Molecular imaging of breast cancer

Breast cancer is the most common type of cancer in women. Imaging techniques play a pivotal role in breast cancer management, especially in lesion detection, treatment planning and evaluation, and prognostication. These imaging techniques have however limitations such as the use of ionizing radiation or radioactive components, limited sensitivity/specificity, and high costs, which motivates development of novel imaging strategies. Molecular imaging comprises visualization and characterization of cellular function and follow-up of molecular and/or biological processes, without perturbing these processes. By visualizing molecular alterations that accompany disease, molecular imaging holds promise to be able to better characterize disease states and extent at an earlier time, with lower required contrast differences than currently used imaging modalities. When fluorophores with a fluorescent spectrum in the near-infrared range (between 650-1000 nm) are applied, no ionizing radiation or radio-active compounds are required, while sufficient tissue penetration is preserved. Conjugation of fluorophores to molecules with affinity for tumor-cell specific processes (such as (therapeutic) monoclonal antibodies or antibody fragments) could improve contrast by increasing local dye concentration and reducing background signals. Development of a desirable molecular imaging tracer with clinical translation potential is however a challenging endeavor, as only a few have entered the clinical setting. In this thesis, we describe several steps in the translational process of molecular imaging tracers and technologies. We first investigate expression patterns of several important molecular targets that are expressed in breast cancer by systematic reviews and meta-analysis. We then identify, produce, and fluorescently label molecules (antibodies and nanobodies) that can bind to breast cancer targets (Carbonic Anhydrase IX and CD44v6) and evaluate, optimize and validate these tracers in vitro and in vivo. We then review current knowledge on registration and fusion of breast optical molecular images with currently used anatomical imaging modalities, which could facilitate optical image interpretation, and show that a clinical optical mammography system is able to visualize a fluorophore (approved for investigations in humans) in nanomolar concentrations with phantom experiments. Then, we show how appropriate patients for molecular imaging trials investigating the Human Epidermal Growth Factor Receptor 2 could be selected based on mammography and breast ultrasound imaging features. Selection of these patients before a diagnostic biopsy has been performed is important, as a biopsy could influence the molecular imaging results. Last, we present a clinical study protocol for investigation of a fluorescent monoclonal antibody directed to the key mediator of neo-angiogenesis in cancer, Vascular Endothelial Growth Factor-A. Molecular imaging could have an application at several stages of breast cancer management. The stability of fluorescent tracers allow for pre-, intra-, and postoperative visualization, with a single administered dose. We are now on the brink of evaluating novel fluorescent tracers and molecular imaging techniques in feasibility trials, and it is expected that several clinical studies applying fluorescent tracers will emerge in the near future. Randomized trials (comparing clinical work-up with or without molecular imaging) will ultimately be required to assess its additional value and role as clinically helpful tool

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

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 and non-malignant 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 multi-scale 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 labelled 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

Fluorescently labeled bevacizumab in human breast cancer: Defining the classification threshold.

In-vivo fluorescently labelled drug (bevacizumab) breast cancer specimen where obtained from patients. We propose a new structured method to determine the optimal classification threshold in targeted fluorescence intra-operative imaging