5-ALA Fluorescence-guided Surgery Research Articles

Fluorescence and immune-cell infiltration of nonneoplastic, postbrachytherapy brain tissue in 5-ALA-guided resection of recurrent anaplastic meningioma: illustrative case.

5-Aminolevulinic acid (5-ALA) fluorescence-guided surgery is a well-established technique for resecting high-grade gliomas. However, its application in meningiomas, especially those previously treated with radiation therapy, remains under investigation. A 48-year-old female with recurrent anaplastic meningioma, World Health Organization grade 3, underwent a right-sided craniotomy using off-label 5-ALA as a surgical adjunct. The patient had previously undergone brachytherapy seed implantation (20 × cesium 131) for tumor management. During the surgery, a large fluorescent tumor mass adjacent to the brachytherapy-treated area was resected, and the prior brachytherapy seeds were removed. Interestingly, the surrounding brain tissue in the irradiated area showed robust 5-ALA fluorescence. Pathological examination confirmed that the fluorescent brain tissue was nonneoplastic and associated with lymphocyte and macrophage infiltration. This case report presents unique 5-ALA fluorescence in nonneoplastic tissue following brachytherapy, which was found during the resection of recurrent anaplastic meningioma. This phenomenon may reflect an intricate interplay among radiation therapy, immune cells, the tumor microenvironment, and 5-ALA metabolism. Given that false-positive findings in fluorescence-guided surgery can lead to unnecessary tissue resection and increased surgical morbidity, further research is warranted to elucidate the mechanisms underlying this phenomenon and its implications for meningioma surgery.

Letter to the Editor. 5-ALA fluorescence-guided surgery as standard of care in the resection of GBM.

Letter to the Editor. 5-ALA fluorescence-guided surgery as standard of care in the resection of GBM.

MODL-23. PAIRED GLIOBLASTOMA CELL CULTURES OF THE FLUORESCENT BULK TUMOR AND NON-FLUORESCENT TUMOR MARGIN DISPLAY DIFFERENTIAL PHENOTYPES AND CELL STATES ACROSS PATIENTS

Abstract Glioblastoma is a devastating disease with a median survival of 8 months after diagnosis. Over 90% of patients have a recurrence that is in contact with the resection cavity. To understand the potential relapse-causing cells of the local infiltrative tumor margin, which have rarely been studied, we have established and analyzed paired cell cultures from the fluorescent bulk tumor and non-fluorescent tumor margin. Tumor samples were resected using 5-ALA fluorescence-guided surgery and employing stringent procedures to separate margin samples from bulk tumor cells. All margin samples were resected at least 1cm beyond the fluorescent border in non eloquent areas as determined intraoperatively. Bulk and margin samples from 13 glioblastoma patients were explanted and 7 paired cultures that could be maintained to at least passage 7 were established. Analysis of self-renewal and orthotopic tumor formation of both acutely dissociated cells and established cultures showed that bulk tumor cells in all cases had a higher capacity than their paired margin cells. Conversely, margin cells always displayed a higher invasive capacity than their paired bulk tumor cells. Combined single nucleus RNA- and ATAC-sequencing was performed on three paired cultures. Analysis of snRNA-seq data showed that the margin cell phenotype was connected with transitions towards more differentiated (AC- and MES-like) cell states compared to paired bulk tumor cells that showed a higher rate of cells in progenitor cell-like states (NPC- and OPC-like). snATAC analysis uncovered that the margin cell phenotype was connected with transcription factor (TF) motifs known to be involved in cell invasion and differentiation, while paired bulk tumor cells were enriched for TF motifs involved in neural development and proliferation. We present a unique collection of margin cell cultures that we believe are more relevant models of the relapse-causing glioblastoma cells than those established from the bulk tumor.

