Development of an intraoperative margin assessment method for breast cancer using a fluorescence probe targeting α-mannosidase: A multicenter study

Abstract Background Accurate intraoperative margin assessment is essential to minimize reoperation in breast-conserving surgery (BCS) for breast cancer (BC). This multicenter study aimed to evaluate a novel fluorescent probe targeting α-mannosidase, referred to as the α-Man probe, as a rapid and reliable alternative to frozen section analysis, which remains the standard technique for intraoperative margin assessment. Methods A total of 274 resected breast tissue samples, including malignant and benign lesions, were analyzed across four institutions. Fluorescence intensity (FI) was measured 5 and 20 min following the application of the α-Man probe. Receiver operating characteristic analysis defined definitive thresholds for changes in FI (ΔFI), maintaining false positive and false negative rates at ≤ 3%. Diagnostic performance was also compared with a γ-glutamyltranspeptidase (GGT)-based fluorescent probe that we have previously developed. Results Using 5-min ΔFI thresholds, positive, uncertain, and negative classifications were 24.1, 53.6, and 22.3%, respectively. At 20 min, these classifications improved to 28.1, 38.3, and 33.6%, respectively. At 5 and 20 min, positive predictive values were 93.9% and 94.8%, whereas negative predictive values were 95.1% and 96.7%, respectively. Compared with the GGT probe, α-Man substantially reduced the uncertain classification rate while maintaining high predictive accuracy. Conclusion This multicenter study demonstrated that a two-step ΔFI-based protocol using the α-Man probe enables accurate, real-time, and pathologist-independent intraoperative margin assessment during BCS. This fluorescence-guided approach is broadly applicable and offers a practical, cost-effective alternative to conventional pathological evaluation. Registration number: jRCT1051240034