Introduction: Fluorescence-guided resection has emerged as a potential tool for defining the often obscure tumor-brain interface during glioblastoma tumor surgery. However while agents such as fluorescein and aminolevulinic acid have been shown to increase the extent of resection, these fluorophores may result in the fluorescence of the surrounding edematous brain. In this study, we have evaluated the specificity of epidermal growth factor conjugated fluorescein (EGF-FL) and have compared it to conventional fluorescein in vitro and in vivo.
Methods: In the in vitro study, U87 (low-EGFR expression) and U87-MG-EGFR (high-EGFR expression) cells were incubated with various concentrations of fluorescein and EGF-FL. In the in vivo study, athymic nude mice were intracranially implanted with U87-MG or U87-MG-EGFR cells. Mice were intravenously administered fluorescein sodium or EGF-FL and then sacrificed.
Results: Cellular fluorescence in U87 cells was highest following treatment with 5ug/mL of EGF-FL, with this fluorescence being significantly greater than that achieved by 15ug/mL of fluorescein (p=0.0014). Also treatment of U87-MG-EGFR cells with 0.5ug/mL of EGF-FL resulted in more cellular fluorescence than 15ug/mL of fluorescein at both 1 and 3 hours (p<0.0001). Evaluation of fluorescein tumor specificity revealed tumor tissue to account for only 68.4% and 59.1% of total fluorescence in low and high EGFR expressing tumors, respectively. Use of EGF-FL in U87 tumors revealed similar tumor specificity as fluorescein (70.9% vs. 68.4%, p=0.71). However, the use of EGF-FL in U87-MG-EGFR tumors revealed a tumor specificity of 93.2% which was significantly greater than that seen for fluorescein (p<0.0001).
Conclusions: EGF-FL is a highly specific fluorophore for the delineation of EGFR-expressing tumors and has comparable efficacy to conventional fluorescein in tumors with low EGFR expression. Due to the established safety profile of fluorescein and the ease of administration, EGF-fluorescein has direct clinical applicability for use in fluorescence-guided resections and requires further study.
Patient Care: Despite modern neurosurgical techniques, achieving maximal resection of glioblastoma (GBM) tumors continues to be a major challenge as tumor borders may be unclear and indistinguishable from the surrounding normal parenchyma. To better define the tumor-brain interface, the intraoperative use of fluorescent agents has emerged as a potential tool. While the most investigated agents including fluorescein and aminolevulinic acid have been shown to increase the rates of gross total resection, these fluorophores may result in the fluorescence of the surrounding edematous brain, potentially resulting in the resection of non-tumor brain tissue. In this study, we have investigated the ability of epidermal growth factor conjugated fluorescein (EGF-FL) to accumulate within GBM cells in vitro and its ability to delineate tumor boundaries in vivo. Additionally, we have compared EGF-FL to conventional fluorescein to evaluate their relative efficacy. This study has demonstrated targeted EGF-FL to be significantly more specific than fluorescein in EGFR-overexpressing tumors, with comparable efficacy in tumors with low EGFR expression. As a result, this agent may more accurately delineate the tumor boundary intraoperatively, allowing for more complete resections which have been shown to result in improved survival. Additionally, as we have shown EGF-FL to specifically accumulate within GBM tumors after intravenous administration, we believe that this is an attractive vehicle for novel therapeutic agents. Currently investigated EGFR-specific agents are unable to adequately penetrate the brain, thereby requiring direct intracereberal administration via convection enhanced delivery (CED). As CED suffers from a variety of limitations including the procedural invasiveness, and unequal and unpredictable intratumoral agent distribution, the creation of easily administered and tumor-specific drugs via conjugation to ligands such as EGF provides an increasingly attractive therapeutic strategy. In conclusion, we believe that this study has demonstrated EGF-FL to be a clinically useful agent for fluorescence-guided resections and has highlighted the potential for ligands to serve as carriers for therapeutic agents.
Learning Objectives: By the conclusion of this session, participants should be able to: 1) Describe the utility of tumor-specific intraoperative fluorescent agents, 2) Discuss, in small groups, the efficacy and limitations of currently used fluorophores, 3) Understand the future implications of the current study.