THE USE OF 5-AMINOLEVULINIC ACID (5-ALA) IN HIGH-GRADE GLIOMA SURGERY, A SINGLE CANADIAN CENTER EXPERIENCE

Abstract 5-Aminolevulinic acid (5-ALA) is a prodrug used to selectively illuminate high-grade glioma (HGG) tissue intra-operatively, shown to nearly double complete resection rates in a 2006 multicentre, phase III clinical trial. Here, we review the history of the 2020 approval of 5-ALA in Canada and present some of the first preliminary results on resection rates, survival analysis, and adverse effects from a single Canadian center. METHODS: We enrolled 76 patients (median age 61 years, 42 male) with suspected HGG amenable to surgical resection to undergo 5-ALA fluorescence-guided surgery between June 2020 and January 2023. Gross total resection was defined by the absence of enhancing l esions on postoperative MRI. We compared the survival distributions of confirmed HGG cases with complete vs. incomplete resection using a log-rank test and Kaplan-Meier statistics. RESULTS: 52 patients were confirmed as having a HGG (grade III or IV) based on a pathological diagnosis. In 32 of these patients (60.3%) a gross total resection was achieved. 46 patients of the initial cohort had their surgery done for more than 180 days. 47.8% had a survival of 600 or more days. CONCLUSIONS: 5-ALA fluorescence-guided surgery resulted in high complete resection rates, comparable to literature with no notable adverse side effects.

P.110 The use of 5-Aminolevulinic acid (5-ALA) in high-grade glioma surgery, a single Canadian center experience

Background: 5-Aminolevulinic acid (5-ALA) is a prodrug used to selectively illuminate high-grade glioma (HGG) tissue intra-operatively, shown to nearly double complete resection rates in a 2006 multicentre, phase III clinical trial. Here, we review the history of the 2020 approval of 5-ALA in Canada and present some of the first preliminary results on resection rates, survival analysis, and adverse effects from a single Canadian center. Methods: We enrolled 76 patients (median age 61 years, 42 male) with suspected HGG amenable to surgical resection between June 2020 and January 2023. Gross total resection was defined by the absence of enhancing lesions on postoperative MRI. We compared the survival distributions of confirmed HGG cases with complete vs. incomplete resection using a log-rank test and Kaplan-Meier statistic. Results: 52 patients were confirmed as having a HGG based on a pathological diagnosis. In 32 of these patients (60.3%) a gross total resection was achieved. 82.76% were still alive at 180 and 270 days, and 72.73% at 360 days. 47.8% had a survival of 600 or more days. Conclusions: 5-ALA fluorescence-guided surgery resulted in high complete resection rates, and improved overall survival comparable to the literature with no notable adverse side effects.

Refining the Intraoperative Identification of Suspected High-Grade Glioma Using a Surgical Fluorescence Biomarker: GALA BIDD Study Report.

Improving intraoperative accuracy with a validated surgical biomarker is important because identifying high-grade areas within a glioma will aid neurosurgical decision-making and sampling. We designed a multicentre, prospective surgical cohort study (GALA-BIDD) to validate the presence of visible fluorescence as a pragmatic intraoperative surgical biomarker of suspected high-grade disease within a tumour mass in patients undergoing 5-aminolevulinic acid (5-ALA) fluorescence-guided cytoreductive surgery. A total of 106 patients with a suspected high-grade glioma or malignant transformation of a low-grade glioma were enrolled. Among the 99 patients who received 5-ALA, 89 patients were eligible to assess the correlation of fluorescence with diagnosis as per protocol. Of these 89, 81 patients had visible fluorescence at surgery, and 8 patients had no fluorescence. A total of 80 out of 81 fluorescent patients were diagnosed as high-grade gliomas on postoperative central review with 1 low-grade glioma case. Among the eight patients given 5-ALA who did not show any visible fluorescence, none were high-grade gliomas, and all were low-grade gliomas. Of the seven patients suspected radiologically of malignant transformation of low-grade gliomas and with visible fluorescence at surgery, six were diagnosed with high-grade gliomas, and one had no tissue collected. In patients where there is clinical suspicion, visible 5-ALA fluorescence has clinical utility as an intraoperative surgical biomarker of high-grade gliomas and can aid surgical decision-making and sampling. Further studies assessing the use of 5-ALA to assess malignant transformation in all diffuse gliomas may be valuable.

Optimization of novel exoscopic blue light filter during fluorescence-guided resection of Glioblastoma.

