Safety and Tumor Specificity of Cetuximab-IRDye800 for Surgical Navigation in Head and Neck Cancer

Abstract

Purpose: Positive margins dominate clinical outcomes after surgical resections in most solid cancer types, including head and neck squamous cell carcinoma. Unfortunately, surgeons remove cancer in the same manner they have for a century with complete dependence on subjective tissue changes to identify cancer in the operating room. To effect change, we hypothesize that EGFR can be targeted for safe and specific real-time localization of cancer.

Experimental design: A dose escalation study of cetuximab conjugated to IRDye800 was performed in patients (n = 12) undergoing surgical resection of squamous cell carcinoma arising in the head and neck. Safety and pharmacokinetic data were obtained out to 30 days after infusion. Multi-instrument fluorescence imaging was performed in the operating room and in surgical pathology.

Results: There were no grade 2 or higher adverse events attributable to cetuximab-IRDye800. Fluorescence imaging with an intraoperative, wide-field device successfully differentiated tumor from normal tissue during resection with an average tumor-to-background ratio of 5.2 in the highest dose range. Optical imaging identified opportunity for more precise identification of tumor during the surgical procedure and during the pathologic analysis of tissues ex vivo. Fluorescence levels positively correlated with EGFR levels.

Conclusions: We demonstrate for the first time that commercially available antibodies can be fluorescently labeled and safely administered to humans to identify cancer with sub-millimeter resolution, which has the potential to improve outcomes in clinical oncology.

Conflict of interest statement

The authors report no conflict of interest.

©2015 American Association for Cancer Research.

Figures

Fig. 1

Trial imaging workflow. Real-time imaging was performed with a wide-field near-infrared imaging system in the clinic on (1) Day 0, 1, and in the (2) operating room on day 3 post cetuximab-IRDye800 infusion. (3) During post-resection processing, resected tissues were imaged with a closed-field near-infrared imaging system. (4) Following histologic preparation, a corresponding slide was imaged in surgical pathology using a fluorescence scanning system.

Fig. 2

Quantification of wide-field fluorescence imaging. Relative fluorescent units (RFU) acquired during wide-field fluorescent imaging of tumor, background and tumor-to-background ratio (TBR) are shown for (a) 2.5mg/m2 cohort, (b) 25mg/m2 cohort, and (c) 62.5mg/m2 cohort. White light and fluorescence images acquired using wide-field imaging device are shown for (d) 2.5mg/m2 cohort, (e) 25mg/m2 cohort, and (f) 62.5mg/m2 cohort. Asterisk denotes significant (p<0.05) increase in tumor RFU compared to background for respective day. Data are RFU and TBR ± SD.

Fig. 3

Intraoperative fluorescence imaging. Shown are (A,D) grayscale fluorescence, (B,E) color map fluorescence, and (C,F) corresponding brightfield acquired using the wide-field device prior to primary tumor resection from patients in the 25mg/m2 dose group undergoing a total glossectomy (A–C) and a wide local excision of the buccal mucosa (D–F).

Fig. 4

Correlation of fluorescence and disease margin. Wide-field fluorescence (A) and brightfield (B) image are shown of resected primary tumor. Gridlines represent whole tissue (4–5mm) sections cut during pathological processing of specimen. Breadloaf sections were fluorescently imaged using closed-field imaging device, then formalin-fixed, sectioned, and H&E stained. Black outline represents tumor deposition as determined by board-certified pathologist. Fluorescent images of whole tissue sections and corresponding H&E stained sections are oriented with mucosal side at top.

Fig. 5

Fluorescent imaging of resected tissue. (A) Mean fluorescent intensity (MFI) are shown for freshly resected tumor, margin, skin, and muscle for the 2.5mg/m2, 25mg/m2, 62.5mg/m2 dose groups acquired using closed-field imaging system. Representative bright field and fluorescence images of tumor, margins, skin, and muscle are shown for the (B–E) 2.5mg/m2 dose group, (F–I) 25mg/m2 dose group, and (J–M) 62.5mg/m2 dose group. Image scaling was kept consistent for representative images at each dose. Asterisk denotes significant (P<0.05) increase in tumor MFI compared to margins, skin, and muscle for respective dose. Scale bars represent 0.5cm or 0.25cm. Data are average MFI ± SD.

Fig. 6

Histopathology and fluorescence localization. (A) Representative H&E image of tumor sections from the 25mg/m2 dose group. Pathology-positive areas of cancer are outlined: T=Tumor, N=Normal adjacent tissue. (B) Fluorescence image of fixed, unstained tumor section shown in panel (A) acquired using fluorescent scanner with inset-magnified area of pathology-positive cancer and (C) EGFR stain. (D) Mean fluorescent intensity (MFI) acquired using fluorescent scanning of fixed unstained tissue sections is shown for tumor, normal adjacent, and muscle. Data are average MFI ± SE. Asterisk denotes significant (P<0.001) increase in tumor MFI compared to normal and muscle for respective dose.