Download Supplemental Material and Methods

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SUPPLEMENTARY MATERIALS AND METHODS / FIGURE LEGENDS
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Trial Design
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Tracer accumulation was defined as fluorescence activity assessed either by detection by the
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optical imaging system in vivo, ex vivo of the excised specimen, or ex vivo fluorescence flatbed
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scanning. The study was to be suspended if any undesired SAE occurred throughout the study or
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if no accumulation of bevacizumab-IRDye800CW could be detected in any of the first seven
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patients. The full study aimed at evaluating a total of 20 patients for bevacizumab-IRDye800CW
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accumulation, with replacement of 10 patients if needed, amounting to a maximum of 30 patients
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who would receive a bevacizumab-IRDye800CW dose. Patients were eligible if they were ≥18
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years of age and had a World Health Organisation (WHO) performance score of 0 - 2. Exclusion
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criteria were the presence of another invasive malignancy, another serious medical condition (as
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determined by the research team), pregnancy or lactation, prior neo-adjuvant chemotherapy, prior
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radiotherapy of the involved area, major surgery within 28 days before initiation of the study
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procedures, prior allergic reaction to immunoglobulins, or the presence of a breast prosthesis in
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the breast with cancer. In the first five patients, a tumor size of at least 15 mm, according to
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standard anatomical imaging data (i.e. mammography, ultrasound and/or dynamic contrast
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enhanced magnetic resonance imaging (DCE-MRI)), was required. From the sixth patient
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onwards, 5 mm diameter was considered sufficient, as based on the data provided by the 89Zr-
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bevacizumab patient study executed at the UMCG at the same time and as such approved by the
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Institutional Review Board (IRB) (19). Routine tumor staging included mammography and
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ultrasound. MRI was performed if better tumor extent definition was needed as standard of care
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in the Netherlands. The Institutional Review Board (IRB) of the University Medical Center
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Groningen (UMCG) approved the study, with local agreement of the University Medical Center
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Utrecht (UMCU). All patients gave written informed consent.
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Bevacizumab-IRDye800CW Preparation and Injection
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The development (i.e. implementation of the manufacturing process and analytical methods
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according to current Good Manufacturing Practice (cGMP), formulation studies, extended
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characterization and stability testing) and safety testing (extended single dose toxicity study in
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mice) was reported for clinical grade bevacizumab-IRDye800CW (27). Both GMP produced
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bevacizumab and IRDye800CW were commercially obtained from the manufacturers. The costs
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of one vial of 4,5 mg bevacizumab-IRDye800CW in a non-commercial academic GMP
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production setting were estimated to be $550. Briefly, bevacizumab and IRDye800CW were
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reacted in a molar ratio of 1:4 in phosphate-buffered saline (PBS, pH 8.5). Thereafter, the product
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was purified by gel filtration using PD-10 Desalting Columns (GE Healthcare). Labeling
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efficiency and purity were determined by validated size-exclusion high-performance liquid
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chromatography. The labeling efficiency was 80-85%, resulting in an average labeling ratio of
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3.3 dyes per antibody with a purity of > 95% after purification. The stability of the tracer was
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verified by storing the product in 0.9% NaCl for 7 days at various conditions (-80, -20 and 4C),
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for which storage at -20C proved to be sufficient. According to FDA/EMEA guidelines (20) we
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adhered to the microdosing concept of imaging studies, as defined by a dose of ≤30 nmol for
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proteins and imaging studies, also described by Kummar et al and the Task Force on
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Methodology for the Development of Innovative Cancer Therapies (MDICT) (21). The molecular
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weight of the protein bevacizumab is 149 kDa. The fluorescent dye has a molecular weight of
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1,166 kDa. With an average conjugation ratio of 1:4 protein:IRDye800CW, the total molecular
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weight is 153,7 kDa. This means for our 4.5 mg tracer dose bevacizumab-IRDye800CW, a
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systemic injection of 26 nmol of bevacizumab-IRDye800CW.
