Abstract Head and neck cancer (HNC) affects thousands globally, with high morbidity rates due to standard treatments like surgery and radiation. Photodynamic therapy (PDT) has shown great promise as a less destructive alternative, selectively targeting tumors while preserving healthy tissue. However, assessing treatment response in the days after PDT is challenging due to significant inflammation and the subsequent vascular shutdown of the tumor. We hypothesize that fluorescence paired‐agent imaging (PAI) can provide early molecular insights within 24 h of PDT to evaluate treatment efficacy. PAI utilizes two fluorescent agents to correct for perfusion‐related changes, allowing for accurate quantification of key signaling proteins. Specifically, we tracked epidermal growth factor receptor (EGFR) response to benzoporphyrin derivative monoacid (BPD)‐PDT at 690 nm, with light fluences ranging from 0 to 100 J/cm 2 . Twenty‐four hours post‐PDT, EGFR concentrations were measured using PAI with ABY‐029 and IRDye 680LT as targeted and untargeted agents, respectively. These findings were compared to histopathology (H&E and EGFR IHC). Our histological results demonstrated that EGFR expression increased with low PDT doses (10 and 25 J/cm 2 ) and decreased below baseline expression with higher doses (50 and 100 J/cm 2 ). Fluorescence intensity of both ABY‐029 and IRDye 680LT was highly variable with treatment dose and was not correlative to tumor response. In contrast, the PAI‐binding potential (BP) corresponded to the varying EGFR expression measured by pathology. In vivo and ex vivo PAI BP was moderately to highly correlative to percent area IHC EGFR expression ( r = 0.65 and 0.54, p < 0.05, respectively) and the in vivo 100 J/cm 2 treatment group demonstrated significantly lower BP than the controls. PAI emerges as a promising tool for tracking early molecular changes in HNC, with potential clinical applications.
Choudhury et al. (2026) studied this question.