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J Thorac Cardiovasc Surg 2005;129:844-850
© 2005 The American Association for Thoracic Surgery
Evolving Technology |
a Cardiac Surgery Research Laboratory, Boston, Mass
b Department of Thoracic Surgery, Boston, Mass
c Brigham & Womens Hospital, and the Division of Hematology/Oncology and Department of Radiology, Beth Israel Deaconess Medical Center, Boston, Mass
d Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Mass
Received for publication May 29, 2004; revisions received July 29, 2004; accepted for publication August 2, 2004. * Address for reprints: John V. Frangioni, MD, PhD, Department of Hematology/Oncology and Department of Radiology, Beth Israel Deaconess Medical Center, 330 Brookline Ave, Room SL-B05, Boston, MA, 02215 (E-mail: jfrangio{at}bidmc.harvard.edu).
| Abstract |
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METHODS: Six Yorkshire pigs underwent thoracotomy and received submucosal, esophageal injection of quantum dots, a novel near-infrared fluorescent lymph tracer designed for retention in sentinel lymph nodes. Six additional pigs underwent thoracotomy and received submucosal esophageal injection of CW800 conjugated to human serum albumin, another novel lymph tracer designed for uptake into distant lymph nodes. Finally, 6 pigs received submucosal injection of the fluorophore-conjugated albumin with an endoscopic needle through an esophagascope. These lymph tracers fluoresce in the near-infrared, permitting visualization of migration to sentinel lymph nodes with a custom intraoperative imaging system.
RESULTS: Injection of the near-infrared fluorescent lymph tracers into the esophagus revealed communicating lymph nodes within 5 minutes of injection. In all 6 pigs that received quantum dot injection, only a single sentinel lymph node was identified. Among pigs that received fluorophore-conjugated albumin injection, in 5 of 12 a single sentinel lymph node was revealed, but in 7 of 12 two sentinel lymph nodes were identified. There was no dominant pattern in the appearance of the sentinel lymph nodes either cranial or caudal to the injection site.
CONCLUSION: Near-infrared fluorescence imaging of sentinel lymph nodes is a novel and reliable intraoperative technique with the power to assist with identification and resection of esophageal sentinel lymph nodes.
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In esophageal cancer, extensive lymphadenectomy may improve survival1; however, it also increases morbidity and mortality.2 Selective removal of involved lymph nodes could minimize the extent of lymphadenectomy yet still provide accurate staging and local control. Unfortunately, current methods of identifying involved lymph nodes, including computed tomography, positron emission tomography, and endoscopic ultrasonography, are only 60% to 80% accurate.3 Similarly, sentinel lymph node (SLN) mapping with vital blue dyes or radioactive tracers is neither sufficient nor adequate in the setting of esophageal cancer. In the thorax, intraoperative mapping with blue dye identifies the SLNs in fewer than 50% of cases because of poor tissue penetration and anthracotic mediastinal lymph nodes.4 Similarly, handheld gamma probes miss more than 50% of radioactive nodes from the esophagus.5 The purpose of this study was to test the ability of two novel near-infrared (NIR) fluorescent lymph tracers to identify SLNs of the esophagus. Our hope was that these findings could provide the basis for advancing further studies of NIR fluorescence technology for clinical applications, namely providing selective removal of esophageal lymph nodes for accurate staging information without the need for extensive lymphadenectomy.
| Material and methods |
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HSA800, designed with a hydrodynamic diameter of 7 nm for migration to distant lymph nodes, will be described in detail elsewhere (Ohnishi et al, unpublished data). Briefly, human serum albumin (HSA) was covalently conjugated to the NIR fluorophore CW800 by means of an amide bond (HSA800). The ratio of CW800 to albumin was 3.4:1. Peak absorbance and emission of HSA800 were 778 nm and 795 nm, respectively, in phosphate buffered saline solution, pH 7.4. A stock solution of 0.8-mg/mL HSA800 in phosphate-buffered saline solution was used for all studies. These fluorescent biologic labels are excellent tracers for SLN mapping because they are highly fluorescent, nonradioactive, and easily visible deep within tissue.
Direct injection of QDs into the submucosa of the esophagus
All protocols were in compliance with the "Guide for the Care and Use of Laboratory Animals" (http://www.nap.edu/catalog/5140.html). Adult, male Yorkshire pigs (mean weight 35 kg) were anesthetized intramuscularly with tiletamine and zolazepam (Telazol, 4.4 mg/kg) and xylazine (2.2 mg/kg) and subsequently with isoflurane (0.5%-5% to effect). The animals were intubated, prepared, and draped in the usual fashion. Pigs underwent a left cervical, right thoracotomy, or left subcostal incision to visualize the upper, middle, or lower esophagus, respectively (n = 2 for each group). Submucosal injection of 100 µL of 0.2-µmol/L NIR QDs in phosphate-buffered saline solution, pH 7.4, into either the upper, middle, or lower esophagus was performed. Injections of the lymph tracer were performed by the same surgeon with identical technique.
Direct or endoscopic injection of HSA800
Six pigs underwent the same procedure except with direct injection of HSA800 (dye concentration 10 µmol/L) into the submucosa of the upper, middle, or lower esophagus (n = 2 for each segment). An additional 6 pigs underwent submucosal injection of 100 µL HSA800 with a rigid esophagascope and an endoscopic injecting needle. Injections of the lymph tracer were performed by the same surgeon with identical technique.