Operative guidelines and use optimization for new surgical exoscopes are not well described in the literature. In this study, we evaluated use of the ORBEYE (Olympus) surgical exoscope system during 5-ALA fluorescence-guided resection of GBMs to optimize workflow and exoscope settings. The ORBEYE exoscope system was fitted with a blue light filter for 5-ALA mediated fluorescence-guided surgery (FGS). Intraoperative images were obtained during 5-ALA FGS in 9 patients with primary or recurrent GBM. The exoscope was set up at constant, increasing focal distances from the target tissue, and light source intensity varied. High-resolution 4K images were captured and analyzed. Comparisons of fluorescence to background were then generated for use optimization. Light intensity did not significantly influence tumor fluorescence (P = 0.878). However, focal distance significantly impacted relative fluorescent intensity (P = 0.007). Maximum average fluorescence was seen consistently at a focal length of 220mm and a light intensity of approximately 75% maximum. Decreasing focal distance from 400mm to 220mm significantly increased visualized fluorescence (P = 0.0038). The ORBEYE surgical exoscope system with blue light filter is a powerful imaging tool for 5-ALA FGS in GBM. The ORBEYE blue filter performs optimally at shorter focal distance with moderate light intensity. Similar to microscope systems, decreasing focal distance significantly influences visualized fluorescence.

5-Aminolevulinic acid for enhanced surgical visualization of high-grade gliomas: a prospective, multicenter study.

Greater extent of resection (EOR) is associated with longer overall survival in patients with high-grade gliomas (HGGs). 5-Aminolevulinic acid (5-ALA) can increase EOR by improving intraoperative visualization of contrast-enhancing tumor during fluorescence-guided surgery (FGS). When administered orally, 5-ALA is converted by glioma cells into protoporphyrin IX (PPIX), which fluoresces under blue 400-nm light. 5-ALA has been available for use in Europe since 2010, but only recently gained FDA approval as an intraoperative imaging agent for HGG tissue. In this first-ever, to the authors' knowledge, multicenter 5-ALA FGS study conducted in the United States, the primary objectives were the following: 1) assess the diagnostic accuracy of 5-ALA-induced PPIX fluorescence for HGG histopathology across diverse centers and surgeons; and 2) assess the safety profile of 5-ALA FGS, with particular attention to neurological morbidity. This single-arm, multicenter, prospective study included adults aged 18-80 years with Karnofsky Performance Status (KPS) score > 60 and an MRI diagnosis of suspected new or recurrent resectable HGG. Intraoperatively, 3-5 samples per tumor were taken and their fluorescence status was recorded by the surgeon. Specimens were submitted for histopathological analysis. Patients were followed for 6 weeks postoperatively for adverse events, changes in the neurological exam, and KPS score. Multivariate analyses were performed of the outcomes of KPS decline, EOR, and residual enhancing tumor volume to identify predictive patient and intraoperative variables. Sixty-nine patients underwent 5-ALA FGS, providing 275 tumor samples for analysis. PPIX fluorescence had a sensitivity of 96.5%, specificity of 29.4%, positive predictive value (PPV) for HGG histopathology of 95.4%, and diagnostic accuracy of 92.4%. Drug-related adverse events occurred at a rate of 22%. Serious adverse events due to intraoperative neurological injury, which may have resulted from FGS, occurred at a rate of 4.3%. There were 2 deaths unrelated to FGS. Compared to preoperative KPS scores, postoperative KPS scores were significantly lower at 48 hours and 2 weeks but were not different at 6 weeks postoperatively. Complete resection of enhancing tumor occurred in 51.9% of patients. Smaller preoperative tumor volume and use of intraoperative MRI predicted lower residual tumor volume. PPIX fluorescence, as judged by the surgeon, has a high sensitivity and PPV for HGG. 5-ALA was well tolerated in terms of drug-related adverse events, and its application by trained surgeons in FGS for HGGs was not associated with any excess neurological morbidity.

