Purpose: To provide proof of principle of safety, breast tumorspecific uptake, and positive tumor margin assessment of the systemically administered near-infrared fluorescent tracer bevacizumab-IRDye800CW targeting VEGF-A in patients with breast cancer.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 coregistration of tumor tissue and healthy tissue.

Multispectral optoacoustic (photoacoustic) tomography (MSOT) is a hybrid modality that can image through several millimeters to centimeters of diffuse tissues, attaining resolutions typical of ultrasound imaging. The method can further identify tissue biomarkers by decomposing the spectral contributions of different photo-absorbing molecules of interest. In this work we investigate the performance of blind source unmixing methods and spectral fitting approaches in decomposing the contributions of fluorescent dyes from the tissue background, based on MSOT measurements in mice. We find blind unmixing as a promising method for accurate MSOT decomposition, suitable also for spectral unmixing in fluorescence imaging. We further demonstrate its capacity with temporal unmixing on real-time MSOT data obtained in-vivo for enhancing the visualization of absorber agent flow in the mouse vascular system.

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 and nonmalignant 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 hybrid multiscale 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 threshold 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 tumor mass. Overall, our results illustrate a quantitative approach to relate fluorescence signals to malignant tissues and improve the theranostic application of fluorescence molecular imaging. Cancer Res; 77(3); 623-31. ©2016 AACR.