Fluorescence 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. In this study, a machine learning-based optomics analysis, which interprets the textural pattern differences in EGFR expression conveyed by fluorescence, was applied to optical ABY-029 fluorescence image data of HNSCC surgical specimens. The study objective was to determine the correlations between optomics method classification performance and tissue inherent EGFR expression level. Fluorescence image data were collected through a Phase 0 clinical trial of ABY-029, which 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 from 12 patients who were stratified into three dose groups (30, 90, and 171 nanomoles). The optomics approach utilized a supervised machine learning pipeline. 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 optomic 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 classifier. A conceptual framework of correlation analysis to evaluate the relationship between optomics tumor classification performance and underlying EGFR expression level was provided, but the present results are underpowered. Some generalized conclusions about the ABY-029 fluorescence optomics method correlating to varied levels of EGFR expression were summarized, suggesting that optomics method using fluorescence molecular imaging data offers a potentially stable image analysis technique for cancer detection for fluorescence-guided surgery applications; however, further study with additional samples is needed to validate this conclusion.
ABY-029, an anti-epidermal growth factor receptor (EGFR) Affibody molecule labeled with IRDye 800CW, has been used in three Phase 0 microdosing clinical trials for fluorescence guided surgery. In May of 2019, the clinical trials were put on hold because the ABY-029 produced under Good Manufacturing Practices (GMP) for human administration had come to the end of term in which the drug product was known to be stable. Stability testing was halted due to limitations in supply of a suitable reference standard and a required test product being discontinued from commercial sale. In order to complete the remaining patients in the three clinical trials, new stability tests were developed and the GMP batch of ABY- 029 drug product tested under the new protocols. The GMP batch of ABY-029 passed all stability tests under the new protocols and the Federal Drug Administration (FDA) has given permission to complete the remaining patients with stability testing of ABY-029 performed for each patient. The tests developed and used to test ABY-029 drug product stability are described here.
Near-infrared (NIR) fluorescence combined with targeting epidermal growth factor receptor (EGFR) overexpression for surgical guidance in many cancers is gaining momentum. ABY-029 is an anti-EGFR Affibody molecule conjugated to IRDye 800CW that is FDA approved as an exploratory Investigational New Drug (eIND 122681). ABY-029 has a short plasma half-life (~15-20 minutes), which allows for administration of the imaging agent and excision surgery to occur on the same day unlike fluorescent antibodies. This fast tissue clearance may provide the means necessary to achieve clinically relevant tumor-to-normal tissue contrast levels using microdosing administration schemes. Pre-clinical studies have indicated that tumor-to-normal tissue contrast peaks between 4-8 hours depending on EGFR expression. Additionally, the No Observable Adverse Effect Level (NOAEL) was determined in pre-clinical toxicity studies to be 1,000X the microdose, or 30 micromole, whereas mild adverse events are common in antibody imaging studies. A number of promising first-in-human clinical Phase 0 trial microdose evaluation of ABY-029 have been initiated for recurrent glioma, soft-tissue sarcoma, and head and neck cancers (NCT0290925, NCT03154411, and NCT03282461, respectively). Here, we provide an update on our experience using ABY-029 for surgical resection at microdose levels and describe tissue contrast and correlation of ABY-029 fluorescence to EGFR tissue expression. Current progress indicates that moderate TBRs (~3-5) are observable at the microdose administration level but increased administration doses (3X and 6X microdoses) are being investigated. In addition, ex vivo tissue fluorescence is highly correlated to EGFR staining in both intensity and spatial localization.
Many tumors for which fluorescence guided surgery (FGS) has been developed are surface tumors, where direct visualization by the surgeon is straightforward. On the other hand, cancers such as soft-tissue sarcomas, are present at a subsurface level. Resection of these sub-surface tumors is performed using ‘wide local excision’ where a single, complete mass is removed with an intact zone of normal tissue (~ 1 cm ‘margin’). We used a phantom model for sarcoma with near infrared fluorophore IRDye800 CW that defined different tissue properties. We compare the detection sensitivity of two commercially available near infrared (NIR) surgical imaging systems, Solaris (Perkin Elmer) and SPY PHI (Novadaq) using the phantom models of sarcoma. We also determine targeted fluorescence signal on both systems for blinded surgical phantom dissection by a surgeon. The fluorescence intensities are higher for Solaris than for SPY-PHI. On average, the fluorescence increased with an increase in intralipid concentration and decreased with an increase in blood concentration. The depth of imaging was higher for Solaris than for SPY PHI. Using the target values, the surgeon successfully dissected all phantoms using Solaris. Using fat phantoms for SPY PHI, the surgeon cut through four out of the total. Further improvement in FGS will improve cancer recurrence and morbidity.
Fluorescence guidance systems are used for different indications, and the relevant range of concentrations and the relevant dye used varies considerably for human clinical trials. Most systems are designed for high concentration ICG imaging, where 0.1 mg/kg is injected IV and perfusion is imaged, yet other applications such as delayed uptake (or second temporal window) ICG imaging can have tissue concentrations an order of magnitude or more below this. Additionally, IRDye800 is used in both antibody imaging and small protein imaging in clinical trials, but injected doses vary from therapeutic to microdose levels. In this study, the sensitivity to both perfusion dose and microdose ranges were tested for ICG and IRDye800CW, using the Spy Elite system (Stryker) and the Solaris (Perkin Elmer). The sensitivity to ICG was significantly different and the sensitivity to IRDye800 was also different but with opposite choice of the optimal system. The concentrations tested ranged from 0.1 mg/kg down to 0.1 ug/kg. The signal to background limited the sensitivity in both cases, and the ambient light effects were significantly different in the two systems. The in vivo testing in lymphatic and vascular transport was assessed to determine limits to detection for vessel size.
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