213 5-Aminolevulinic Acid for Enhanced Surgical Visualization of High-Grade Gliomas: A Prospective, Multicenter Study

INTRODUCTION: Greater extent of resection is associated with longer overall survival in patients with high grade gliomas (HGG). 5-aminolevulinic acid (5-ALA) can increase EOR by improving intraoperative visualization of contrast-enhancing tumor during fluorescence-guided surgery (FGS). 5-ALA is converted by glioma cells into protoporphyrin IX (PPIX), which fluoresces under blue light. 5-ALA has been available for use in Europe since 2010, but only recently gained FDA approval as an intraoperative agent for HGG tissue. METHODS: This single-arm, prospective study included adults aged 18-80 with Karnofsky performance status (KPS)>60 and an MRI diagnosis of suspected new or recurrent resectable HGG. Intraoperatively, 3-5 samples per tumor were taken and their fluorescence status recorded by the surgeon. Patients were followed for six weeks postoperatively for adverse events, changes in the neurological exam, and KPS. We performed multivariate analyses of the outcomes of KPS decline, EOR, and residual enhancing tumor volume to identify predictive patient and intraoperative variables. RESULTS: Sixty-nine patients underwent 5-ALA FGS, providing 275 tumor samples for analysis. PPIX fluorescence had a sensitivity of 96.5%, specificity of 29.4%, positive predictive value (PPV) for HGG of 95.4%, and diagnostic accuracy of 92.4%. Drug-related adverse events occurred at a rate of 22%. Serious adverse events occurred at a rate of 4.3%. There were 2 unrelated deaths. KPS was significantly lower at 48 hours and 2 weeks, but was not different at 6 weeks postoperatively. Complete resection of enhancing tumor occurred in 51.9% of patients. Smaller preoperative tumor volume and use of intraoperative MRI predicted lower residual tumor volume. CONCLUSION: PPIX fluorescence, as judged by the surgeon, has a high sensitivity and PPV for HGG. 5-ALA was well-tolerated, and its application by trained surgeons in FGS for HGGs was not associated with any excess neurological morbidity.

Contemporary intraoperative visualization for GBM with use of exoscope, 5-ALA fluorescence-guided surgery and tractography.

Maximal safe resection is the primary goal of glioma surgery. By incorporating improved intraoperative visualization with the 3D exoscope combined with 5-ALA fluorescence, in addition to neuronavigation and diffusion tensor imaging (DTI) fiber tracking, the safety of resection of tumors in eloquent brain regions can be maximized. This video highlights some of the various intraoperative adjuncts used in brain tumor surgery for high-grade glioma.In this case, the authors highlight the resection of a left posterior temporal lobe high-grade glioma in a 33-year-old patient, who initially presented with seizures, word-finding difficulty, and right-sided weakness. They demonstrate the multiple surgical adjuncts used both before and during surgical resection, and how multiple adjuncts can be effectively orchestrated to make surgery in eloquent brain areas safer for patients. Patient consent was obtained for publication.The video can be found here: https://stream.cadmore.media/r10.3171/2021.10.FOCVID21174

Standardized intraoperative 5-ALA photodynamic therapy for newly diagnosed glioblastoma patients: a preliminary analysis of the INDYGO clinical trial

Glioblastoma (GBM) is the most aggressive malignant primary brain tumor. The unfavorable prognosis despite maximal therapy relates to high propensity for recurrence. Thus, overall survival (OS) is quite limited and local failure remains the fundamental problem. Here, we present a safety and feasibility trial after treating GBM intraoperatively by photodynamic therapy (PDT) after 5-aminolevulinic acid (5-ALA) administration and maximal resection. Ten patients with newly diagnosed GBM were enrolled and treated between May 2017 and June 2018. The standardized therapeutic approach included maximal resection (near total or gross total tumor resection (GTR)) guided by 5-ALA fluorescence-guided surgery (FGS), followed by intraoperative PDT. Postoperatively, patients underwent adjuvant therapy (Stupp protocol). Follow-up included clinical examinations and brain MR imaging was performed every 3months until tumor progression and/or death. There were no unacceptable or unexpected toxicities or serious adverse effects. At the time of the interim analysis, the actuarial 12-months progression-free survival (PFS) rate was 60% (median 17.1months), and the actuarial 12-months OS rate was 80% (median 23.1months). This trial assessed the feasibility and the safety of intraoperative 5-ALA PDT as a novel approach for treating GBM after maximal tumor resection. The current standard of care remains microsurgical resection whenever feasible, followed by adjuvant therapy (Stupp protocol). We postulate that PDT delivered immediately after resection as an add-on therapy of this primary brain cancer is safe and may help to decrease the recurrence risk by targeting residual tumor cells in the resection cavity. Trial registration NCT number: NCT03048240. EudraCT number: 2016-002706-39.