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After release of the final product by the certified qualified person at the UMCG GMP facility,
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patients received 4.5 mg bevacizumab-IRDye800CW at a concentration of 1 mg/ml as
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intravenous bolus injection 3 days before scheduled surgery, based on the preclinical
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pharmacokinetic data obtained in breast cancer tumor bearing mice injected with bevacizumab-
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IRDye800CW (26). After injection, the infusion line was flushed with 0.9% NaCl and patients
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were observed and vital signs were monitored for the occurrence of adverse events for 4 hours
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(patient 1-9) or 1 hour (patient 10-20) after tracer administration. Immediately after injection, as
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well as on day 3 (directly prior to surgery) and on day 10-14 after surgery, blood was drawn for
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bevacizumab-IRDye800CW whole blood measurement (immediately after injection, day 3, and
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day 10-14).
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Optical Imaging System
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At both participating centers, a similar near-infrared (NIR) optical imaging system was used for
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intra-operative and fluorescence imaging of the excised specimen. The optical imaging system
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was developed by the Technical University Munich (TUM)/Helmholtz Center) and has been
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described previously (23,26,30). In brief, the excitation wavelength was created by a 750 nm-
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300mW continuous wave diode laser (BWF2-750-0, B&W Tek) guided through a widening lens
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(F260SMA-B, Thorlabs Inc) and a diffuser (DG10-220, Thorlabs Inc). The field of view (FOV)
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was illuminated by a 250 W halogen lamp (KL-2500LCD, Schott AG), guided through a short
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pass filter (E700SP-2P, Chroma) to prevent illumination crosstalk to the fluorescence channel.
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The FOV was imaged by a motorized lens (CVO GAZ11569M, Goyo Optical), separated in
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visible and NIR fluorescence parts by a dichroic beam splitter (T760lpxr, Chroma) and relayed to
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appropriate CCD sensors accordingly (MAP10100100, Thorlabs Inc). The NIR fluorescence
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image was guided through a band pass filter (ET810/90, Chroma) for eliminating non-
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fluorescence parts of the spectrum and acquired by a scientific deep-cooled (-70°C) back3
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illuminated CCD Camera (iXon DU-888, Andor Technology). The color-reflection image was
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guided through a shortpass filter (E700SP-2P, Chroma) and acquired by a 12-bit bayer-patterned
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color camera (pixelfly QE 275xs PCO AG). For mitigation of specula reflections on wet tissue
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surfaces in the color image the white-light illumination and color camera reflection channel were
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equipped with linear-polarizers (21003b, Chroma) in a cross-polarization geometry. The heat
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disposal for the deep-cooled CCD camera was achieved by additional liquid cooling. Both, color
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and fluorescence images where acquired, processed, stored and displayed simultaneously at video
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rate. The acquisition was performed by a standard PC where image correlation, de-bayering,
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mapping to visualization dynamic range and image fusion are performed at video rates using the
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Graphics Processing Unit (GPU). All imaging parameters including the zoom, iris and focus of
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the motorized lens are controllable by a lightweight, user-friendly, GPU-accelerated acquisition
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and visualization software. The color reflectance image, the raw florescence image or a fused
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false-color overlay image are displayed instantly in video rate and latency-free at the build-in
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screens of the operational theater. A synchronized multi-CPU system (Dell Computers) controls
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the camera system and can process raw data and image registration and rendering simultaneously.
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The field-of-view (FOV) can be varied from 3 x 3 cm to 15 x 15 cm, with corresponding
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resolution varying from 30 x 30 µm to 150 x 150 µm per pixel for a total of 512 x 512 pixels per
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fluorescence image. Color and fluorescence images were simultaneously collected by the two
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cameras and processed. Custom made software was used for imaging processing and post-
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processing.
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Sterile transparent drapes (Carl Zeiss Vision BV) covered the camera-head and the arm
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during surgery. As the system was a prototype, no CE marking was obtained. Instead, the device
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was verified for laser safety and leak currents as tested at the TUM and by the Medical Devices
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Unit at the UMCG and UMCU. Both imaging devices were approved for intraoperative
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application in humans. For standardization purposes of imaging data between both hospitals, a
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standard concentration series of indocyanin green (ICG, ICG-PULSION, PULSION Medical
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Systems) for each imaging session was evaluated, which made comparison between both systems
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and centers possible.
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Ex Vivo Analyses of Tumor Samples
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Imaging fresh surgical specimen. After bread-loaf slicing of the fresh surgical breast
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specimen and before formalin fixation, all slices were imaged with the optical imaging system for
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determination of fluorescence signal in tumor and surrounding healthy breast tissue.