Identification and resection of the SLNs
The NIR-fluorescence imaging system has been described elsewhere in detail.7 Briefly, it is composed of a 400- to 700-nm "white" light (0.5 mW/cm2) and a 725- to 775-nm NIR fluorescence excitation light (5 mW/cm2). Images of white light and NIR fluorescence can be displayed separately and merged. The entire apparatus is suspended on an articulated arm over the surgical field, thus permitting noninvasive and nonintrusive imaging. Real-time video images, refreshed 15 times per second, and zooming capability allowed precise localization and dissection as needed.
The migration of the lymph tracers from the injection site either cranially or caudally was imaged in real time. Selected pigs were imaged 4 hours after injection of QDs and HSA800 to detect new fluorescence or loss of fluorescence in lymph nodes of the esophagus. SLNs and control nonfluorescing lymph nodes were dissected under NIR-fluorescence image guidance, embedded with Tissue-Tek OCT compound (Fisher Scientific, Pittsburgh, Pa), frozen in liquid nitrogen, and sectioned at 6 µm. Alternate cuts were examined by hematoxylin and eosin staining and NIR fluorescence imaging. The NIR fluorescence was visualized through a modified microscope with custom-designed optics, as described previously elsewhere.8
Statistical analysis
Associations between categorical parameters were assessed with Fisher exact and paired Student t tests.
| Results |
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| Discussion |
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There are several advantages of NIR fluorescence technology for SLN identification in the esophagus. Both QDs and HSA800 are powerful lymph tracers that fluoresce brightly against a low background and distinctly from the injection site. These NIR-fluorescent lymph tracers do not leak from tissues, nor do they stain the field of dissection. Communicating lymph nodes can be seen 5 minutes to 4 hours later, thus providing great flexibility of preoperative or intraoperative administration. With NIR-fluorescent imaging, the entire surgical field can be imaged in real-time for in situ SLNs, thus minimizing sampling error. NIR fluorescence imaging can provide magnified images to assist with precise and complete resection of the SLN without having to handle another instrument, such as a gamma probe. NIR-fluorescing tracers withstand histologic processing, thus providing another degree of certainty in the identification of SLNs.
SLN mapping with NIR technology could pose some of the same challenges as SLN mapping with technetium Tc 99m or vital blue dyes. Tumor within a lymph node could potentially alter lymphatic flow, deny access of lymph tracer to a positive node, and possibly create a false-negative SLN. However, studies with the breast have found that tumor burden does not increase the false-negative rate.12 Tumor burden can decrease the success of finding SLNs. Therefore failure to identify a SLN or suspicious nodes found at the time of SLN biopsy warrants complete lymphadenectomy. These studies and practices developed for breast cancer will have to be explored in esophageal cancer.
NIR-fluorescent lymph tracers
The optimal size of lymph tracers is controversial but thought to be in the range of 4 to 100 nm.13,14 Because esophageal cancer appears to metastasize to distant lymph nodes, suggesting a broad distribution of drainage or skip metastasis,15 a small, mobile particle such as HSA800 may be an ideal tracer for SLN mapping. QDs, with a larger diameter, migrated only to the first draining lymph node. In contrast, HSA800 could be found in two draining nodes in 7 of 12 pigs. In 1 case, HSA800 identified a lymph node and also partially traveled out to identify a second lymph node. Because HSA800 is smaller than QDs, it may have access to smaller lymphatics that QDs cannot traverse. The clinical significance of a second SLN in the esophagus will have to be determined with future studies. However, the presence of multiple communicating nodes and even multiple lymphatic patterns does not preclude the application of accurate SLN identification in the esophagus. Multiple SLNs are phenomenon accepted in SLN mapping of breast and melanoma.16 In fact, the identification of multiple SLNs improved the false-negative rate to 4.3% from 14.3% when only one SLN was identified.17 The ability of HSA800 to identify multiple nodes may make it a better lymph tracer to limit false-negative results; however, specific studies directed at this question will have to be performed.
A potential limitation of QDs is their unknown toxicity. The individual metals comprising the inorganic core and shell have known toxic effects, especially at concentrations higher than those used in our study. The toxicity of these metals when complexed as salts in the core of a QD is unknown. In our studies, there were no signs of acute toxicity, namely changes in heart rate, rhythm, blood pressure, or oxygen saturation. HSA800 has greater potential for immediate clinical application because it is purely organic and is the product of nontoxic components. However, studies directed at establishing the toxicity, if any, of these tracers must still be performed before applying the technology to human patients.
Last, our data corroborate earlier studies showing an unpredictable drainage pattern of the esophagus.18 Injections of the lymph tracers, whether direct or endoscopic, were performed by the same surgeon with identical technique. Yet, regardless of tracer size, mode of injection, or location of injection, there was no consistent pattern of drainage from injection sites. These findings emphasize the need for a reliable, sensitive, patient-specific method for identifying SLNs of the esophagus. NIR-fluorescence imaging is not only a promising method for identifying SLNs but also a powerful instrument for image-guided dissection.
Advancing this technology to SLN biopsy in human beings could have clinical impact on both staging and treatment in esophageal cancer. Selective removal of the SLNs could provide staging information without the morbidity and mortality of routine extensive lymphadenectomy. If the SLN proved positive, aggressive treatment options such as extended lymphadenectomy or more aggressive adjuvant therapy could be appropriately offered. In conclusion, our studies show the feasibility of patient-specific identification of SLNs of the esophagus with NIR fluorescence technology. However, further studies are needed to apply this technique in humans.
| Footnotes |
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