11 Fluorescein and High-Grade Gliomas

The ability of 5-aminolevulinic acid (5-ALA) to provide safe and accurate, real-time identification of diffusely infiltrative glioblastoma (GBM) tumors, with associated improvements in extent of resection (EOR), has encouraged the investigation of additional fluorescent agents with similar capabilities.1 This is particularly important as safe, maximal resection is widely accepted to be an independent predictor of prognosis in high-grade gliomas (HGGs).^2,3,4,5

Fluorescein sodium is a green fluorescent compound that accumulates in areas of malignancy, vascular leaking defects, pooling defects, and abnormal vasculature or neovascularization.⁶ Originally described as a method for intraoperative guidance during intracranial tumor resection in 1947 by George E. Moore,⁶ the use of fluorescein has recently received renewed interest as its accumulation in regions of blood–brain barrier (BBB) breakdown can be used as an intraoperative, real-time method of accurately labeling tumor for resection.⁷

Compared with 5-ALA, fluorescein is inexpensive, easy to use, and associated with minimal side effects.⁸ Furthermore, the recent introduction of operative microscopes fitted with fluorescein-specific filters has facilitated improved intraoperative visualization of fluorescein-stained tissue at lower doses than traditionally used, and, as a result, has accelerated interest in its use for the resection of HGGs with promising results.^{9,10,11,12,13,14,15,16,17,18,19} However, definitive studies clearly outlining the benefits of fluorescein have not been performed.

This chapter will review the presumed mechanism of action of fluorescein for labeling tumor, specific technical considerations of its use, evidence supporting its use in HGGs, and potential future applications of fluorescein guidance for the resection of HGGs.

11.2 Molecular Mechanism of Fluorescein Staining in HGG

Fluorescein sodium (NaC₂₀H₁₀Na₂O₅; molecular weight = 376 g/mol) is a salt form of the synthetic organic fluorophore fluorescein.⁸ Fluorescein’s peak absorption spectrum occurs at 465 to 490 nm with an emission peak at 500 to 530 nm permitting autofluorescence in white light and easy observation by the naked eye.^9,15 In contrast to 5-ALA, which is metabolized within tumor cells into the fluorescent byproduct protoporphyrin IX (PpIX),⁴ intravenously administered fluorescein is delivered systemically through the bloodstream where it nonpreferentially extravasates into regions of increased vascular permeability. Fluorescein then accumulates in the extracellular spaces of these regions of BBB breakdown caused by central nervous system (CNS) diseases such as HGGs.⁷ Interestingly, while normal astrocytes have been shown to demonstrate some degree of intracellular fluorescein uptake, tumor cells have not.¹² This exclusion of fluorescein from glioma cells is believed to be the result of upregulation of organic anion efflux transporters such as multidrug resistance protein 1 (MRP1), of which fluorescein has been shown to be a substrate.^20,21,22

Following the intravenous administration of fluorescein, circulating fluorescein is rapidly cleared from regions of normal healthy brain with intact vasculature. The resultant fluorescein-mediated identification of HGGs is derived from the differential identification of fluorescent drug in the extracellular space surrounding glioma pathology as compared with regions with a functional BBB ( Fig. 11.1).

11.3 Technical Considerations

The timing and dose of fluorescein administration remains nonstandardized, and the length of time before physical factors such as oxidation and drug metabolism affect fluorescence intensity remains poorly defined. Of particular concern is the length of time during which fluorescein is preferentially retained in pathologic tissues before it diffuses through and indiscriminately labels edematous peritumoral tissues, which also remains unknown.²³ The importance of timing is relevant as early surgical manipulation of normal tissue immediately following administration of intravenous fluorescein results in messy extravasation of the fluorescent dye if sufficient time for clearance by normal tissue is not provided.^24,25

