Maximal safe resection is a key strategy for improving patient prognosis in the management of brain tumors. Intraoperative fluorescence guidance has emerged as a standard in the surgery of high-grade gliomas. The administration of 5-aminolevulinic acid prior to surgery induces tumor-specific accumulation of protoporphyrin IX, which emits red fluorescence under blue-light illumination. The technology, however, is substantially limited for low-grade gliomas and weakly tumor-infiltrated brain, where low protoporphyrin IX concentrations are outweighed by tissue autofluorescence. In this context, fluorescence lifetime imaging has shown promise to distinguish spectrally overlapping fluorophores. We integrated frequency-domain fluorescence lifetime imaging in a surgical microscope and combined it with spatially registered fluorescence spectroscopy, which can be considered a research benchmark for sensitive protoporphyrin IX detection. Fluorescence lifetime maps and spectra were acquired for a representative set of fresh ex-vivo brain tumor specimens (low-grade gliomas n = 15, high-grade gliomas n = 80, meningiomas n = 41, and metastases n = 35). Combining the fluorescence lifetime with fluorescence spectra unveiled how weak protoporphyrin IX accumulations increased the lifetime respective to tissue autofluorescence. Infiltration zones (4.1ns ± 1.8ns, p = 0.017) and core tumor areas (4.8ns ± 1.3ns, p = 0.040) of low-grade gliomas were significantly distinguishable from non-pathologic tissue (1.6ns ± 0.5ns). Similarly, fluorescence lifetimes for infiltrated and reactive tissue as well as necrotic and core tumor areas were increased for high-grade gliomas and metastasis. Meningioma tumor specimens showed strongly increased lifetimes (12.2ns ± 2.5ns, p = 0.005). Our results emphasize the potential of fluorescence lifetime imaging to optimize maximal safe resection in brain tumors in future and highlight its potential toward clinical translation.

5-Aminolevulinic acid (5-ALA) is a fluorescent dye that after metabolization to Protoporphyrin IX (PpIX) by the heme biosynthesis pathway typically leads to visible fluorescence in WHO grade IV but not grade II gliomas. The exact mechanism for high PpIX levels in WHO grade IV gliomas and low PpIX levels in WHO grade II gliomas is not fully clarified. To detect relevant changes in mRNA expression, we performed an in-silico analysis of WHO grade II and IV glioma sequencing datasets provided by The Cancer Genome Atlas (TCGA) to investigate mRNA expression levels of relevant heme biosynthesis genes: Solute Carrier Family 15 Member 1 and 2 (SLC15A1 and SLC15A2), Aminolevulinate-Dehydratase (ALAD), Hydroxymethylbilane-Synthase (HMBS), Uroporphyrinogen-III-Synthase (UROS), Uroporphyrinogen-Decarboxylase (UROD), Coproporphyrinogen-Oxidase (CPOX), Protoporphyrinogen-Oxidase (PPOX), ATP-binding Cassette Subfamily B Member 6 (ABCB6)/G Member 2 (ABCG2) and Ferrochelatase (FECH). Altogether, 258 WHO grade II and 166 WHO grade IV samples were investigated. The mRNA expression levels showed significant differences in 8 of 11 examined genes between WHO grade II and IV gliomas. Significant differences in mRNA expression included increases of HMBS, UROD, FECH and PPOX as well as decreases of SLC15A2, ALAD, UROS and ABCB6 in WHO IV gliomas. Since the majority of changes was found in directions that might actually impair PpIX accumulation in WHO grade IV gliomas, additional studies are needed to analyze the corresponding factors of the heme biosynthesis also on protein level.