Heterogeneity Matters: Different Regions of Glioblastoma Are Characterized by Distinctive Tumor-Supporting Pathways.

Simple Summary5-ALA Fluorescence Guided Surgery aims at extending the boundaries of glioblastoma (GBM) resection. It is based on the use of a fluorescent dye, 5-aminolevulinic acid (5-ALA). Depending on the fluorescence levels, it is possible to distinguish the core of the tumor, the infiltrating borders and the healthy tissue. Since GBM progression is supported by tumor cells and their interaction with the surrounding microenvironment, we hypothesized that 5-ALA intensity could identify microenvironments with different tumor supporting properties. Taking advantage of glioma-associated stem cells; a human in vitro model of the glioma microenvironment, we demonstrate that all regions of the tumor support the tumor growth, but through different pathways. This study highlights the importance of understanding the TME to obtain key information on GBM biology and develop new therapeutic approaches.The glioblastoma microenvironment plays a substantial role in glioma biology. However, few studies have investigated its spatial heterogeneity. Exploiting 5-ALA Fluorescence Guided Surgery (FGS), we were able to distinguish between the tumor core (ALA+), infiltrating area (ALA-PALE) and healthy tissue (ALA−) of the glioblastoma, based on the level of accumulated fluorescence. The aim of this study was to investigate the properties of the microenvironments associated with these regions. For this purpose, we isolated glioma-associated stem cells (GASC), resident in the glioma microenvironment, from ALA+, ALA-PALE and ALA− samples and compared them in terms of growth kinetic, phenotype and for the expression of 84 genes associated with cancer inflammation and immunity. Differentially expressed genes were correlated with transcriptomic datasets from TCGA/GTEX. Our results show that GASC derived from the three distinct regions, despite a similar phenotype, were characterized by different transcriptomic profiles. Moreover, we identified a GASC-based genetic signature predictive of overall survival and disease-free survival. This signature, highly expressed in ALA+ GASC, was also well represented in ALA PALE GASC. 5-ALA FGS allowed to underline the heterogeneity of the glioma microenvironments. Deepening knowledge of these differences can contribute to develop new adjuvant therapies targeting the crosstalk between tumor and its supporting microenvironment.

Improved Diagnostic Imaging of Brain Tumors by Multimodal Microscopy and Deep Learning.

Fluorescence-guided surgery is a state-of-the-art approach for intraoperative imaging during neurosurgical removal of tumor tissue. While the visualization of high-grade gliomas is reliable, lower grade glioma often lack visible fluorescence signals. Here, we present a hybrid prototype combining visible light optical coherence microscopy (OCM) and high-resolution fluorescence imaging for assessment of brain tumor samples acquired by 5-aminolevulinic acid (5-ALA) fluorescence-guided surgery. OCM provides high-resolution information of the inherent tissue scattering and absorption properties of tissue. We here explore quantitative attenuation coefficients derived from volumetric OCM intensity data and quantitative high-resolution 5-ALA fluorescence as potential biomarkers for tissue malignancy including otherwise difficult-to-assess low-grade glioma. We validate our findings against the gold standard histology and use attenuation and fluorescence intensity measures to differentiate between tumor core, infiltrative zone and adjacent brain tissue. Using large field-of-view scans acquired by a near-infrared swept-source optical coherence tomography setup, we provide initial assessments of tumor heterogeneity. Finally, we use cross-sectional OCM images to train a convolutional neural network that discriminates tumor from non-tumor tissue with an accuracy of 97%. Collectively, the present hybrid approach offers potential to translate into an in vivo imaging setup for substantially improved intraoperative guidance of brain tumor surgeries.

