specificity, and accuracy values of 91, 76, and 83%, whereas TCP estimates via characteristic lipid profiles yielded 76, 85, and 81%, respectively. Assessment of IDH mutation status of 71 core biopsies yielded sensitivity, specificity, and accuracy values of 89, 100, and 94%. Ongoing validation of the methodology is being performed at a second institution, where we have collected 282 biopsies from 36 patients. IDH mutation assessment of the first 15 patients indicate 100% sensitivity, specificity, and accuracy. Conclusion This study represents the first and largest study using DESI-MS for the intraoperative evaluation of IDH status and TCP measurement in gliomas. Prospectively, we propose to modify our DESI-MS system to allow estimation of IDH mutation status and TCP in surgical cavities without the need for a biopsy by placing a surgical material along the margin and transferring material from the blot to a microscope slide prior to DESI-MS analysis. We envision molecular analysis by DESI-MS as a complementary technique to histopathology capable of providing additional clinical information in near real-time.