Procedures
tumor, ®, Concomitant neck dissections, head and neck pathologist, maxillary carcinoma
SENTINEL NODE, STERILE, TUMOR, TONGUE TUMOR
Clinical-grade cetuximab-800CW was produced at the Good Manufacturing Practice licensed facilities at the University Medical Centre Groningen. Briefly, commercially available cetuximab (Erbitux®) 5 mg/mL was conjugated to IRDye800CW NHS Ester (LI-COR Biosciences, Lincoln, NE, USA) under regulated conditions with a dye:antibody ratio of 2:1. The solution was purified using PD-10 buffer at 1 mg/ml exchange columns (GE Healthcare, Chicago, IL, USA). Cetuximab-800CW was formulated in a sodium-phosphate buffer at 1 mg/mL concentration and sterile filled into injection vialsPatients received the study drugs intravenously, preceded by 2 mg clemastine according to standard of care cetuximab treatment. We administered 75 mg of unlabeled cetuximab to prevent rapid plasma clearance and occupy off-target receptors, followed by 15 mg cetuximab-800CW after 1 hTwo days after tracer administration, surgery was performed according to standard of care in our hospital. Routinely, CT and/or MRI were preoperatively available, on which the primary resection was planned. The tumor was removed with an estimated clinical margin of 1 cm. Fresh frozen sectioning of the margins was not performed routinely. Concomitant neck dissections or sentinel node biopsies were performed based on cTN classification, according to Dutch guidelines. In the case of clinical suspicion of an irradical resection intraoperatively, an extra resection was performed and attached to the primary specimen in the same setting. The tumor was removed with a margin of clinically uninvolved tissue, taken approximately 1 cm based on visual and tactile information to aim for a histopathological margin of >5 mm. The SurgVision Explorer Air® (SurgVision GmbH, Munich, Germany) was used for in vivo imaging and benchmarked using a fluorescence phantom prior to surgeryThe freshly excised surgical specimen was imaged in what is referred to as the ‘back table phase’. Ex vivo, specimen-driven imaging allows for controllable imaging parameters, resulting in consistent and reproducible images which were used for intraoperative margin analysisImaging was performed intraoperatively immediately after resection. For reliable fluorescence imaging results, the camera was placed perpendicular to the tissue of interest. All surgical planes containing a deep margin were imaged; therefore, the number of acquired images per specimen depended on the size and complexity of the specimen. For instance, a small tongue tumor specimen would result in one image containing a deep margin, and a more complex resection of a maxillary carcinoma could result in five (Fig. All images were scaled to the maximum fluorescence intensity observed in the tumor of each patient, as variations in tumor biology exist with each patient. A region of interest was drawn around each identified fluorescent spot on the excised specimen. A background region of interest was drawn on the same fluorescence image that included adjacent tissue of the same origin (e.g., connective tissue, muscle) without the fluorescent spot. A signal-to-background ratio (SBR) was calculated by dividing the mean fluorescence intensity of the spot by the mean fluorescence intensity of the background. When a fluorescent spot was identified with only one of the devices, the SBR of this fluorescent spot was used for analysis. In case a fluorescent spot was found using both imaging devices, the highest SBR (i.e., from either the Pearl-Trilogy® or the SurgVision Explorer Air®) was used for definitive analysis to make sure that no at-risk margins were missed. If no fluorescent spot was identified in the margin, the SBR was set at one. Fluorescence imaging and analysis were performed in approximately 5 min, depending on specimen size and complexity.After completing the fluorescence imaging protocol, the surgical specimen was submitted to the Department of Pathology and formalin-fixed for at least 24 h according to the standard of care. The formalin-fixed specimen was inked for orientation purposes and serially sliced into 3–4 mm thick tissue slices. All tissue slices were imaged in the Pearl-Trilogy® to obtain cross-sectional fluorescence images of the tumor that allow for correlation with final histopathology. After paraffin embedding, a 4 µm section was cut from each tissue slice and stained with hematoxylin & eosin (H&E). A head and neck pathologist, blinded for fluorescence imaging results, delineated the tumor and determined the tumor margins in each 4 µm tissue section, according to the standard of care. The final margin status was classified as tumor-positive (<1 mm), close (1–5 mm) or tumor-negative (≥ 5 mm), as defined by the Royal College of Pathologists