5-Aminolevulinic Acid–Guided Surgery for Recurrent Supratentorial Pediatric Neoplasms

The use of 5-aminolevulinic acid (5-ALA) in pediatric neuro-oncology is considered off-label, and little data are available on its use in tumor recurrence surgery. Here we present our experience with 5-ALA fluorescence-guided surgery for recurrent supratentorial tumors in the pediatric population. Eleven pediatric patients presenting with recurrence of a supratentorial high-grade malignancy (5 glioblastoma [GBM], 6 non-GBM) underwent 5-ALA-assisted surgery. Biopsy specimens were obtained from pathological and normal-appearing areas of the tumor margin. From the margin of the tumor displaying solid fluorescence, a total of 36 samples were obtained. All of these histological samples were found to harbor tumor cells. From areas of vague enhancement, a total of 49 histological samples were taken, of which 38 samples (77%) harbored tumor cells. There was no significant difference in the percentage of biopsy-positive vague fluorescent areas between the GBM cases (80%) and non-GBM cases (75%). A total of 59 biopsy specimens were taken from the tumor margin that appeared completely negative for fluorescence. On analysis, 24 (40.7%) of these specimens demonstrated tumor cells. There was no significant difference in the number of false-negative biopsies between the GBM group (40%) and the non-GBM group (41%). The positive predictive value of solid fluorescence is high in recurrent disease but is substantially lower in areas of vague fluorescence. The rate of false-negative fluorescence is high. 5-ALA should be considered as an adjuvant in revision surgery with the aforementioned caveats in mind.

Spatial heterogeneity of cell-matrix adhesive forces predicts human glioblastoma migration.

BackgroundGlioblastoma (GBM) is a highly aggressive incurable brain tumor. The main cause of mortality in GBM patients is the invasive rim of cells migrating away from the main tumor mass and invading healthy parts of the brain. Although the motion is driven by forces, our current understanding of the physical factors involved in glioma infiltration remains limited. This study aims to investigate the adhesion properties within and between patients’ tumors on a cellular level and test whether these properties correlate with cell migration.MethodsSix tissue samples were taken from spatially separated sections during 5-aminolevulinic acid (5-ALA) fluorescence-guided surgery. Navigated biopsy samples were collected from strongly fluorescent tumor cores, a weak fluorescent tumor rim, and nonfluorescent tumor margins. A microfluidics device was built to induce controlled shear forces to detach cells from monolayer cultures. Cells were cultured on low modulus polydimethylsiloxane representative of the stiffness of brain tissue. Cell migration and morphology were then obtained using time-lapse microscopy.ResultsGBM cell populations from different tumor fractions of the same patient exhibited different migratory and adhesive behaviors. These differences were associated with sampling location and amount of 5-ALA fluorescence. Cells derived from weak- and nonfluorescent tumor tissue were smaller, adhered less well, and migrated quicker than cells derived from strongly fluorescent tumor mass.ConclusionsGBM tumors are biomechanically heterogeneous. Selecting multiple populations and broad location sampling are therefore important to consider for drug testing.

