Abstract 2055: Modulated near-infrared fluorescence light imaging of primary tumor margins, cancer positive lymph nodes, and freshly excised human cancers with imaging agent targeting EpCAM
Authors: Zhu Banghe , Harvey Barrett , Aldrich Melissa , Wu Grace , Wilganowski Nathaniel , Robinson Holly , Pinkston Kenneth , Peng Gao , Zhang Songlin , Karni Ron , Sevick-Muraca Eva M. 
Published: May 2015

Abstract
We developed an epithelial cancer-specific, imaging agent labeled for near-infrared florescence (NIRF) imaging together with an intraoperative surgical device for sensitive detection of tumor margins and local metastatic lesions within the surgical field of view. The drug/device combination was tested on an orthotopic model and on freshly resected, human cancerous tissues. To maximize device performance, we illuminated tissue surfaces with modulated near-infrared (NIR) excitation light (785 nm) and collected fluroescent light (830 nm) via using a NIR sensitive image intensifier modulated at with 90 degree phase delays added between 3 consecutively acquired images. These phase-delayed images were collected by a CCD and processed in near-real time to provide images of fluroescence amplitude with significantly higher contrast and without significant reduction in signal to noise ratio as compared to conventional images acquired without modulation. To develop a universal imaging agent for use with the device, a high affinity monoclonal antibody (mAb) against epithelial cell adhesion molecule (EpCAM) was selected from a previously generated panel and labeled with NIRF dye, IRDye800. The labeled mAb was characterized for conserved biological activity.
Upon dual-labeling this lead mAb candidate for both PET and NIRF, we showed the accuracy of planar NIRF imaging (96%) to be superior to PET (93%) for detecting cancer positive lymph nodes 24 hours following 40 ug i.v. injection in an orthotopic model of PC3 human metastatic prostate cancer 10-12 weeks post-implantation. Compared to NIRF labeled isotype control mAb, the labeled anti-EpCAM mAb accurately detected primary tumor margins as indicated by both pathology and far-red fluorescent protein reporter, iRFP (excitation 690 nm/ emission 720 nm), that was stably expressed in implanted PC3 cells. Furthermore, modulated light imaging provided greater accuracy in detecting margins compared to the widely used conventional fluorescent collection techniques. Upon immersing freshly excised human squamous cell carcinoma tissues from individual patients in dilute solutions of labeled anti-EpCAM and labeled isotype control and rinsing, we used the device to demonstrate consistent and specific binding of labeled anti-EpCAM mAb to cancer sites. In addition, cancer margins detected by NIRF were pathologically confirmed.
When combined with a high-affinity imaging agent targeting an antigen overexpressed on nearly all epithelial cancers, NIRF imaging can be used to accurate detect margins as well as cancer positive LNs. Finally, the modulated NIRF imaging can be deployed for open surgeries as well as endoscopic and robotic surgeries wherein surgical field illumination would otherwise destroy molecular imaging contrast.
Citation Format: Banghe Zhu, Barrett Harvey, Melissa Aldrich, Grace Wu, Nathaniel Wilganowski, Holly Robinson, Kenneth Pinkston, Gao Peng, Songlin Zhang, Ron Karni, Eva M. Sevick-Muraca. Modulated near-infrared fluorescence light imaging of primary tumor margins, cancer positive lymph nodes, and freshly excised human cancers with imaging agent targeting EpCAM. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2055. doi:10.1158/1538-7445.AM2014-2055