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Subsequently, small (2-6 mm) biopsies from tumor tissue and normal breast parenchyma were
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taken from the bread-loaf slices, snap frozen and stored in dark conditions at −80C, provided the
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tumor was large enough and no interference with standard processing procedures was anticipated.
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Subsequently, the specimen was fixed in formalin and embedded in paraffin (FFPE) as a routine
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procedure.
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Imaging FFPE blocks and slides. FFPE blocks were available for all patients, including
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tumor tissue and surrounding tissue. We used the Odyssey® CLX fluorescence scanning system
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(LI-COR Biosciences Inc.) for assessment of fluorescence in the NIR region and location in the
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FFPE blocks and/or tissue slides. Additionally, whole blocks were imaged by the intra-operative
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real-time optical imaging camera to compare both techniques.
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Bevacizumab-IRDye800CW and VEGF-A quantification
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For bevacizumab-IRDye800CW quantification in tumor tissue, adjacent margin tissue and
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normal breast tissue, each biopsy was weighed and subsequently disrupted with a TissueLyser II
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system (Qiagen) using pre-cooled Eppendorf holders, 5 mm stainless steel beads, and
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RIPA buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1%
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SDS) supplemented with a complete EDTA-free mini tablet protease inhibitor cocktail (Roche
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Applied Science). The homogenates were then diluted in series (1:2 dilution steps) in 96-well
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plates, with the same buffer used for homogenization of the biopsies. In parallel, the
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dilution series of the probe were also made using RIPA buffer. The intensity of the
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NIR fluorescence of the samples and standard probe was detected at 800 nm with the Odyssey
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scanner using the following settings: intensity 7, focus offset 3.5, quality: medium, resolution 169
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μm. Subsequently, the concentration of the probe present in the homogenates was extrapolated
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from the calibration curves made with the standard probe, using the software Prism 6 (GraphPad
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Software Inc.). These concentration values were used to calculate the concentration of the tracer
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per mg of tissue biopsy (ng/ml/mg) (31).
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For quantification of VEGF-A expression (pg/mL) in the surgical specimen, a VEGF-A
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Enzyme-Linked Immuno Sorbent (ELISA) assay was performed according to the manufacturer
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instructions (Quantikine). One to three random samples were collected from the surgical
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specimen of the primary tumor, margin tumor-surrounding tissue, and surrounding (normal)
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tissue. Tissue was lyzed manually using RIPA buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1
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mM EDTA, 1% Triton X-100, 0.1% SDS) supplemented with a complete EDTA-free mini tablet
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protease inhibitor cocktail (Roche Applied Science). Thereafter, whole lysate mixtures were first
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measured on the Odyssey scanner at the 800 nm channel to determine the intensity of the
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NIR fluorescence of each sample using the following settings: intensity 7, focus offset 3.5,
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quality: medium, resolution 169 μm. Subsequently, lysates were centrifuged to spin down cellular
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debris, and supernatants were used to determine VEGF-A levels by ELISA. Results of VEGF-A
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measurement were normalized for the total weight of the tissue sample before sample analysis.
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Tumor lysates of three patients were also analyzed by sodium dodecyl sulfate polyacrylamidegel
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electrophoresis (SDS-PAGE), to ensure the complete compound bevacizumab-IRDye800CW
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was present in the primary breast tumor.