TMIC-09. MULTIMODAL VISIBLE LIGHT OPTICAL COHERENCE MICROSCOPY AND FLUORESCENCE IMAGING OF GLIOBLASTOMA REGIONAL SAMPLES

Abstract Optical coherence tomography (OCT) is an imaging modality based on the inherent backscattering of light within different tissue types. OCT was introduced in the early 1990s and has since become a standard diagnostic tool in ophthalmology. Images can be acquired non-destructively, in real time and three dimensions at micrometer resolution. Only recently, OCT has been increasingly recognized in other fields such as neuroimaging. Here we present a multimodal imaging approach using a custom-built visible light optical coherence microscope (OCM) combined with a fluorescence imaging mode for the evaluation of different tumor compartments in glioblastoma (GB) samples retrieved during 5-aminolevulinic acid (5-ALA) fluorescence-guided surgery. 18 biopsies of ten GB patients were imaged using the visible light OCM system, providing the three-dimensional morphologic structure of the tissue on a cellular level (axial resolution in brain tissue 0.88 µm, penetration depth 100 µm). Attenuation coefficients, i.e., indicators for light penetration and scattering, were calculated for each sample. Tumor-specific contrast enhanced by 5-ALA was evaluated in co-registered images from the fluorescence channel. Samples were ultimately processed for histopathologic work-up and compared to OCM findings. Three different groups of biopsies could be defined based on quantitative 5-ALA fluorescence [normalized between 0 and 1], attenuation [mm-1] and histological H&E stainings: tumor core (n=8; fluorescence [median ± standard deviation]=0.72±0.13, attenuation [median ± standard deviation]=3.9±0.66), infiltration zone (n=6; fluorescence=0.5±0.19, attenuation=4.4±0.5), and adjacent brain parenchyma (n=4; fluorescence=0.3±0.12, attenuation=5.0±0.79). Concurrent increase in fluorescence intensity and cell density was significantly associated with tissue malignancy (tumor core: 3849±1028 nuclei/mm²; brain parenchyma: 1364±236 nuclei/mm². p=0.024). Furthermore, a negative correlation between fluorescence and attenuation coefficient was detected (r=0.51, p=0.032). Aforementioned results suggest that this multimodal imaging setup is a promising approach for non-destructively investigating the three-dimensional morphologic structure at microscopic resolution whilst at the same time leveraging tumor-specific contrast through 5-ALA fluorescence.

SURG-05. EXPERIENCE PROFILING OF FLUORESCENCE-GUIDED SURGERY FOR GLIOMAS

Abstract Numerous studies reported a usefulness of 5-ALA fluorescence-guided surgery (FGS) in high grade gliomas. However, fluorescence pattern and intensity is variable among gliomas. In this study, we report our extensive experiences of FGS in various gliomas focusing on epidemiological data of fluorescence pattern. A total of 827 histologically proven glioma patients out of 900 brain tumor patients who had undergone FGS using 5-ALA during the period of 8.5 years between July 2010 and January 2019 were analyzed. Indication for FGS in glioma surgery harbored any evidence of possible high-grade foci at presumed gliomas in preoperative magnetic resonance images (MRI). Among the 827 gliomas, the number of cases corresponding to 2016 World Health Organization (WHO) grade IV, III, II, and I are 528 (58.7%), 193 (21.4%), 87 (9.7%) and 19 (2.1%), respectively. In terms of fluorescence rate, grade IV gliomas showed positive fluorescence in 95.4% of cases including strong intensity in 85.6%. Grade III gliomas showed fluorescence in about half of cases (55.0%), while 45.0% of cases did not show any fluorescence. Anaplastic oligodendroglioma had more positive rate (63.9%) than anaplastic astrocytoma (46.2%). Both grade II and I gliomas still showed positive fluorescence in about one-fourths of cases (24.1% and 26.3%, respectively). Among them ependymoma and pilocytic astrocytoma were fluorescence-prone tumors. This epidemiological data of 5-ALA fluorescence in various grades of gliomas provides fundamental reference to clinical application of FGS using 5-ALA in glioma surgery.

Novel Exoscope System for 5-ALA Fluorescence-Guided Surgery for Gliomas

Novel Exoscope System for 5-ALA Fluorescence-Guided Surgery for Gliomas

Quantification of PpIX-fluorescence of cerebral metastases: a pilot study.

