MRI and amino acid PET detection of whole-brain tumor burden.

Background: [18F]fluciclovine amino acid PET has shown promise for detecting brain tumor regions undetected on conventional anatomic MRI scans. However, it remains unclear which of these modalities provides a better assessment of the whole brain tumor burden. This study quantifies the performance of [18F]fluciclovine PET and MRI for detecting the whole brain tumor burden. Methods: Thirteen rats were orthotopically implanted with fluorescently transduced human glioblastoma cells. Rats underwent MRI (T1- and T2-weighted) and [18F]fluciclovine PET. Next brains were excised, optically cleared, and scanned ex vivo with fluorescence imaging. All images were co-registered using a novel landmark-based registration to enable a spatial comparison. The tumor burden identified on the fluorescent images was considered the ground truth for comparison with the in vivo imaging. Results: Across all cases, the PET sensitivity for detecting tumor burden (median 0.67) was not significantly different than MRI (combined T1+T2-weighted) sensitivity (median 0.61; p=0.85). However, the combined PET+MRI sensitivity (median 0.86) was significantly higher than MRI alone (41% higher; p=0.004) or PET alone (28% higher; p=0.0002). The specificity of combined PET+MRI (median=0.91) was significantly lower compared with MRI alone (6% lower; p=0.004) or PET alone (2% lower; p=0.002). Conclusion: In these glioblastoma xenografts, [18F]fluciclovine PET did not provide a significant increase in tumor burden detection relative to conventional anatomic MRI. However, a combined PET and MRI assessment did significantly improve detection sensitivity relative to either modality alone, suggesting potential value in a combined assessment for some tumors.

5-aminolevulinic acid, fluorescein sodium, and indocyanine green for glioma margin detection: analysis of operating wide-field and confocal microscopy in glioma models of various grades.

Introduction: Surgical resection remains the first-line treatment for gliomas. Several fluorescent dyes are currently in use to augment intraoperative tumor visualization, but information on their comparative effectiveness is lacking. We performed systematic assessment of fluorescein sodium (FNa), 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PpIX), and indocyanine green (ICG) fluorescence in various glioma models using advanced fluorescence imaging techniques. Methods: Four glioma models were used: GL261 (high-grade model), GB3 (low-grade model), and an in utero electroporation model with and without red fluorescence protein (IUE +RFP and IUE -RFP, respectively) (intermediate-to-low-grade model). Animals underwent 5-ALA, FNa, and ICG injections and craniectomy. Brain tissue samples underwent fluorescent imaging using a wide-field operative microscope and a benchtop confocal microscope and were submitted for histologic analysis. Results: Our systematic analysis showed that wide-field imaging of highly malignant gliomas is equally efficient with 5-ALA, FNa, and ICG, although FNa is associated with more false-positive staining of the normal brain. In low-grade gliomas, wide-field imaging cannot detect ICG staining, can detect FNa in only 50% of specimens, and is not sensitive enough for PpIX detection. With confocal imaging of low-intermediate grade glioma models, PpIX outperformed FNa. Discussion: Overall, compared to wide-field imaging, confocal microscopy significantly improved diagnostic accuracy and was better at detecting low concentrations of PpIX and FNa, resulting in improved tumor delineation. Neither PpIX, FNa, nor ICG delineated all tumor boundaries in studied tumor models, which emphasizes the need for novel visualization technologies and molecular probes to guide glioma resection. Simultaneous administration of 5-ALA and FNa with use of cellular-resolution imaging modalities may provide additional information for margin detection and may facilitate maximal glioma resection.

Imaging Glioblastoma With 18F-Fluciclovine Amino Acid Positron Emission Tomography.

INTRODUCTION: Conventional methods of imaging brain tumors fail to assess metabolically active tumor regions, which limits their capabilities for tumor detection, localization, and response assessment. Positron emission tomography (PET) with 18F-fluciclovine (fluciclovine) provides regional assessment of amino acid uptake in tumors that could overcome some of the limitations of conventional imaging. However, the biological basis of enhanced fluciclovine uptake is insufficiently characterized in brain tumors, which confounds clinical interpretation and application. This study sought to address this gap by correlating multiple biologic quantities with fluciclovine PET uptake across a range of human glioblastoma xenograft models. METHODS: Thirty-one rats underwent orthotopic implantations with one of five different human glioblastoma cell lines. After tumors were established, fluciclovine PET and magnetic resonance imaging (MRI) scans were performed. The fluciclovine tumor-to-normal-brain (TN) uptake ratio was used to quantify fluciclovine uptake. MRI scans were used to assess tumor volume and gadolinium enhancement status. Histologic assessments quantified tumor cell proliferation, tumor cell density, and tumor cell amino acid transporters (LAT1 and ASCT2). Multivariate linear regression models related fluciclovine uptake with the other measured quantities. RESULTS: Within the multivariate regression, the fluciclovine TN uptake ratio (measured 15 to 35 minutes after fluciclovine injection) was most strongly associated with tumor ASCT2 levels (ß=0.64; P=0.001). The fluciclovine TN uptake ratio was also significantly associated with tumor volume (ß=0.45; P=0.001) and tumor enhancement status (ß=0.40; P=0.01). Tumor cell proliferation, tumor cell density, and LAT1 levels were not significantly associated with fluciclovine uptake in any of the multivariate models. In general, both enhancing and non-enhancing tumors could be visualized on fluciclovine PET images, with the median TN uptake ratio across the five tumor lines being 2.4 (range 1.1 to 8.9). CONCLUSIONS: Increased fluciclovine PET uptake was associated with increased levels of the amino acid transporter ASCT2, suggesting fluciclovine PET may be useful for assessing brain tumor amino acid metabolism. Fluciclovine PET uptake was elevated in both enhancing and non-enhancing tumors but the degree of uptake was greater in larger tumors and tumors with enhancement, indicating these variables could confound fluciclovine metabolic measurements if not accounted for.

