Previously, Motomura et al (11
) injected 25 mg of indocyanine green in 5 mL of diluent in the breast parenchyma peritumorally to identify stained LNs. Later, Kitai et al (12
) demonstrated the use of subareolar administration of 25 mg indocyanine green and a light-emitting-diode–charged-coupled device system to collect fluorescence for guiding SLNM in breast cancer patients following intradermal injection. In contrast to these studies, we are not
attempting to develop new methods for SLNM in breast cancer patients, as current techniques by using radiocolloid and isosulfan dye show excellent SLN identification rates (13
). Rather, we seek to show the feasibility of NIR optical imaging following microdose (≤100 μg) administration of indocyanine green to use comparable fluorophore concentrations in molecularly targeted agents to noninvasively assess nodal status in humans.
We found initially that doses of indocyanine green less than 1.0 μg were insufficient for adequate NIR imaging in 15 of 15 patients. With doses of 10–100 μg however, we were able to successfully image lymphatic drainage pathways and SLNs in eight of nine women. Our results with low doses of indocyanine green suggest the feasibility of future NIR molecular imaging for nodal staging with microdose administration of protein- and peptide-labeled NIR agents in humans. Microdosing is expected to result in a low probability of adverse events, as is achieved with positron emission tomographic imaging agents, and facilitate translation of NIR molecular imaging (7
). We believe the opportunity to employ molecularly targeted NIR conjugates for guiding surgical resection of cancer-positive LNs has the potential to improve the accuracy and morbidity of nodal staging, not only in breast, but other cancers as well.
The NIR dye used in this feasibility study was nonspecific and passed through the lymphatic system, as evidenced by the trend of negative correlation between the number of resected LNs that were fluorescent and the time elapsed between NIR fluorophore administration and LN resection. Consequently, we were restricted to early time imaging whereby the high photon count rate from superficial lymphatics and injection sites saturated the CCD and limited the noninvasive detection of deeper LNs. We believe that the use of target-specific NIR-labeled imaging agents that pass from superficial lymphatics, but are retained in cancer-positive LNs, would not create saturation of the camera and would enable noninvasive nodal staging by signals needed for deeper tissue imaging.
Indocyanine green dyes do not have reactive groups for conjugation to targeting moieties, but on conjugating other NIR fluorophores such as cypate (14
), Cy7 (15
), or IRDye800 CW (16
) with targeting peptides or proteins, the microdosing limits of 100 μg or less of labeled protein or 30 nmol or less of labeled biologic agent (7
) could be achieved. Indeed, for the minimum detectable signal offered by 10 μg of indocyanine green shown in this study, the minimum molar ratio of NIR dye (with molecular weight and fluorescent yield similar to that of indocyanine green) to antibody would be an easily achievable 0.43, while labeling ratios of dye to antibody that retains binding affinity can be expected to be between 1.0 and 7.0.
Additional improvements to reduce the noise by reducing excitation light leakage with proper filtering and optics (17
) can further improve sensitivity. Finally, fluorescence tomography, as recently described and demonstrated by others (18
), can be used to more accurately locate diseased LNs in three dimensions.
To our knowledge, ours is the first description of functional NIR lymph imaging in humans. Unexpectedly, we discovered that the lymph trafficking hypothesized to result from lymphangion contraction (19
) exists in humans and can be directly imaged. Although angiographic and scintigraphic techniques are current reference standards of lymph imaging, neither offer the ability to dynamically image lymph function with temporal resolutions of milliseconds or less.
Owing to the high photon count rate associated with NIR fluorescence and the ability to perform measurements with millisecond integration times, we discovered that the lymph propulsion characteristic of lymphangions occurs, even if not consistently, within the lymphatics draining the breast tissue. We believe that the use of short integration times of an NIR-sensitive camera and optics enabled imaging of lymph propulsion, and we hypothesized that the ability to monitor lymph trafficking could enable noninvasive diagnostic imaging of lymph function in patients with lymphatic disorders. In our study of women undergoing SLNM, we could not discern the reasons for the variability of lymph propulsion among patients and note that insufficiency in lymph function is not measured or known as a risk factor for breast cancer–related lymphedema following nodal resection.
There were several limitations in our study. First, the small number of patients in the dose escalation trial prevented a direct comparison of whether radioactivity, blue dye, or NIR fluorophores demarked the resected tumor. The inability to control the time between administration of the nonspecific imaging agents and surgical resection also probably contributed to variability of results, especially because indocyanine green drains freely through the lymphatic system. In addition, we believe that excitation light leakage through rejection filters prevented imaging of deep lymphatics when less than 10 μg of indocyanine green were administered.
Nonetheless, we demonstrated that noninvasive NIR optical imaging by using microdose administration of an NIR fluorophore is feasible in humans. Since indocyanine green cannot be conjugated to target peptides and antibodies, new dyes with reactive groups, greater quantum efficiencies, and excitation and emission spectra similar to indocyanine green must be employed. Future directions include conjugation of NIR fluorophores to target specific markers of cancer-positive LNs, improvement of agent delivery systems for supraclavicular and internal mammary LN detection and study of normal and abnormal lymph function.