Radiologically suspected low-grade gliomas (LGG) represent a special challenge for the neurosurgeon during surgery due to their histopathological heterogeneity and indefinite tumor margin. Therefore, new techniques are required to overcome these current surgical drawbacks. Intraoperative visualization of brain tumors with assistance of 5-aminolevulinic acid (5-ALA) induced protoporphyrin IX (PpIX) fluorescence is one of the major advancements in the neurosurgical field in the last decades. Initially, this technique was exclusively applied for fluorescence-guided surgery of high-grade glioma (HGG). In the last years, the use of 5-ALA was also extended to other indications such as radiologically suspected LGG. Here, we discuss the current role of 5-ALA for intraoperative visualization of focal malignant transformation within suspected LGG. Furthermore, we discuss the current limitations of the 5-ALA technology in pure LGG which usually cannot be visualized by visible fluorescence. Finally, we introduce new approaches based on fluorescence technology for improved detection of pure LGG tissue such as spectroscopic PpIX quantification fluorescence lifetime imaging of PpIX and confocal microscopy to optimize surgery.

The prediction of the individual prognosis of low-grade glioma (LGG) patients is limited in routine clinical practice. Nowadays, 5-aminolevulinic acid (5-ALA) fluorescence is primarily applied for improved intraoperative visualization of high-grade gliomas. However, visible fluorescence is also observed in rare cases despite LGG histopathology and might be an indicator for aggressive tumor behavior. The aim of this study was thus to investigate the value of intraoperative 5-ALA fluorescence for prognosis in LGG patients. We performed a retrospective analysis of patients with newly diagnosed histopathologically confirmed LGG and preoperative 5-ALA administration at two independent specialized centers. In this cohort, we correlated the visible intraoperative fluorescence status with progression-free survival (PFS), malignant transformation-free survival (MTFS) and overall survival (OS). Altogether, visible fluorescence was detected in 7 (12%) of 59 included patients in focal intratumoral areas. At a mean follow-up time of 5.3 ± 2.9 years, patients with fluorescing LGG had significantly shorter PFS (2.3 ± 0.7 vs. 5.0 ± 0.4 years; p = 0.01), MTFS (3.9 ± 0.7 vs. 8.0 ± 0.6 years; p = 0.03), and OS (5.4 ± 1.0 vs. 10.3 ± 0.5 years; p = 0.01) than non-fluorescing tumors. Our data indicate that visible 5-ALA fluorescence during surgery of pure LGG might be an already intraoperatively available marker of unfavorable patient outcome and thus close imaging follow-up might be considered.

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Diffusely infiltrating gliomas are characterized by a variable clinical course, and thus novel prognostic biomarkers are needed. The heme biosynthesis cycle constitutes a fundamental metabolic pathway and might play a crucial role in glioma biology. The aim of this study was thus to investigate the role of the heme biosynthesis mRNA expression signature on prognosis in a large glioma patient cohort. Glioma patients with available sequencing data on heme biosynthesis expression were retrieved from The Cancer Genome Atlas (TCGA). In each patient, the heme biosynthesis mRNA expression signature was calculated and categorized into low, medium, and high expression subgroups. Differences in progression-free and overall survival between these subgroups were investigated including a multivariate analysis correcting for WHO grade, tumor subtype, and patient age and sex. In a total of 693 patients, progression-free and overall survival showed a strictly monotonical decrease with increasing mRNA expression signature subgroups. In detail, median overall survival was 134.2 months in the low, 79.9 months in the intermediate, and 16.5 months in the high mRNA expression signature subgroups, respectively. The impact of mRNA expression signature on progression-free and overall survival was independent of the other analyzed prognostic factors. Our data indicate that the heme biosynthesis mRNA expression signature might serve as an additional novel prognostic marker in patients with diffusely infiltrating gliomas to optimize postoperative management.