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Immunohistochemistry
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FFPE blocks of tumor tissue and normal healthy breast tissue were available for all patients and
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negative controls. Secondary use of excised breast cancer specimen from four patients
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undergoing breast-conserving surgery as negative controls was approved by the IRB and the
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Department of Pathology. Paraffin sections were cut at 4 μm, mounted on silane-coated slides
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and dried overnight at 37 °C. All specimens were examined by one pathologist using
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standard H/E staining. Additionally, (I)HC staining was performed for VEGF, CD34 and
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collagen. Before (I)HC staining, slides were deparaffinized using xylene, and the 800 nm NIR
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fluorescence signal was measured on the Odyssey scanner. The tissue blocks and slides were
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scanned with the following settings. For blocks: wavelength 800 nm, intensity 6, focus offset 1,
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quality: highest, resolution 21 μm. For slides (4 μm and 10 μm where appropriate): wavelength
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800 nm, intensity 9, focus offset 0, quality: highest, resolution 21 μm. Subsequently, CD34 was
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stained using monoclonal antibody anti-CD34 clone Qbend10 (Immunotech) and the OptiView
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detection kit on the Ventana Benchmark Ultra. Collagen was stained with Sirius red. For VEGF-
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A staining we used the following protocol. First, slides were rehydrated via graded
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alcohols. Endogenous peroxidase blocking was performed during 15 minutes with a phosphate
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citrate buffer containing 5% hydrogen peroxide, followed by heat induced antigen retrieval in 0.1
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M 95-99 °C sodium citrate (pH 6.0) for 20 minutes on a hot plate. After washing steps, sections
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were incubated overnight at 4°C with polyclonal rabbit anti-human VEGF-A (RB9031,
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ThermoScientific) in a concentration of 0.2 µg/mL (1:1000). Tissue was stained using poly-HRP-
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anti Ms/Rb/Rt IgG (BrightVision) for 30 minutes at room temperature, followed by visualization
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with diaminobenzidine for 10 minutes. PBS was used throughout for washing. Finally, sections
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were counterstained with Mayer’s hematoxylin, dehydrated in alcohol and xylene and
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coverslipped. Resection margins were defined according to the Dutch guidelines: a free margin is
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that tumor does not reach into any of the surgical margins in an adequately processed sample.
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There is focal spread in a surgical margin if the tumor (invasive carcinoma and/or DCIS) reaches
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into a single limited area (≤ 4 mm) in an inked margin (33).
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Fluorescence microscopy
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Additional microscopic evaluation of NIR fluorescence signal was performed on
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additional 4 μm sections, with counterstaining of the nuclei (Hoechst staining, 33258; Invitrogen)
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and mounting with Kaiser’s glycerine (modified), using an inverted laser-scanning microscope
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(DMI6000B, Leica Biosystems GmbH). To optimize NIR visualization, the setup was equipped
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with additional accessories, including a highly sensitive monochrome DFC365 FX fluorescence
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camera (1.4M Pixel CCD, Leica Biosystems GmbH), an optimal NIR filter set (two band-pass
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filters 850-90m-2p and a long-pass emission filter HQ800795LP; Chroma Technology Corp®), a
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highly sensitive NIR LED light source ranging up to 900 nm (X-Cite® 200DC, Excelitas™
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Technologies) and LAS-X software (Leica Biosystems GmbH).
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Multiplex Advanced Pathology Imaging (MAPI) methodology
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For ex vivo correlation of the fluorescence scans obtained by the optical imaging system
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and the Odyssey imaging system, a workflow methodology was developed for cross-correlation
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of fluorescence scans with IHC for VEGF-A, CD34 (microvessel density) and presence of
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collagen (Sirius Red staining) in a multiplex overlay (Supplemental Fig. S6 and Supplemental
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Movie S1). After whole slide NIR fluorescence scanning on the Odyssey imaging system, IHC
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stained slides were scanned on a digital slide scanner (ScanScope XT) at 20x resolution (0.50
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µm/pixel). Color deconvolution with vectors H DAB (ImageJ version 1.48) was used to separate
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DAB and hematoxylin color layers. DAB color layers were converted to red-black images using
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auto threshold. NIRF Odyssey images of the same slide were converted green-black images also
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with auto threshold. NIRF images and IHC images of the same slide where registered to show
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expression of bevacuzimab-IRDye800CW in the H/E stained histology slides and identify co-
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localization of the bevacuzimab-IRDye800CW NIRF signal and VEGF-A or collagen.
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Statistical Analysis
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Given the early phase nature of this exploratory study, the emphasis was on a high power (1-β) to
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detect potentially clinically relevant bevacizumab-IRDye800CW breast tumor accumulation in
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patients with primary breast cancer. Tracer accumulation was defined as fluorescence activity
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assessed either by detection by the optical imaging system in vivo, or ex vivo of the excised breast
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cancer specimen, bread-loaf slices, paraffin embedded tissue blocks, or fluorescence flatbed
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scanning of tissue slides. This was done to avoid erroneous dismissal of a successful compound
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in the early stage of its evaluation. Accordingly, if no accumulation was detected in any of the
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first seven consecutive patients of stage 1, we could dismiss a true accumulation rate of 35% or
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larger with a β of 5% or smaller, and a true accumulation rate of 48% or larger at the ≤ 1% β
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level. Similarly, the study was powered at 99.99% to continue after the first seven patients given
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a true accumulation rate of 75%. If the study continued to its full size in the second stage with a
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total of 20 evaluable patients for accumulation of the bevacizumab-IRDye800CW tracer, the
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margin of error of the accumulation rate estimate (as calculated by the Score method using α =
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5%) would be at most 20% for an observed accumulation rate of 50%, yielding an accumulation
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rate estimate of 0.50 with a 95% confidence interval (CI) of 0.30 to 0.70. As one patient in our
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study did not show accumulation, this related to an accumulation rate of 0.95 (95%CI: 76-99%).
