Optical imaging, and especially NIR fluorescence imaging, has several advantages for oncologic surgery. Autofluorescence of tissue and blood is relatively low in the 800 nm wavelength range.2,8
By adding an exogenous fluorophore, a bright spot on a black background is thus created, permitting high sensitivity detection of any desired target within the surgical field. The imaging system described in this study is a stable optical platform that utilizes safe NIR diode excitation, provides simultaneous overlay of color anatomy and NIR fluorescence, is positioned safely above the patient without requiring any contact, and has no moving parts.
Since even NIR light is scattered in living tissue, the depth of light penetration might be expected to be a major obstacle to optical imaging in surgery; however, this doesn't seem to be a major issue. First, the surgeon can follow the lymphatic channels from the injection site towards the SLN, so the direction is always known. Second, because tissue background is so low, tracers can be detected below relatively thick (≈0.5−1 cm) tissue6
and below up to 5 cm of lung.13
Of course, on the surface of the tissue, the appearance of the SLN will be “blurred” by scatter and appear larger than it really is (see, for example, ), but its exact location, and therefore the exact location of the surgical incision, will still be known. Finally, it will likely be possible to adapt newer optical techniques, such as frequency-domain photon migration (reviewed in 25,26
), to our imaging system to improve detectability into the 2−4 cm range.
This study essentially completes pre-clinical development of NIR fluorescent SLN mapping and resection. Using large animal model systems, we have now demonstrated optimized protocols for SLN mapping of spontaneous melanoma, skin,6,10
Using this technology, the need for radioactivity and blue dye is eliminated, and the SLN is typically identified within seconds.
Our studies also found important differences among lymphatic tracers; these differences have implications for the translation of this technology to the clinic. Organic tracers, like those from this study based on albumin, are advantageous because of their availability and biocompatibility. Inorganic/organic hybrid NIR QDs are brighter and remarkably stable to photobleaching, but have yet to undergo proper toxicological screening. From this study, it also appears that type II NIR QDs with a purely anionic coating bind serum proteins and shift their size to 20 nm and greater, whereas zwitterionic molecules with the same concentration of anions, do not. This is an important finding because the small agents, such as HSA800 and ICG:HSA, often identify lymphatic branches (see 16
for detailed description and of this study) and small SLNs, whereas we have never seen branching with NIR QDs and other large HD tracers. The importance of this point cannot be overstated. As shown in and , the other (distal) SLN was extremely small (only 2 mm) yet harbored metastatic cells. For eventual use in humans, the goal must be to find all SLNs, regardless of their size and the size of the connecting lymphatic channels. Since flow in lymphatic channels is pulsatile with heartbeat and respiration, and channels are often collapsed, the smaller HD particles might have an advantage. Moreover, since the SLN is identified within seconds using NIR fluorescence imaging, the probability of tracer flow through the SLN to the next tier is minimized. At least in the case of the Sinclair melanoma model, SLNs from all 12 primary tumors studied harbored either micro- or macrometastases, suggesting an extremely low false-negative rate for all three lymphatic tracers.
The clinical translation of this technology for SLN mapping is imminent. At the present time, the ICG:HSA formulation offers the significant advantage that both components are already FDA-approved for other indications, the amount of each component needed for SLN mapping is orders of magnitude lower than the doses of each used for these other indications, and total fluorescence yield in a serum environment is reasonably high. Within the organic class of SLN tracers, HSA800 would provide an additional 2.2-fold improvement in fluorescent yield, but HSA800, like ICG:HSA, would have a hydrodynamic diameter that permits passage through the SLN if too much time elapses after injection.10,14
Quantum dots, although still years away from clinical translation, provide the highest fluorescence yield, as well as a definable hydrodynamic diameter that ensures complete retention in the first lymph encountered.6,10,13,15-17,20
Newer NIR fluorescent contrast agents, such as HSA800,10
are being developed at a relatively high rate, and the future promises NIR fluorescent contrast agent targeted to a variety of cancers.27
Indeed, tumor-specific targeting should permit real-time assessment of tumor margins and identification of occult metastases within the surgical field. The technology can also help the surgeon to avoid nerves, blood vessels, etc. during cancer resection. The availability of tissue- and disease-targeted NIR fluorophores will likely have a lag of 3−5 years due to a variety of regulatory factors (reviewed in 28
). Nevertheless, ICG:HSA, and ICG alone,5,29,30
will undoubtedly have many clinical applications and will hopefully fill the void.
It should be emphasized that the NIR fluorescence imaging system described in this study might eventually play a dual role in patient management. It not only provides the surgeon with real-time image guidance during any oncologic procedure, but also assists the surgical pathologist. First, the specimen can be trimmed under image guidance. Second, the particular area of interest, e.g., sinus entry point of the SLN, can be found and sectioned at high-resolution to find micrometastases. Finally, since NIR fluorophores withstand frozen sectioning,6,16
a NIR fluorescence microscope31
can be used to correlate the resected target with at least three other fluorescence channels of biologic markers.