Correlation of Tumor Hypoxia Metrics Derived from 18F-Fluoromisonidazole Positron Emission Tomography and Pimonidazole Fluorescence Images of Optically Cleared Brain Tissue.

18F-fluoromisonidazole (FMISO) positron emission tomography (PET) is a widely used noninvasive imaging modality for assessing hypoxia. We describe the first spatial comparison of FMISO PET with an ex vivo reference standard for hypoxia across whole tumor volumes. Eighteen rats were orthotopically implanted with C6 or 9L brain tumors and made to undergo FMISO PET scanning. Whole brains were excised, sliced into 1-mm-thick sections, optically cleared, and fluorescently imaged for pimonidazole using an in vivo imaging system. FMISO maximum tumor uptake, maximum tumor-to-cerebellar uptake (TCmax), and hypoxic fraction (extracted 110 minutes after FMISO injection) were correlated with analogous metrics derived from pimonidazole fluorescence images. FMISO SUVmax was not significantly different between C6 and 9L brain tumors (P = .70), whereas FMISO TCmax and hypoxic fraction were significantly greater for C6 tumors (P < .01). FMISO TCmax was significantly correlated with the maximum tumor pimonidazole intensity (ρ = 0.76, P < .01), whereas FMISO SUVmax was not. FMISO tumor hypoxic fraction was significantly correlated with the pimonidazole-derived hypoxic fraction (ρ = 0.78, P < .01). Given that FMISO TCmax and tumor hypoxic fraction had strong correlations with the pimonidazole reference standard, these metrics may offer more reliable measures of tumor hypoxia than conventional PET uptake metrics (SUVmax). The voxel-wise correlation between FMISO uptake and pimonidazole intensity for a given tumor was strongly dependent on the tumor's TCmax (ρ = 0.81, P < .01) and hypoxic fraction (ρ = 0.85, P < .01), indicating PET measurements within individual voxels showed greater correlation with pimonidazole reference standard in tumors with greater hypoxia.

A practical method for multimodal registration and assessment of whole-brain disease burden using PET, MRI, and optical imaging.

Many neurological diseases present with substantial genetic and phenotypic heterogeneity, making assessment of these diseases challenging. This has led to ineffective treatments, significant morbidity, and high mortality rates for patients with neurological diseases, including brain cancers and neurodegenerative disorders. Improved understanding of this heterogeneity is necessary if more effective treatments are to be developed. We describe a new method to measure phenotypic heterogeneity across the whole rodent brain at multiple spatial scales. The method involves co-registration and localized comparison of in vivo radiologic images (e.g. MRI, PET) with ex vivo optical reporter images (e.g. labeled cells, molecular targets, microvasculature) of optically cleared tissue slices. Ex vivo fluorescent images of optically cleared pathology slices are acquired with a preclinical in vivo optical imaging system across the entire rodent brain in under five minutes, making this methodology practical and feasible for most preclinical imaging labs. The methodology is applied in various examples demonstrating how it might be used to cross-validate and compare in vivo radiologic imaging with ex vivo optical imaging techniques for assessing hypoxia, microvasculature, and tumor growth.

Progress in Confocal Laser Endomicroscopy for Neurosurgery and Technical Nuances for Brain Tumor Imaging With Fluorescein.

Background: Previous studies showed that confocal laser endomicroscopy (CLE) images of brain tumors acquired by a first-generation (Gen1) CLE system using fluorescein sodium (FNa) contrast yielded a diagnostic accuracy similar to frozen surgical sections and histologic analysis. We investigated performance improvements of a second-generation (Gen2) CLE system designed specifically for neurosurgical use. Methods: Rodent glioma models were used for in vivo and rapid ex vivo CLE imaging. FNa and 5-aminolevulinic acid were used as contrast agents. Gen1 and Gen2 CLE images were compared to distinguish cytoarchitectural features of tumor mass and margin and surrounding and normal brain regions. We assessed imaging parameters (gain, laser power, brightness, scanning speed, imaging depth, and Z-stack [3D image acquisition]) and evaluated optimal values for better neurosurgical imaging performance with Gen2. Results: Efficacy of Gen1 and Gen2 was similar in identifying normal brain tissue, vasculature, and tumor cells in masses or at margins. Gen2 had smaller field of view, but higher image resolution, and sharper, clearer images. Other advantages of the Gen2 were auto-brightness correction, user interface, image metadata handling, and image transfer. CLE imaging with FNa allowed identification of nuclear and cytoplasmic contours in tumor cells. Injection of higher dosages of FNa (20 and 40 mg/kg vs. 0.1-8 mg/kg) resulted in better image clarity and structural identification. When used with 5-aminolevulinic acid, CLE was not able to detect individual glioma cells labeled with protoporphyrin IX, but overall fluorescence intensity was higher (p < 0.01) than in the normal hemisphere. Gen2 Z-stack imaging allowed a unique 3D image volume presentation through the focal depth. Conclusion: Compared with Gen1, advantages of Gen2 CLE included a more responsive and intuitive user interface, collection of metadata with each image, automatic Z-stack imaging, sharper images, and a sterile sheath. Shortcomings of Gen2 were a slightly slower maximal imaging speed and smaller field of view. Optimal Gen2 imaging parameters to visualize brain tumor cytoarchitecture with FNa as a fluorescent contrast were defined to aid further neurosurgical clinical in vivo and rapid ex vivo use. Further validation of the Gen2 CLE for microscopic visualization and diagnosis of brain tumors is ongoing.