Background and Objectives: Complete neurosurgical resection of intracranial meningiomas is essential to avoid residual tumor tissue and thus minimize the risk of tumor recurrence. However, local recurrence of meningiomas is not uncommon mainly due to insufficient intraoperative detection of residual tumor tissue within the tumor bulk or peritumoral tissue such as bone and satellite lesions. Although 5-aminolevulinic acid (5-ALA) induced fluorescence was found to visualize the majority of meningiomas, no comprehensive histopathological assessment of fluorescing samples from the tumor bulk and peritumoral tissue is available. The aim of our study was thus to histopathologically analyze a large series of tissue samples derived from meningioma surgery to assess the positive predictive value (PPV) of visible 5-ALA fluorescence. Study Design/Materials and Methods: In this study, we retrospectively investigated a series of tissue samples with visible 5-ALA fluorescence collected during surgery of intracranial meningiomas from the tumor bulk and peritumoral tissue including the bone flap, dura/dural tail, arachnoidea, adjacent cortex, and satellite lesions. The tumor diagnosis was established according to the World Health Organization (WHO) criteria and all collected fluorescing samples were screened for presence of tumor tissue to calculate the PPV. Results: Altogether, 191 tissue samples with visible 5-ALA fluorescence derived during surgery of 85 meningiomas (63 WHO grade I, 17 WHO grade II, and 5 WHO grade III) were included. In detail, 158 samples from the tumor bulk and 33 specimens from the peritumoral tissue were investigated. According to histopathological analysis, the PPV of 5-ALA fluorescence was significantly higher in samples from the tumor bulk (100%) as compared with peritumoral tissue (73%; P < 0.001). With regard to peritumoral tissue, tumor tissue was present in most fluorescing samples from the satellite lesions (100%), the bone flap (92%), arachnoidea (83%), and dura/dural tail (75%). In contrast, tumor tissue was absent in the majority of samples from fluorescing cortex (six of seven samples; 86%). However, distinct reactive tissue alterations were found in all six tumor-free fluorescing cortex samples and additional vascular proliferation in two cases. Conclusion: In this largest series to date, visible 5-ALA fluorescence is characterized by a high PPV detecting tumor bulk and peritumoral tissue in intracranial meningiomas. Thus, 5-ALA fluorescence supports the neurosurgeon in identifying residual tumor tissue at relevant surgical sites to optimize meningioma surgery and minimize the risk of local recurrence.

INTRODUCTION Molecular classification has transformed the management of brain tumors by enabling more accurate prognostication and personalized treatment. Access to timely molecular diagnostic testing for brain tumor patients is limited, complicating surgical and adjuvant treatment and obstructing clinical trial enrollment. OBJECTIVE We aim to develop a rapid (&lt; 90 seconds), AI-based diagnostic screening system that can provide molecular classification of diffuse gliomas and report its use in a prospective, multicenter, international clinical trial of diffuse glioma patients (n = 153). METHODS By combining stimulated Raman histology (SRH), a rapid, label-free, non-consumptive, optical imaging method, and deep learning-based image classification, we are able to predict the molecular genetic features used by the World Health Organization (WHO) to define the adult-type diffuse glioma taxonomy, including IDH-1/2, 1p19q-codeletion, and ATRX loss. We developed a multimodal deep neural network training strategy that uses both SRH images and large-scale, public diffuse glioma genomic data in order to achieve optimal molecular classification performance. RESULTS One institution was used for model training (University of Michigan) and four institutions (NYU, UCSF, Medical University of Vienna, and University Hospital Cologne) were included for prospective patient enrollment and model testing. Using our system, called DeepGlioma, we achieved an average molecular genetic classification accuracy of 93.2% and identified the correct diffuse glioma molecular subgroup with 91.5% accuracy. DeepGlioma outperformed conventional IDH1-R132H immunohistochemistry (94.2% versus 91.4% accuracy, respectively) as a first-line molecular diagnostic screening method for diffuse gliomas, detecting canonical and non-canonical IDH mutations with high accuracy. CONCLUSION Our results demonstrate how artificial intelligence and optical histology can be used to provide a rapid and scalable alternative to wet lab methods for the molecular diagnosis of brain tumor patients during surgery.