Abstract / Summary
Abstract Antibody delivery to solid tumors is often assumed to be uniform, yet this has not been systematically evaluated in human tissues. Optically labeled antibodies such as panitumumab-IRDye800CW (Pan800) remain detectable in FFPE specimens, enabling direct assessment of in vivo drug distribution. We aimed to validate macroscopic (21–337 μm/pixel) fluorescence measurements against microscopy (0.3 μm/pixel) and to characterize multi-scale heterogeneity of Pan800-derived fluorescence using a high-throughput, slide-free fluorescence imaging approach. A total of 946 FFPE blocks from seventeen patients who received systemic Pan800 were analyzed using near-infrared fluorescence imaging. Selected blocks were sectioned for fluorescence microscopy validation. Fluorescence intensities were compared between cancer-positive and -negative blocks, decalcified versus non-decalcified specimens, and across blocks within and between patients. Across resolutions (21–337 μm), maximum fluorescence intensity (FIₘₐₓ) discriminated cancer-positive from -negative blocks, while low-resolution scanning enabled rapid acquisition (~ 1 min/block). Macroscopic fluorescence signals correlated with microscopy at the section level (R 2 = 0.73), within patients (R 2 = 0.66), and across patients (R 2 = 0.66). Decalcification significantly reduced fluorescence at the block level (median FI max : 875 vs 287; P < 0.0001), and this reduction was confirmed in patient-level paired analyses when stratified by cancer status (P < 0.05). After excluding decalcified samples, FIₘₐₓ successfully differentiated cancer-positive from cancer-negative blocks (AUC = 0.78), with higher accuracy in lymph nodes than primary tumors. Marked intra- and inter-patient heterogeneity in Pan800-derived fluorescence was observed. Macroscopic fluorescence correlates with microscopic measurements and pathologically confirmed cancer presence, validating a high-throughput method for Pan800 distribution in clinical specimens. Signal heterogeneity precludes inference from single sections, supporting multi-block evaluation and a hierarchical workflow integrating screening with targeted microscopy. Clinical trial registration: NCT#04511078, first posted on 08/12/2020, https://clinicaltrials.gov/study/NCT04511078 ; NCT#05945875, first posted on 07/14/2023, https://clinicaltrials.gov/study/NCT05945875 .