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Similarly, for an observed accumulation rate of 75%, the margin of error would be 18%, yielding
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an accumulation rate estimate of 0.75 (95% CI: 0.53-0.89). The full-study sample size thus
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allowed reasonably precise estimates of the bevacizumab-IRDye800CW accumulation rate.
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Sample size considerations pertained only to bevacizumab-IRDye800CW tumor accumulation, as
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the expected incidence of SAEs was so low that the study size needed to formally address and
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estimate SAE incidence associated with the compound was prohibitively large. Nevertheless,
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given a true SAE incidence rate of 2.5%, the chance of observing an SAE in the study
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approached 50%, which is close to 75% for a true SAE incidence rate of 5.0%. Data-analysis of
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secondary endpoints was exploratory in nature. The secondary outcome measures of the clinical
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study included the detection ability of the optical imaging system camera of the fluorescent signal
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from bevacizumab-IRDye800CW during surgery and to assess and compare the presence of a
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fluorescent signal in breast cancer tissue and normal tissue by assessing the images during and
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after surgery, the accumulation, tissue distribution, localization and quantification of the tracer in
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resected tumor tissue, and surrounding healthy tissue, as measured by fluorescence imaging in
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vivo and ex vivo. Correlations of intraoperative imaging to ex vivo quantitative measures of
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fluorescence, tracer distribution, and IHC analyses of surgical specimens were investigated for
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the purposes of co-localization and correlation (Pearson correlation) and for determination of
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MFI obtained by fluorescence flatbed scans in regions of tumor, stroma and healthy surrounding
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tissue (paired t-test or Wilcoxon signed rank test for paired data and the unpaired t-test for
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unpaired data). Data are presented as a mean ± standard deviation (SD). A two-sided P value of
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less than 0.05 was considered significant (SPSS, version 19.0; IBM).
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SUPPLEMENTAL FIGURE LEGENDS / MOVIE
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Supplemental Figure S1. Near-Infrared Optical Imaging of Positive Tumor Margin. In one
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of the two patients with a positive margin (see also Fig. 1), the bevacizumab-IRDye800CW tracer
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could be detected ex vivo by optical imaging of the excised lump (panel A white-light, panel B
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fluorescence, panel C overlay). Bread-loaf slicing (panel D-F) and the paraffin block (panel G-I)
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is shown for the corresponding fluorescent signal. Hematoxylin/eosin (H/E) stain (panel J),
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fluorescence flatbed scanning (panel K) and image overlay (panel L) is shown for the 4 μm slide
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(red/white/black dashed line = tumor outline, white/black arrow = positive tumor margin).
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Supplemental Figure S2. Representative Fluorescence Flatbed Scans of Paraffin Blocks of all
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Patients (N=20). Of all included patients, representative paraffin blocks are depicted, including 5
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negative controls of patients with breast cancer (i.e. tumor present but without injection of
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bevacizumab-IRDye800CW, last column). Tumor areas are demarcated by dotted lines. *
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(UMCU4) = skin, ** (UMCG9) = fibroadenoma. Fluorescence is depicted by mean fluorescence
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intensity (MFI). Macroscopic Segmentation of Fluorescence Tissue Localization and Target-
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to-Background Ratios (TBR). The mean TBR is depicted (** = P < 0.001) in a separate panel.
