Dual-tracer fluorescence imaging and surgical resection of metastatic lymph nodes in breast cancer: sensitivity, specificity, and first-in-human results

Abstract Contrast-enhanced fluorescence imaging is under intense research and development for real-time molecular imaging, tumor margin delineation, and lymph node mapping during surgery. However, current fluorescent tracers and imaging devices cannot reliably differentiate between metastatic and nonmetastatic lymph nodes. Here we report a dual-tracer approach based on the synergistic use of two fluorescent tracers for highly sensitive and specific detection of metastatic lymph nodes in breast cancer. The fluorescent tracers are indocyanine green (ICG), a tumor-agnostic dye that emits fluorescent light in the near-infrared spectrum, and 5-aminolevulinic acid (5-ALA), a co-factor in the biosynthesis of a natural fluorophore called protoporphyrin-9 (Pp-IX). These tracers have distinct and complementary mechanisms for seeking out permeabilized microstructures and metabolically active cells in the heterogenous environments of lymphatic nodes. When analyzed using 2D Youden’s statistics, we find that the dual-tracer approach provides a major improvement over traditional single-tracer/single-color methods. For both animal xenograft models and human breast cancer patients, we have achieved nearly 100% sensitivity in detecting metastatic lymph nodes, while conserving 81.3% and 89.7% of histologically negative lymph nodes in animal models and in human patients, respectively. Pathological staining and microscopic imaging data further reveal that each tracer has distinct localization patterns in detecting metabolically active tumor cells, necrotic regions, and altered lymphatic microstructures, and that these patterns are strongly correlated with the progression of lymph node metastasis. Graphical Abstract