J. Fletcher1, J. Warram1, Y. Hartman1, E. De Boer1, E. Rosenthal1 1University Of Alabama School Of Medicine,Otolaryngology,Birmingham, ALABAMA, USA
Introduction: Fluorescent-based techniques are being introduced to guide surgical excision of cancer. Antibodies can be covalently conjugated to near-infrared (NIR) dyes to permit real-time, optical localization of cancer in the surgical setting. Successful antibody-dye combinations are not identified by total tumor accumulation, but by the greatest difference between tumor and normal tissue. While this strategy has the potential to achieve complete resection, the accurate characterization of study drugs is essential to understanding binding kinetics of antibody-NIR dye candidates for this technique.
Methods: Using tissues obtained from a dose-escalation clinical trial assessing the safety of cetuximab conjugated to a NIR dye (cetuximab-IRDYe800) in patients with head and neck cancer, a novel methodology was explored to normalize for optical-based attenuation to accurately quantify drug uptake within tissues. Tumor and muscle (n=4) specimens were systematically homogenized and a SDS-PAGE assay was performed on cell lysate (40ug) from each sample. In addition, a serial dilution (0.02ug-0.1ng) of cetuximab-IRDye800 was run on a separate gel to serve as a standard. Using a specialized NIR fluorescent scanner designed to image IRDye800 (Odyssey, LICOR, Lincoln, NE), the gels were imaged and mean fluorescent intensity (MFI) from cetuximab-IRDye800 (150kd) bands were quantified in tumor and patient-matched muscle. MFI were compared to the standard curve to determine percentage of cetuximab-IRDye800 injected dose per gram of tissue (%ID/g).
Results: In the lowest cetuximab-IRDye800 dose group, average %ID/g for tumor (2.8×10-6 MFI) was found to be 8-fold greater than %ID/g for muscle (3.5×10-7 MFI). In the higher dose group (2.5-fold dose increase), average %ID/g for tumor (3.2×10-6 MFI) was found to be 3-fold greater than %ID/g for muscle (1.0×10-6 MFI).
Conclusion: Using this technique, the lower cetuximab-IRDye800 dose was shown to be optimal to provide the greatest difference between tumor and muscle tissue.