Assessing Intraoperative Tumor-to-Background Ratios Across Different Subsites of the Oral Cavity Using an...

Background/Objectives: Intraoperative fluorescence imaging (FI) with tumor-targeted tracers offers a promising approach to improve surgical precision in cancer surgery. cRGD-ZW800-1, an integrin-targeted fluorescent tracer, has previously demonstrated safety, tumor specificity, and utility in detecting inadequate margins in oral cancer. During this study, we observed variability in background fluorescence between different subsites of the oral cavity. Therefore, this study aimed to systematically evaluate intraoperative in vivo and ex vivo mucosal contrast ratios across various oral cavity subsites using FI with cRGD-ZW800-1. Methods: Thirty-one patients with oral squamous cell carcinoma underwent intraoperative FI following intravenous injection of cRGD-ZW800-1 at least 18 h preoperatively. In vivo imaging was performed using the Quest Spectrum platform. In addition, ex vivo FI of the resected specimen was performed using the Pearl Trilogy Small Animal Imaging System. As these ex vivo images were obtained under uniform and controlled acquisition conditions, they allow for direct comparison with the intraoperative fluorescence signals. Fluorescence intensities and tumor-to-background ratios (TBRs) were assessed per oral subsite using manually drawn regions of interest (ROIs) on the tumor and adjacent healthy mucosa using Quest's Spectrum Software, version 4.8.2, (in vivo images) and the Pearl's integrated software ImageStudio version 6.2 (ex vivo images). A TBR ≥ 1.5 was considered sufficient. Results: Under uniform imaging settings, all samples exhibited adequate contrast (TBR ≥ 2.3), allowing clear tumor visualization and precise evaluation of mucosal margins on final histopathology. Notably, intraoperative in vivo contrast in the posterior located maxillary alveolar process was comparatively lower, which was attributable to suboptimal imaging conditions and subsite-specific background fluorescence. Conclusions: Our findings indicate that, although contrast varies across different oral subsites, all specimens exhibited sufficient ex vivo mucosal contrast to allow reliable tumor delineation. As in vivo imaging may be affected by subsite-specific background fluorescence and inherent limitations of intraoperative imaging geometry, fluorescence signals should be interpreted in conjunction with standard visual and tactile assessment. Due to anatomical constraints, different oral subsites may appear within the same field of view, which can influence perceived signal intensity. Therefore, intraoperative ex vivo fluorescence evaluation is recommended for signal interpretation.