BackgroundMohs micrographic surgery is a procedure used for non-melanoma skin cancers that has 97-99% cure rates largely owing to 100% margin analysis enabled byen facesectioning with real-time, iterative histologic assessment. However, the technique is limited to small and aggressive tumors in high-risk areas because the histopathological preparation and assessment is very time intensive. To address this, paired-agent imaging (PAI) can be used to rapidly screen excised specimens and identify tumor positive margins for guided and more efficient microscopic evaluation.MethodsA mouse xenograft model of human squamous cell carcinoma (n= 8 mice, 13 tumors) underwent PAI. Targeted (ABY-029, anti-epidermal growth factor receptor (EGFR) affibody molecule) and untargeted (IRDye 680LT carboxylate) imaging agents were simultaneously injected 3-4 h prior to surgical tumor resection. Fluorescence imaging was performed on main, unprocessed excised specimens anden facemargins (tissue sections tangential to the deep margin surface). Binding potential (BP) – a quantity proportional to receptor concentration – and targeted fluorescence signal were measured for each, and respective mean and maximum values were analyzed to compare diagnostic ability and contrast. The BP and targeted fluorescence of the main specimen and margin samples were also correlated with EGFR immunohistochemistry (IHC).ResultsPAI consistently outperformed targeted fluorescence alone in terms of diagnostic ability and contrast-to-variance ratio (CVR). Mean and maximum measures of BP resulted in 100% accuracy, while mean and maximum targeted fluorescence signal offered 97% and 98% accuracy, respectively. Moreover, maximum BP had the greatest average CVR for both main specimen and margin samples (average 1.7 ± 0.4 times improvement over other measures). Fresh tissue margin imaging improved similarity with EGFR IHC volume estimates compared to main specimen imaging in line profile analysis; and margin BP specifically had the strongest concordance (average 3.6 ± 2.2 times improvement over other measures).ConclusionsPAI was able to reliably distinguish tumor from normal tissue in freshen facemargin samples using the single metric of maximum BP. This demonstrated the potential for PAI to act as a highly sensitive screening tool to eliminate the extra time wasted on real-time pathological assessment of low-risk margins.