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Supplemental Figure S3. Microscopic Biodistribution of Bevacizumab-IRDye800CW. Tissue
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slides of 4 μm were scanned on a flatbed scanner for mean fluorescence intensity, followed by
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hematoxylin/eosin (H/E) staining and microscopy (2x panel A and 20x panel B) for co-
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localization at the microscopic level of fluorescence intensities (panel C and D) in a tumor sprout
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surrounded by blood vessels and collagen-rich stroma (rectangular). Color and fluorescence
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images were superimposed (panel E and F). Localization of Fluorescence Bevacizumab-
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IRDye800CW in Human Primary Breast Cancer. Near-infrared fluorescence microscopy of 4
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µm tissue slides (panel G: [BLUE] = Hoechst stain, [RED] = NIR channel / bevacizumab-
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IRDye800CW, white arrows = tumor margin) and subsequently stained for hematoxylin/eosin
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(panel H, black arrows = tumor margin).
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Supplemental Figure S4. Co-localization of Bevacizumab-IRDye800CW, hematoxylin/eosin
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(H/E), VEGF-A, Collagen and CD34 in Breast Cancer and a Satellite Lesion. A fresh excised
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breast tumor specimen with a satellite lesion was processed with multiplex advanced pathology
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imaging (MAPI) for co-localization of white light images (white-light panel A, H/E panel E,
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VEGF-A staining panel I, collagen panel M, CD34 panel Q), black-and-white bevacizumab-
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IRDye800CW fluorescence (panel B and green pseudocolor in panel C, G, K, O and S),
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segmentation (panel F), deconvolution (VEGF-A panel J, collagen panel N, CD34 panel R) and
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overlay images (panel D: white-light + fluorescence, panel H: H/E and fluorescence, panel L:
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VEGF-A and fluorescence, panel P: collagen and fluorescence and panel T: CD34). In panel T an
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overlay of CD34 (blue color) bevacizumab-IRDye800CW fluorescence (green) and VEGF-A
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(red) is depicted.
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Supplemental Figure S5. Co-localization of Bevacizumab-IRDye800CW, hematoxylin / eosin
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(H/E), VEGF-A, Collagen and CD34 in Ductal Carcinoma In Situ (DCIS). A fresh excised
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breast tumor specimen with DCIS was processed with multiplex advanced pathology imaging
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(MAPI) for co-localization of white light images (white-light panel A, H/E panel E, VEGF-A
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staining panel I, collagen panel M, CD34 panel Q), black-and-white bevacizumab-IRDYe800CW
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fluorescence (panel B and green pseudocolor in panels C, G, K, O and S), segmentation (panel
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F), deconvolution (VEGF-A panel J, collagen panel N, CD34 panel R) and overlay images (panel
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D: white-light + fluorescence, panel H: H/E and fluorescence, panel L: VEGF-A and
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fluorescence, panel P: collagen and fluorescence and panel T: CD34). In panel T and overlay of
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CD34 (blue color) bevacizumab-IRDye800CW fluorescence (green) and VEGF-A (red) is
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depicted.
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Supplemental Figure S6. Intraoperative and Ex Vivo Optical Imaging of Axillary Lymph
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Nodes. Intraoperative optical imaging of the axillary dissection (white-light panel A, black-and-
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white fluorescence panel B (lymph nodes encircled white), pseudocolor overlay panel C).
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Paraffin-embedded tissue block (white light panel D, pseudocolor fluorescence panel E,
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hematoxylin/eosin staining panel F). VEGF-A IHC staining (panel G), red deconvolution (panel
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H), bevacizumab-IRDye800CW flatbed scanning (panel I), green deconvolution (panel J) and
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VEGF-A / bevacizumab-IRDye800CW overlay (panel K).
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Supplemental Figure S7. Co-localization of Hematoxylin / Eosin (H/E), VEGF-A, and
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Bevacizumab-IRDye800CW in Tumor-negative Lymph Nodes. Two excised lymph nodes
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were processed for co-localization of white light images (panel A), pseudocolor fluorescence
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(panel B) and Hematoxylin/eosin (H/E, panel C). VEGF-A staining (panel D) red deconvolution
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(panel E), bevacizumab-IRDye800CW flatbed scanning (panel F), green deconvolution (panel
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G), and overlay VEGF-A / bevacizumab-IRDye800CW (panel H).
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Supplemental Movie S1. Explanatory Movie of Multiplex Advanced Pathology Imaging
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(MAPI). Multiplex pathology imaging of white-light microscopy, bevacizumab-IRDye800CW
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fluorescence imaging (green pseudocolor), VEGF-A (red pseudocolor), collagen (red
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pseudocolor) and CD34 (blue pseudocolor) in breast cancer, including overlay images is shown
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in a stepwise manner for reasons of clarity.
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