A Protease-Activated Fluorescent Probe Allows Rapid Visualization of Keratinocyte Carcinoma during Excision

Keratinocyte carcinomas, including basal and squamous cell carcinomas, are the most common human cancers worldwide. While 75% of all keratinocyte carcinoma (4 million annual cases in the United States) are treated with conventional excision, this surgical modality has much lower cure rates than Mohs micrographic surgery, likely due to the bread-loaf histopathologic assessment that visualizes <1% of the tissue margins. A quenched protease-activated fluorescent probe 6qcNIR, which produces a signal only in the protease-rich tumor microenvironment, was topically applied to 90 specimens ex vivo immediately following excision. Puzzle-fit analysis was used to correlate the fluorescent images with histology. Probe-dependent fluorescent images correlated with cancer determined by conventional histology. Point-of-care fluorescent detection of skin cancer had a clinically relevant sensitivity of 0.73 and corresponding specificity of 0.88. Importantly, clinicians were effectively trained to read fluorescent images within 15 minutes with reliability and confidence, resulting in sensitivities of 62%-78% and specificities of 92%-97%. Fluorescent imaging using 6qcNIR allows 100% tumor margin assessment by generating en face images that correlate with histology and may be used to overcome the limitations of conventional bread-loaf histology. The utility of 6qcNIR was validated in a busy real-world clinical setting, and clinicians were trained to effectively read fluorescent margins with a short guided instruction, highlighting clinical adaptability. When used in conventional excision, this approach may result in higher cure rates at a lower cost by allowing sameday reexcision when needed, reducing patient anxiety and improving compliance by expediting postsurgical specimen assessment. Significance: A fluorescent-probe-tumor-visualization platform was developed and validated in human keratinocyte carcinoma excision specimens that may provide simple, rapid, and global assessment ofmargins during skin cancer excision, allowing same-day reexcision when needed.