5-ALA fluorescence-guided surgery (FGS) is a major advance in neuro-oncological surgery. So far, Protoporphyrin IX (PpIX)-fluorescence has been observed in about half of cerebral metastases resected with routinely equipped microscopes during 5-ALA FGS. The aim of the present pilot study was to quantify PpIX-induced fluorescence of cerebral metastases with a spectrometer. We hypothesize that non-fluorescing metastases under the operating microscope may have spectrometrically measurable levels of fluorescence. A second aim was to analyze correlations between quantified 5-ALA fluorescence and histology or primary tumor type, respectively. Standard FGS was performed in all patients. The fluorescence intensity of the metastasis was semi-quantitatively determined in vivo by a senior surgeon using a special surgical microscope equipped for FGS. A systematic spectrometric ex vivo evaluation of tumor specimens and PpIX-induced fluorescence was performed using a spectrometer connected by optic fibers to a handheld probe. Quantification of 5-ALA-derived fluorescence was measured in a standardized manner with direct contact between mini-spectrometer and metastasis. The difference between the maximum PpIX-fluorescence at 635nm and the baseline fluorescence was defined as the PpIX fluorescence intensity of the metastasis and given in arbitrary units (AU). Diagnosis of a cerebral metastasis was confirmed by histopathological analysis. A total of 29 patients with cerebral metastases were included. According to neuropathological analysis, 11 patients suffered from non-small cell lung cancer, 10 patients from breast cancer, 6 patients from cancer originating in the gastro-intestinal tract, 1 patient suffered from a malignant melanoma and one patient from renal cancer. The mean age was 63years (37-81years). 15 patients were female, 14 patients male. 13 cerebral metastases were considered as ALA-positive by the surgeon. In nine metastases, 5-ALA fluorescence was not visible to the naked eye and could only be detected using the spectrometer. The threshold for an ALA signal rated as "positive" by the surgeon was PpIX fluorescence above 1.1 × 106 AU. The mean PpIX fluorescence of all analyzed cerebral metastases was 1.29 × 106 ± 0.23 × 106 AU. After quantification, we observed a significant difference between the mean 5-ALA-derived fluorescence in NSCLC and breast cancer metastases (Mean Diff: - 1.2 × 106; 95% CI of difference: - 2.2 × 106 to - 0.15 × 106; Šidák-adjusted p = 0.026). In our present pilot series, about half of cerebral metastases showed a 5-ALA fluorescence invisible to the naked eye. Over 50% of these non-fluorescent metastases show a residual 5-ALA fluorescence which can be detected and quantified using a spectrometer. Moreover, the quantified 5-ALA signal significantly differed with respect to the primary tumor of the corresponding cerebral metastasis. Further studies should evaluate the predictive value of the 5-ALA signal and if a quantified 5-ALA signal enables a reliable intraoperative differentiation between residual tumor tissue and edematous brain-in particular in metastases with a residual fluorescence signal invisible to the naked eye.

Identification of PEPT2 as an important candidate molecule in 5-ALA-mediated fluorescence-guided surgery in WHO grade II/III gliomas.

5-aminolevulinic acid (5-ALA) fluorescence-guided surgery (FGS) appears to be a promising treatment for glioma. However, 5-ALA-mediated fluorescence cannot always be detected in grade II/III gliomas. We hypothesized that gene expression patterns in the Protoporphyrin IX (PpIX) synthesis pathway may be associated with intraoperative fluorescence status of grade II/III gliomas, and then attempted to identify the key molecule of 5-ALA-mediated fluorescence. Using 50 surgically obtained specimens, which were diagnosed as grade II and III gliomas, we analyzed gene expression within the PpIX synthesis pathway to identify candidate molecules according to intraoperative 5-ALA-mediated fluorescence status. The most likely candidate gene was selected and confirmed by protein expression analysis. To evaluate the biological function of the molecule in PpIX synthesis, functional analysis was performed using specific, small interference (si)RNA in the SW-1783human grade III glioma cell line. Among the genes involved in the porphyrin synthesis pathway, the mRNA expression of Peptide transporter 2 (PEPT2) in FGS fluorescence-positive gliomas was significantly higher than that in fluorescence-negative gliomas. Protein expression of PEPT2 was also significantly higher in the fluorescence-positive gliomas, which was confirmed by western blot analysis and immunofluorescence analysis. The siRNA-mediated downregulation of the mRNA and protein expression of PEPT2 led to decreased PpIX fluorescence intensity, as confirmed by fluorescence spectrum analysis. The results suggest PEPT2 is an important candidate molecule in 5-ALA-mediated FGS in grade II/III gliomas. As the overexpression of PEPT2 was associated with higher PpIX fluorescence intensity, PEPT2 may improve fluorescence-guided resection in grade II/III gliomas.