Contact immunofluorescence: a novel methodology for intraoperative ex vivo margin assessment using fluorescent EGFR-targeting nanobodies

Background
In surgical oncology, obtaining negative resection margins remains one of the key prognostic factors in a multitude of tumor types. Recent advancements like fluorescence-guided surgery (FGS) have revolutionized intraoperative margin assessment. However, current clinical trials implementing FGS display a lack in sensitivity, specificity and usability with the currently used molecular tracers. To address this gap, we propose a novel guidance tool utilizing a fluorescently labeled nanobody (Nb) targeting the tumor-upregulated epidermal growth factor receptor (EGFR). This tool aims to help the surgeon in identifying tumoral tissue with high sensitivity and specificity on excised malignancies during surgery. In this study, we optimized the protocol, resulting in a clinically relevant and applicable tool.
Material and methods
Patients undergoing tumorectomy for a malignancy of the head and neck (n=6) or breast (n=7) were included. Freshly excised specimens with tumoral tissue and surrounding control tissue were cut into small samples and incubated in a solution of 50-200nM of the EGFR-targeting Nb 7D12 labeled with the fluorophore s775z, alone or combined with a blocking solution containing a 100x molar excess of unlabeled Nb. The incubated samples were then washed in fresh PBS and imaged using a flatbed fluorescent imaging scanner, followed by standard histopathological processing. Multiple incubation and washing times were tested, ranging from 4 hours to 5 minutes. Additional EGFR-immunohistochemistry was performed to correlate fluorescence with EGFR-expression.
Results (For Surgical Trial Proposals fill in 'the Feasibility', for Surgical Trial in Progress fill in the 'Current status')
A higher fluorescent signal was observed in the tumoral tissue compared to its surrounding healthy tissue with an average tumor-to-background ratio (TBR) of 2,49 (±0,45) when incubated for 10’ at a concentration of 150 nM. Histological analysis of the tissue slices confirmed a correlation of histology and fluorescence, displaying the potential of this methodology in correctly identifying tumoral tissue, and hence in identifying positive resection margins.
Conclusions (For Surgical Trial in Progress and Surgical Trial Proposals fill in 'NA')
We here display a novel methodology allowing the intraoperative imaging of specific targets using fluorescent Nbs. While this study displays the tool’s potential in targeting EGFR, Nbs can be developed against almost all cell surface-expressed proteins, allowing endless flexibility in deciding which target to use for each individual cancer type. In addition, no tracer needs to be administered to the patient, requiring no GMP-production of the product nor expensive medicinal clinical trials. Consequently, this method could help in identifying positive resection margins in nearly all tumor types, overcoming the current difficulties of the currently used frozen sections.