Currently, the most accepted protocol is to administer fluorescein following induction of anesthesia, just prior to incision, as this appears to allow excellent contrast between pathologic and normal tissue while avoiding extravasation of dye from surgically manipulated normal capillary beds in the period of time it takes an experienced surgeon to perform a craniotomy and expose a tumor.^{11,12,14,24,25,26} Using this schedule, the sensitivity, specificity, and positive predictive value of fluorescein for identifying malignant glioma tissue is retained throughout a 1- to 4-hour window postinjection and suggests that the administration of fluorescein prior to incision permits sufficient time for drug clearance from normal brain while permitting adequate extravasation within pathologic tissue and avoidance of unintentional extravasation through interrupted capillary beds.14 Unfortunately, the effects of prolonged surgical procedures on the accuracy of fluorescein remain to be determined.

Fig. 11.1 Intraoperative appearance of fluorescein under white light (a) and YELLOW 560 (b) during resection of GBM in patient treated with 3 mg/kg fluorescein following anesthesia induction. (c) Intraoperative neuronavigation correlates to region of fluorescence staining. (d) Representative tumor pathology from fluorescent region.

The optimal dose of fluorescein also remains poorly defined. Initial studies utilizing fluorescein relied on identification of tissue staining under operative white light with direct visualization of dyed tissue, and required higher doses of intravenous fluorescein up to 20 mg/kg. These higher doses are associated with common side effects such as nausea, vomiting, and urticaria in 0.01 to 9.24% of patients according to published series and serious adverse reactions such as bronchospasm and laryngeal edema occurring rarely.15,16,17,27,28,29 Additionally, seizures have been reported with intrathecal fluorescein injection when used for identifying cerebrospinal fluid (CSF) leaks and therefore may be possible with extracellular extravasation of fluorescein into CSF spaces.^30,31,32

The subsequent development and wide adoption of specialized microscopes with fluorescent filters that allow easy switching between white-light and fluorescence modes has permitted a reduction in dose to between 2 and 5 mg/kg, with subsequent reduced risk of dose-related adverse reactions. These doses permit identification of fluorescein staining that is imperceptible under white light.^24,25 Furthermore, available fluorescein filters are bright enough to provide adequate visualization of anatomy under fluorescent illumination, thus allowing the surgeon to perform tumor resection under the filter without the absolute need to switch to visible light.¹⁴

Despite advances in microscopy, there is currently no standardized way to view fluorescein. While fluorescent microscopy has facilitated lower doses and improved discrimination of fluorescent tissue, the ability to visualize fluorescein-stained tissue using white-light illumination is appealing, particularly in settings with limited resources. Even more variability exists when considering fluorescence microscopy. Various fluorescent filters have become commercially available, for example, the YELLOW 560 system (Carl Zeiss) or the FL560 System (Leica Microsystems), with variable wavelength absorption resulting in a lack of uniformity between existing and future studies. As a result, findings may not be generalizable depending on the fluorescence system used.

11.4 The Influence of Fluorescein-Guided Resection on Extent of Resection and Outcomes in High-Grade Glioma

Safe, maximal tumor resection improves symptoms, quality of life, progression-free survival (PFS), and overall survival (OS) in HGGs.⁴ Given the success of 5-ALA for improving EOR, a number of studies have attempted to use fluorescein to improve EOR in malignant gliomas.^{4,9,10,12,13,14,15,16,17,18,19}

The first modern studies describing fluorescein-guided resection of HGGs predated the popularization of intraoperative fluorescence microscopy ( Table 11.1).^15,16,17,18 These studies utilized high doses of intravenous fluorescein and relied on the ability to identify fluorescein-stained tissues under white light. In general, these studies all confirmed that fluorescein-guided resection of grossly visible, stained tissue under white-light illumination improved the surgeon’s ability to completely resect regions of contrast-enhancing tumor as compared with conventional microsurgical techniques, with rates of gross total resection (GTR) ranging from 80 to 100% in regions amenable to GTR.^15,16,17,18

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