Purpose-To prospectively demonstrate the feasibility of using indocyanine green, a nearinfrared (NIR) fluorophore at the minimum dose needed for noninvasive optical imaging of lymph nodes (LNs) in breast cancer patients undergoing sentinel lymph node mapping (SLNM).Materials and Methods-Informed consent was obtained from 24 women (age range, 30-85 years) who received intradermal subcutaneous injections of 0.31-100 μg indocyanine green in the breast in this IRB-approved, HIPAA-compliant, dose escalation study to find the minimum microdose for imaging. The breast, axilla, and sternum were illuminated with NIR light and the fluorescence generated in the tissue was collected with an NIR-sensitive intensified chargedcoupled device. Lymphoscintigraphy was also performed. Resected LNs were evaluated for the presence of radioactivity, blue dye accumulation, and fluorescence. The associations between the resected LNs that were fluorescent and (a) the time elapsed between NIR fluorophore administration and resection and (b) the dosage of NIR fluorophores were tested with the Spearman rank and Pearson product moment correlation tests, respectively.Results-Lymph imaging consistently failed with indocyanine green microdosages between 0.31 and 0.77 μg. When indocyanine green dosages were 10 μg or higher, lymph drainage pathways from the injection site to LNs were imaged in eight of nine women; lymph propulsion was observed in seven of those eight. When propulsion in the breast and axilla regions was present, the mean apparent velocities ranged from 0.08 to 0.32 cm/sec, the time elapsed between "packets" of propelled fluid varied from 14 to 92 seconds. In patients who received 10 μg of indocyanine green or more, a weak negative correlation between the fluorescence status of resected LNs and the time © RSNA, 2008 Address correspondence to E.M.S. (evas@bcm.tmc.edu). Supplemental material: http://radiology.rsnajnls.org/cgi/content/full/2463070962/DC1 Author contributions:Guarantor of integrity of entire study, E.M.S.; study concepts/study design or data acquisition or data analysis/interpretation, all authors; manuscript drafting or manuscript revision for important intellectual content, all authors; manuscript final version approval, all authors; literature research, all authors; clinical studies, all authors; statistical analysis, all authors; and manuscript editing, all authors See Materials and Methods for pertinent disclosures. NIH Public Access Author ManuscriptRadiology. Author manuscript; available in PMC 2011 September 3. Conclusion-NIR fluorescence imaging of lymph function and LNs is feasible in humans at microdoses that would be needed for future molecular imaging of cancer-positive LNs.Currently, standard-of-care staging of breast cancer requires surgical resection of the first tumor-draining, or sentinel, lymph node (SLN) for subsequent pathologic examination (1). If the SLN is cancerous, then additional lymph nodes (LNs) in the axillary basin are subsequently removed for accurate staging. Recently, the a...

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Targeted fluorescent molecular imaging probes may provide an optimal means of detecting disease. Stable, organic fluorophores can be repeatedly excited in vivo by propagated light and consequentially can provide large signal-to-noise ratios (SNRs) for image detection of target tissues. In the literature, many small animal imaging studies are performed with a red excitable dye, Cy5.5, conjugated to the targeting component. We report the comparison of the in vivo fluorescent imaging performance of a near-IR (NIR) and a red-excitable dye. Epidermal growth factor (EGF) was conjugated with Cy5.5 [excitation/emission (ex/em), 660710 nm] or IRDye 800CW (ex/em: 785830 nm) for imaging EGF receptor (EGFr) positive (MDA-MB-468) and/or negative (MDA-MB-435) human breast cancer cell lines in subcutaneous xenograft models. The conjugates were injected intravenously at 1-nmol-dye equivalent with and without anti-EGFr monoclonal antibody C225, preadministered 24 h prior as a competitive ligand to EGFr. Our images show that while both agents target EGFr, the EGF-IRDye 800CW evidenced a significantly reduced background and enhanced the tumor-to-background ratio (TBR) compared to the EGF-Cy5.5. Immunohistochemistry shows that EGF causes activation of the EGFr signaling pathway, suggesting that prior to use as a targeting, diagnostic agent, potential deleterious effects should be considered.

These clinical research studies demonstrate the potential of NIR fluorescence imaging as a diagnostic measure of functional lymphatics and as a new tool in translational research studies to decipher the role of the lymphatic system in cancer and other diseases.

Near-infrared (NIR) fluorescence imaging clinical studies have been reported in the literature with six different devices that employ various doses of indocyanine green (ICG) as a non-specific contrast agent. To date, clinical applications range from (i) angiography, intraoperative assessment of vessel patency, and tumor/metastasis delineation following intravenous administration of ICG, and (ii) imaging lymphatic architecture and function following subcutaneous and intradermal ICG administration. In the latter case, NIR fluorescence imaging may enable new discoveries associated with lymphatic function due to (i) a unique niche that is not met by any other conventional imaging technology and (ii) its exquisite sensitivity enabling high spatial and temporal resolution. Herein, we (i) review the basics of clinical NIR fluorescence imaging, (ii) survey the literature on clinical application of investigational devices using ICG fluorescent contrast, (iii) provide an update of non-invasive dynamic lymphatic imaging conducted with our FDPM device, and finally, (iv) comment on the future NIR fluorescence imaging for non-invasive and intraoperative use given recent demonstrations showing capabilities for imaging following microdose administration of contrast agent.