Surgical excision is the primary treatment for solid tumors in oral squamous cell carcinomas, where achieving a healthy tissue margin of >5 mm is the goal. However, current clinical methods of assessing surgical margins cannot provide assessment of the whole margins intraoperatively (while the patient is still on the operating table) and while recent intraoperative fluorescence-guided surgery approaches have shown promise for detected “positive” inadequate margins (<1 mm), they have had limited success in the detection of “close” inadequate margins (1-5 mm), in patients injected with cetuximab-IRDye 800CW prior to surgery. Here, a dual aperture fluorescence ratio (dAFR) approach presented previously by our group is expanded upon, where herein we present a version of the analysis where the measurements are normalized by a background signal. We compare this additional approach directly against a single aperture view fluorescence (sAF) and pathology measurements of margin thickness in specimens from five patients and a total 14 margin locations (1 positive, 7 close, and 6 clear margins). The area under the curve of the receiver operating characteristic, representing the ability to detect close compared to clear margins was found to be 1.0 and 0.6 using dAFR and sAF, respectively, with the improvements in dAFR being statistically significant (p < 0.01). We demonstrate that the addition of a background normalization can account for noise and low signal in narrow aperture images.
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