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Abstract Introduction With the application of high-resolution 3D 7 Tesla Magnetic Resonance Spectroscopy Imaging (MRSI) in high-grade gliomas, we previously identified intratumoral metabolic heterogeneities. In this study, we evaluated the potential of 3D 7 T-MRSI for the preoperative noninvasive classification of glioma grade and isocitrate dehydrogenase (IDH) status. We demonstrated that IDH mutation and glioma grade are detectable by ultra-high field (UHF) MRI. This technique might potentially optimize the perioperative management of glioma patients. Methods We prospectively included 36 patients with WHO 2021 grade 2–4 gliomas (20 IDH mutated, 16 IDH wildtype). Our 7 T 3D MRSI sequence provided high-resolution metabolic maps (e.g., choline, creatine, glutamine, and glycine) of these patients’ brains. We employed multivariate random forest and support vector machine models to voxels within a tumor segmentation, for classification of glioma grade and IDH mutation status. Results Random forest analysis yielded an area under the curve (AUC) of 0.86 for multivariate IDH classification based on metabolic ratios. We distinguished high- and low-grade tumors by total choline (tCho) / total N-acetyl-aspartate (tNAA) ratio difference, yielding an AUC of 0.99. Tumor categorization based on other measured metabolic ratios provided comparable accuracy. Conclusions We successfully classified IDH mutation status and high- versus low-grade gliomas preoperatively based on 7 T MRSI and clinical tumor segmentation. With this approach, we demonstrated imaging based tumor marker predictions at least as accurate as comparable studies, highlighting the potential application of MRSI for pre-operative tumor classifications.

Measurement(s) Cancer Histology • Cellularity Measurement • Total Sample Tissue Area • brain neoplasm Technology Type(s) Hematoxylin and Eosin Staining Method • digital curation • Histology and Immunohistochemistry Shared Resource Factor Type(s) age • sex • location Sample Characteristic - Organism Homo sapiens Machine-accessible metadata file describing the reported data: https://doi.org/10.6084/m9.figshare.16652272

PurposeModern techniques for improved tumor visualization have the aim to maximize the extent of resection during brain tumor surgery and thus improve patient prognosis. Optical imaging of autofluorescence is a powerful and non-invasive tool to monitor metabolic changes and transformation in brain tumors. Cellular redox ratios can be retrieved from fluorescence emitted by the coenzymes reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD). Recent studies point out that the influence of flavin mononucleotide (FMN) has been underestimated.Experimental designFluorescence lifetime imaging and fluorescence spectroscopy were performed through a modified surgical microscope. We acquired 361 flavin fluorescence lifetime (500-580 nm) and fluorescence spectra (430-740 nm) data points on freshly excised different brain tumors: low-grade gliomas (N=17), high-grade gliomas (N=42), meningiomas (N=23), metastases (N=26) and specimens from the non-tumorous brain (N=3).ResultsProtein-bound FMN fluorescence in brain tumors did increase with a shift toward a more glycolytic metabolism (R=-0.87). This increased the average flavin fluorescence lifetime in tumor entities with respect to the non-tumorous brain. Further, these metrics were characteristic for the different tumor entities and showed promise for machine learning based brain tumor classification.ConclusionsOur results shed light on FMN fluorescence in metabolic imaging and outline the potential for supporting the neurosurgeon in visualizing and classifying brain tumor tissue during surgery.

ObjectiveThe intraoperative visualization of adult-type diffuse gliomas with 5-aminolevulinic acid (5-ALA) induced fluorescence is widely used in the neurosurgical field. While visible 5-ALA induced fluorescence is found in the majority of high-grade gliomas, most low-grade gliomas lack visible fluorescence during surgery. Recently, the heme biosynthesis pathway was identified as crucial influencing factor for presence of visible fluorescence since it metabolizes 5-ALA to fluorescing Protoporphyrin IX (PpIX). However, the exact alterations within the heme biosynthesis pathway resulting in visible 5-ALA induced fluorescence in gliomas are still unclear. The aim of the present study was thus to compare the mRNA and protein expression of promising intramitochondrial heme biosynthesis enzymes/transporters in glioma tissue samples of different fluorescence behavior.MethodsA total of 19 strongly fluorescing and 21 non-fluorescing tissue samples from neurosurgical adult-type diffuse gliomas (WHO grades II-IV) were included in the current analysis. In these samples, we investigated the mRNA expression by quantitative real time PCR and protein expression using immunohistochemistry of the intramitochondrial heme biosynthesis enzymes Coproporphyrinogen Oxidase (CPOX), Protoporphyrinogen Oxidase (PPOX), Ferrochelatase (FECH), and the transporter ATP-binding Cassette Subfamily B Member 2 (ABCG2).ResultsRegarding mRNA expression analysis, we found a significantly decreased ABCG2 expression in fluorescing specimens compared to non-fluorescing samples (p = 0.001), whereas no difference in CPOX, PPOX and FECH was present. With respect to protein expression, significantly higher levels of CPOX (p = 0.005), PPOX (p < 0.01) and FECH (p = 0.003) were detected in fluorescing samples. Similar to mRNA expression analysis, the protein expression of ABCG2 (p = 0.001) was significantly lower in fluorescing samples.ConclusionDistinct alterations of the analyzed heme biosynthesis factors were found primarily on protein level. Our data indicate that heme biosynthesis pathway activity in general is enhanced in fluorescing gliomas with upregulation of PpIX generating enzymes and decreased ABCG2 mediated PpIX efflux outweighing the also increased further metabolization of PpIX to heme. Intramitochondrial heme biosynthesis factors thus constitute promising pharmacological targets to optimize intraoperative 5-ALA fluorescence visualization of usually non-fluorescing tumors such as low-grade gliomas.

Precise intraoperative brain tumor visualization supports surgeons in achieving maximal safe resection. In this sense, improved prognosis in patients with high-grade gliomas undergoing protoporphyrin IX fluorescence-guided surgery has been demonstrated. Phase fluorescence lifetime imaging in the frequency-domain has shown promise to distinguish weak protoporphyrin IX fluorescence from competing endogenous tissue fluorophores, thus allowing for brain tumor detection with high sensitivity. In this work, we show that this technique can be further improved by minimizing the crosstalk of autofluorescence signal contributions when only detecting the fluorescence emission above 615 nm. Combining fluorescence lifetime and spectroscopic measurements on a set of 130 ex vivo brain tumor specimens (14 low- and 56 high-grade gliomas, 39 meningiomas and 21 metastases) coherently substantiated the resulting increase of the fluorescence lifetime with respect to the detection band employed in previous work. This is of major interest for obtaining a clear-cut distinction from the autofluorescence background of the physiological brain. In particular, the median fluorescence lifetime of low- and high-grade glioma specimens lacking visual fluorescence during surgical resection was increased from 4.7 ns to 5.4 ns and 2.9 ns to 3.3 ns, respectively. While more data are needed to create statistical evidence, the coherence of what was observed throughout all tumor groups emphasized that this optimization should be taken into account for future studies.