Use of monoclonal antibody-IRDye800CW bioconjugates in the resection of breast cancer

Abstract

Background: Complete surgical resection of breast cancer is a powerful determinant of patient outcome, and failure to achieve negative margins results in reoperation in between 30% and 60% of patients. We hypothesize that repurposing Food and Drug Administration-approved antibodies as tumor-targeting diagnostic molecules can function as optical contrast agents to identify the boundaries of malignant tissue intraoperatively.

Materials and methods: The monoclonal antibodies bevacizumab, cetuximab, panitumumab, trastuzumab, and tocilizumab were covalently linked to a near-infrared fluorescence probe (IRDye800CW) and in vitro binding assays were performed to confirm ligand-specific binding. Nude mice bearing human breast cancer flank tumors were intravenously injected with the antibody-IRDye800 bioconjugates and imaged over time. Tumor resections were performed using the SPY and Pearl Impulse systems, and the presence or absence of tumor was confirmed by conventional and fluorescence histology.

Results: Tumor was distinguishable from normal tissue using both SPY and Pearl systems, with both platforms being able to detect tumor as small as 0.5 mg. Serial surgical resections demonstrated that real-time fluorescence can differentiate subclinical segments of disease. Pathologic examination of samples by conventional and optical histology using the Odyssey scanner confirmed that the bioconjugates were specific for tumor cells and allowed accurate differentiation of malignant areas from normal tissue.

Conclusions: Human breast cancer tumors can be imaged in vivo with multiple optical imaging platforms using near-infrared fluorescently labeled antibodies. These data support additional preclinical investigations for improving the surgical resection of malignancies with the goal of eventual clinical translation.

Keywords: Antibody; Breast cancer; Fluorescence; Near-infrared; Optical imaging.

Copyright © 2014 Elsevier Inc. All rights reserved.

Figures

Fig. 1

(A) Western blot of 2LMP receptor expression. EGFR was the most strongly expressed within the 2LMP cell line. VEGF showed moderate expression, HER2/neu had low-level expression, and IL-6R expression was not demonstrated. (B–F) Antigen binding assays. Nonlinear regression curves demonstrating preservation of antigen specificity after conjugation to IRDye800 for bevacizumab (Avastin), anti-VEGF (B); cetuximab (Erbitux), anti-EGFR (C); panitumumab (Vectibix), anti-EGFR (D); tocilizumab (Actemra), anti-IL-6R (E); and trastuzumab (Herceptin), anti-HER2/neu (F).

Fig. 2

(A–F) Daily imaging of tumors. 2LMP flank tumors were imaged daily with both SPY and Pearl modalities. TBRs peaked at 6–9 d on SPY and 13–16 d on Pearl. SPY and Pearl images from the day of peak TBR on each modality are shown adjacent to the TBR graphs for the antibody-IRDye800 bioconjugates. Cetuximab and panitumumab provided the highest TBR. Tocilizumab provided the TBR similar to control IgG. Error bars indicate the standard deviation. (Color version of figure is available online.)

Fig. 3

(A) Uptake of antibody-IRDye800 within 2LMP flank tumors at the time of resection. Surgical resections were performed in real-time with SPY and confirmed with Pearl. (B and C) TBRs for resected tumors on SPY and Pearl. Cetuximab and panitumumab gave the highest TBR on both modalities. Error bars indicate the standard error. *Significance at P

Fig. 4

Traditional hematoxylin and eosin and fluorescent microscopy of 2LMP tumors and normal tissue (muscle). Odyssey images permit distinction between neoplastic tissue and normal tissue based on the fluorescence intensity. Fluorescent microscopy images (x60) show the pattern of antibodye-IRDye800 uptake within the tumors compared with normal tissue. (Color version of figure is available online.)

Fig. 5

(A) Serial tumor resection with panitumumab-IRDye800. Tumor was weighed, imaged, and resected in half until it could no longer be grossly divided. Panitumumab-IRDye800 allowed detection of the smallest fragment of tumor (0.5 mg). Note that tumor was imaged within the tumor bed to account for background fluorescence. (B) TBR of tumor sections by SPY and Pearl. Fluorescence decreased with decreasing tumor size, but was still greater than background with only 0.5 mg of tumor remaining. (Color version of figure is available online.)