Tumor-specific uptake of fluorescent bevacizumab-IRDye800CW microdosing in patients with primary breast cancer:a phase I feasibility study

PURPOSE: to provide proof of principle of safety, breast tumor-specific uptake and positive tumor margin assessment of the systemically administered near-infrared fluorescent (NIRF) tracer bevacizumab-IRDye800CW targeting vascular endothelial growth factor (VEGF)-A in breast cancer patients. EXPERIMENTAL DESIGN: Twenty patients with primary invasive breast cancer eligible for primary surgery received 4.5 mg bevacizumab-IRDye800CW as intravenous bolus injection. Safety aspects were assessed as well as tracer uptake and tumor delineation during surgery and ex vivo in surgical specimens using an optical imaging system. Ex vivo multiplexed histopathology analyses were performed for evaluation of biodistribution of tracer uptake and co-registration of tumor tissue and healthy tissue. RESULTS: None of the patients experienced adverse events. Tracer levels in primary tumor tissue were higher compared to those in the tumor margin (P < 0.05) and healthy tissue (P < 0.0001). VEGF-A tumor levels also correlated with tracer levels (r = 0.63, P < 0.0002). All but one tumor showed specific tracer uptake. Two out of 20 surgically excised lumps contained microscopic positive margins detected ex vivo by fluorescent macro- and microscopy and confirmed at the cellular level. CONCLUSIONS: Our study shows that systemic administration of the bevacizumab-IRDye800CW tracer is safe for breast cancer guidance and confirms tumor and tumor-margin uptake as evaluated by a systematic validation methodology. The findings are a step towards a phase II dose-finding study aimed at in vivo margin assessment and point to a novel drug assessment tool that provides a detailed picture of drug distribution in tumor tissue

Towards clinically translatable NIR fluorescence molecular guidance for colonoscopy

White-light surveillance colonoscopy is the standard of care for the detection and removal of premalignant lesions to prevent colorectal cancer, and the main screening recommendation following treatment for recurrence detection. However, it lacks sufficient diagnostic yield, exhibits unacceptable adenoma miss-rates and is not capable of revealing functional and morphological information of the detected lesions. Fluorescence molecular guidance in the near-infrared (NIR) is expected to have outstanding relevance regarding early lesion detection and heterogeneity characterization within and among lesions in these interventional procedures. Thereby, superficial and sub-surface tissue biomarkers can be optimally visualized due to a minimization of tissue attenuation and autofluorescence by comparison with the visible, which simultaneously enhance tissue penetration and assure minimal background. At present, this potential is challenged by the difficulty associated with the clinical propagation of disease-specific contrast agents and the absence of a commercially available endoscope that is capable of acquiring wide-field, NIR fluorescence at video-rates. We propose two alternative flexible endoscopic fluorescence imaging methods, each based on a CE certified commercial, clinical grade endoscope, and the employment of an approved monoclonal antibody labeled with a clinically applicable NIR fluorophore. Pre-clinical validation of these two strategies that aim at bridging NIR fluorescence molecular guidance to clinical translation is demonstrated in this study. (C) 2013 Optical Society of Americ

Molecular Fluorescence Endoscopy Targeting Vascular Endothelial Growth Factor A for Improved Colorectal Polyp Detection

Small and flat adenomas are known to carry a high miss-rate during standard white-light endoscopy. Increased detection rate may be achieved by molecular fluorescence endoscopy with targeted near infrared (NIR) fluorescent tracers. The aim of this study was to validate vascular endothelial growth factor A (VEGF-A) and epidermal growth factor receptor (EGFR)-targeted fluorescent tracers during ex vivo colonoscopy with an NIR endoscopy platform. Methods: VEGF-A and EGFR expression was determined by immunohistochemistry on a large subset of human colorectal tissue samples-48 sessile serrated adenomas/polyps, 70 sporadic high-grade dysplastic adenomas, and 19 hyperplastic polyps and tissue derived from patients with Lynch syndrome-78 low-grade dysplastic adenomas, 57 high-grade dysplastic adenomas, and 31 colon cancer samples. To perform an ex vivo colonoscopy procedure, 14 mice with small intraperitoneal EGFR-positive HCT116(luc) tumors received intravenous bevacizumab-800CW (anti-VEGF-A), cetuximab-800CW (anti-EGFR), control tracer IgG-800CW, or sodium chloride. Three days later, 8 resected HCT116(luc) tumors (2-5 mm) were stitched into 1 freshly resected human colon specimen and followed by an ex vivo molecular fluorescence colonoscopy procedure. Results: Immunohistochemistry showed high VEGF-A expression in 79%-96% and high EGFR expression in 51%-69% of the colorectal lesions. Both targets were significantly overexpressed in the colorectal lesions, compared with the adjacent normal colon crypts. During ex vivo molecular fluorescence endoscopy, all tumors could clearly be delineated for both bevacizumab-800CW and cetuximab-800CW tracers. Specific tumor uptake was confirmed with fluorescent microscopy showing, respectively, stromal and cell membrane fluorescence. Conclusion: VEGF-A is a promising target for molecular fluorescence endoscopy because it showed a high protein expression, especially in sessile serrated adenomas/polyps and Lynch syndrome. We demonstrated the feasibility to visualize small tumors in real time during colonoscopy using a NIR fluorescence endoscopy platform, providing the endoscopist a wide-field red flag technique for adenoma detection. Clinical studies are currently being performed in order to provide in-human evaluation of our approach

Threshold Analysis and Biodistribution of Fluorescently Labeled Bevacizumab in Human Breast Cancer

In vivo tumor labeling with fluorescent agents may assist endoscopic and surgical guidance for cancer therapy as well as create opportunities to directly observe cancer biology in patients. However, malignant andnonmalignant tissues are usually distinguished on fluorescence images by applying empirically determined fluorescence intensity thresholds. Here, we report the development of fSTREAM, a set of analytic methods designed to streamline the analysis of surgically excised breast tissues by collecting and statistically processing hybridmultiscale fluorescence, color, and histology readouts toward precision fluorescence imaging. fSTREAM addresses core questions of how to relate fluorescence intensity to tumor tissue and how to quantitatively assign a normalized thresh-old that sufficiently differentiates tumor tissue from healthy tissue. Using fSTREAM we assessed human breast tumors stained in vivo with fluorescent bevacizumab at microdose levels. Showing that detection of such levels is achievable, we validated fSTREAM for high-resolution mapping of the spatial pattern of labeled antibody and its relation to the underlying cancer pathophysiology and tumor border on a per patient basis. We demonstrated a 98% sensitivity and 79% specificity when using labeled bevacizumab to outline the tumormass. Overall, our results illustrate a quantitative approach to relate fluorescence signals to malignant tissues and improve the theranostic application of fluorescence molecular imaging. (C) 2016 AACR