Background and Objectives Fluorescence‐guided surgery using epidermal growth factor receptor (EGFR) targeting has been performed successfully in clinical trials using a variety of fluorescent agents. We investigate ABY‐029 (anti‐EGFR Affibody® molecule labeled with IRDye 800CW) compared with a small‐molecule perfusion agent, IRDye 700DX carboxylate, in a panel of soft‐tissue sarcomas with varying levels of EGFR expression and vascularization. Methods Five xenograft soft‐tissue sarcoma cell lines were implanted into immunosuppressed mice. ABY‐029 and IRDye 700DX were each administered at 4.98 μM. Fluorescence from in vivo and ex vivo (fresh and formalin‐fixed) fixed tissues were compared. The performance of three fluorescence imaging systems was assessed for ex vivo tissues. Results ABY‐029 is retained longer within tumor tissue and achieves higher tumor‐to‐background ratios both in vivo and ex vivo than IRDye 700DX. ABY‐029 fluorescence is less susceptible to formalin fixation than IRDye 700DX, but both agents have disproportional signal loss in a variety of tissues. The Pearl Impulse provides the highest contrast‐to‐noise ratio, but all systems have individual advantages. Conclusions ABY‐029 demonstrates promise to assist in wide local excision of soft‐tissue sarcomas. Further clinical evaluation of in situ or freshly excised ex vivo tissues using fluorescence imaging systems is warranted.

Background: Current practices for fluorescence-guided cancer surgery utilize a single fluorescent agent, but homogeneous distribution throughout the tumor is difficult to achieve. We hypothesize that administering a perfusion and a moleculartargeted agent at their optimal administration-to-imaging time will improve wholetumor contrast. Experimental Design: Mice bearing subcutaneous xenograft human synovial sarcomas were administered indocyanine green (ICG) (3 mg/kg) or ABY-029 (48.7 μg/kg)-an epidermal growth factor receptor-targeted Affibody molecule-alone or in combination. Fluorescence contrast and signal distribution were compared between treatment groups. Two commercial fluorescence imaging systems were tested for simultaneous imaging of ICG and ABY-029. Results: ABY-029 has a moderate positive correlation with viable tumor (ρ = 0.2 ± 0.4), while ICG demonstrated a strong negative correlation (ρ = −0.6 ± 0.1). The contrast-to-variance ratio was highest in the ABY-029 +ICG (2.5 ± 0.8), compared to animals that received ABY-029 (2.3 ± 0.8) or ICG (2.0 ± 0.5) alone. Moreover, the combination of ABY-029 + ICG minimizes the correlation between viable tumor and fluorescence intensity (ρ = −0.1 ± 0.2) indicating the fluorescence signal distribution is more homogeneous throughout the tumor milieu. Conclusion: Dual-agent imaging utilizing a single channel in a commercial fluorescence-guided imaging system tailored for IRDye 800CW is a promising method to increase tumor contrast in a clinical setting.

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Microdose administration of ABY-029, an anti-epidermal growth factor receptor Affibody molecule conjugated to IRDye 800CW, is being studied in a Phase 0 trial for resection of soft-tissue sarcomas. The excised tissue of a single patient in the microdose administration group was imaged with both a wide-field fluorescence surgical system and a flat-bed scanning fluorescence imaging system. Here the resultant fluorescence from a breadloaf section of the primary tumor specimen and six region-specific tissue samples collected from that breadloaf are compared using these two imaging systems – a flatbed, black-box, fluorescence scanning system, the Odyssey CLx, and a open-air, wide-field, pre-clinical surgical imaging system, the Solaris. Florescence signal is compared using a variety of methods including: mean, standard deviation, variance, tumor-to-background ratio, biological-variance ratio, and contrast-to-noise ratio. The images produced from the Odyssey scanner have higher signal variance but more accurately represent the EGFR expression in small tissue sections. The Solaris system has higher depth sensitivity and volume averaging, and as such has lower signal variation and higher contrast-to-noise ratio.

BackgroundFluorescence molecular imaging using ABY-029, an epidermal growth factor receptor (EGFR)-targeted, synthetic Affibody peptide labeled with a near-infrared fluorophore, is under investigation for surgical guidance during head and neck squamous cell carcinoma (HNSCC) resection. However, tumor-to-normal tissue contrast is confounded by intrinsic physiological limitations of heterogeneous EGFR expression and non-specific agent uptake.ObjectiveIn this preliminary study, radiomic analysis was applied to optical ABY-029 fluorescence image data for HNSCC tissue classification through an approach termed “optomics.” Optomics was employed to improve tumor identification by leveraging textural pattern differences in EGFR expression conveyed by fluorescence. The study objective was to compare the performance of conventional fluorescence intensity thresholding and optomics for binary classification of malignant vs. non-malignant HNSCC tissues.Materials and MethodsFluorescence image data collected through a Phase 0 clinical trial of ABY-029 involved a total of 20,073 sub-image patches (size of 1.8 × 1.8 mm2) extracted from 24 bread-loafed slices of HNSCC surgical resections originating from 12 patients who were stratified into three dose groups (30, 90, and 171 nanomoles). Each dose group was randomly partitioned on the specimen-level 75%/25% into training/testing sets, then all training and testing sets were aggregated. A total of 1,472 standardized radiomic features were extracted from each patch and evaluated by minimum redundancy maximum relevance feature selection, and 25 top-ranked features were used to train a support vector machine (SVM) classifier. Predictive performance of the SVM classifier was compared to fluorescence intensity thresholding for classifying testing set image patches with histologically confirmed malignancy status.ResultsOptomics provided consistent improvement in prediction accuracy and false positive rate (FPR) and similar false negative rate (FNR) on all testing set slices, irrespective of dose, compared to fluorescence intensity thresholding (mean accuracies of 89% vs. 81%, P = 0.0072; mean FPRs of 12% vs. 21%, P = 0.0035; and mean FNRs of 13% vs. 17%, P = 0.35).ConclusionsOptomics outperformed conventional fluorescence intensity thresholding for tumor identification using sub-image patches as the unit of analysis. Optomics mitigate diagnostic uncertainties introduced through physiological variability, imaging agent dose, and inter-specimen biases of fluorescence molecular imaging by probing textural image information. This preliminary study provides a proof-of-concept that applying radiomics to fluorescence molecular imaging data offers a promising image analysis technique for cancer detection in fluorescence-guided surgery.