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Ann Thorac Surg 2005;79:269-277
© 2005 The Society of Thoracic Surgeons
a Division of Cardiac Surgery, Department of Surgery, Brigham and Women's Hospital, Boston, Massachusetts, USA
b Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
c Division of Hematology/Oncology
d Department of Radiology, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA
Accepted for publication June 4, 2004.
* Address reprint requests to Dr Soltesz, Department of Surgery, Brigham and Women’s Hospital, 75 Francis St, Boston, MA02115 (E-mail: esoltesz{at}partners.org).
Presented at the Fortieth Annual Meeting of The Society of Thoracic Surgeons, San Antonio, TX, Jan 26–28, 2004.
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Abstract |
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METHODS: Invisible near-infrared (NIR) light penetrates relatively deeply into tissue and background autofluorescence is low. We have developed a NIR fluorescence imaging system that simultaneously displays color video and NIR images of the surgical field. We recently engineered 15 nm nonradioactive NIR fluorescent quantum dots (QDs) as optimal lymphotrophic optical probes. The introduction of these QDs into lung tissue allows real-time visualization of draining lymphatic channels and nodes.
RESULTS: In 12 Yorkshire pigs (mean weight 35 kg) we demonstrated that 200 pmol of NIR QDs injected into lobar parenchyma accurately maps lymphatic drainage and the SLN. All SLNs were strongly fluorescent and easily visualized within 5 minutes of injection. In 14 separate injections QDs localized to a mediastinal node, whereas in 2 injections QDs localized to a hilar intraparenchymal node. Histologic analysis in all cases confirmed the presence of nodal tissue.
CONCLUSIONS: We report a highly sensitive rapid technique for SLN mapping of the lung. This technique permits precise real-time imaging and therefore overcomes many limitations of currently available techniques.
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Introduction |
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The introduction of sentinel lymph node (SLN) mapping and biopsy revolutionized the assessment of nodal status regarding the spread of neoplasms, particularly melanoma and breast cancer [4]. The underlying hypothesis of SLN mapping is that the first lymph node to receive lymphatic drainage from a tumor site will contain tumor cells if there has been direct lymphatic spread [5]. Current techniques with regard to the identification of the SLN involve radioguided lymphatic mapping and/or visualization of the nodes with vital blue dyes [6].
Recent studies of SLN mapping have confirmed its feasibility in patients with NSCLC [7]. Several studies have indicated that the SLN accurately reflects the tumor status of regional mediastinal nodes exhibiting greater than 80% accuracy [8–11]. Present methods of SLN mapping for other organs are, however, not readily amenable for use with respect to staging NSCLC. The ideal method should be accurate, rapid, noninvasive, and potentially usable in a thoracoscopic setting. None of the current methods fulfill all of these criteria [12]. In the lung intraoperative mapping with blue dye has been determined to identify the SLN in fewer than 50% of patients because tissue penetration of the dye is poor and mediastinal lymph nodes are often anthracotic [13]. The use of radiolabeled tracers has improved the detection rate and accuracy of pulmonary SLN mapping, but the high radioactivity of the primary injection site may interfere with intraoperative in vivo detection of nearby nodes. If radioisotopes are injected intraoperatively the time period required for the tracer to migrate to the SLN may delay the operative procedure. Conversely preoperative injection of the radiocolloid tracer necessitates CT-guided injection anywhere from 6–24 hours before surgery.
We recently developed a completely portable near-infrared (NIR) fluorescence imaging system that permits real-time intraoperative SLN mapping using optimized quantum dots (QDs) [14–16]. QDs are bright fluorescent semiconductor nanocrystals that contain an inorganic core of metal and an outer soluble organic coating. These fluorescent biological labels are exceptional probes for SLN mapping because they are highly fluorescent, nonradioactive, and easily visible deep within tissue [17]. In this present study we investigated the feasibility of using QDs for mapping pulmonary lymphatic drainage and guiding excision of the SLN.
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Material and Methods |
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NIR Fluorescent Lymphatic Tracer Preparation
The complete synthesis of quantum dots used in this study has been previously described [14]. Briefly we synthesized type-II NIR fluorescent semiconductor nanocrystals (QDs) with a hydrodynamic diameter of 15–20 nm, a maximal absorption cross-section, a 840–860 nm fluorescence emission, an aqueous quantum yield greater than or equal to 13%, and a stable oligomeric phosphine coating. The hydrodynamic parameters of these QDs were engineered to maximize both rapid uptake into lymphatics and retention in the first draining lymph node.
Surgical Preparation
Adult Yorkshire pigs (n = 12, mean weight 35 kg) of either sex were used in this study. Animals were obtained from EM Parsons (EM Parsons & Sons, Inc., Hadley, MA). All animals received humane care in compliance with institutional protocols and the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health (National Institutes of Health publication 85 to 23, revised 1985). Investigators complied with the 1996 NRC Guide for the Care and Use of Laboratory Animals.
General anesthesia was induced with 5 mg of intramuscular tamsulosin (Telazol) and 5% isoflurane/6L O2. Animals were endotracheally intubated and ventilated with 100% oxygen and anesthesia was maintained with 1.5%–2% isoflurane. Continuous oxygen saturation and heart rate were monitored throughout the experiment. An intravenous catheter was placed in the left marginal ear vein and maintenance intravenous fluid was infused. The left internal jugular vein was cannulated for central venous access as necessary. Either a median sternotomy or a wide clam-shall thoracotomy was used to gain access to the thoracic cavity for lymphatic mapping.
Lymphatic Mapping and SLN Identification
Pulmonary lymphatic mapping was performed by injecting 100 µl of either 2 µmol/L NIR QDs in phosphate buffered saline (PBS), pH 7.4, or 4 ml of 1% (17.6 mmol/L) isosulfan blue (Lymphazurin; BenVenue Laboratories Inc., Bedford, OH) intraparenchymally using a tuberculin syringe. Injections were positioned approximately 1.5 cm deep into the medial surface of the right and left upper and lower lobes. Completely reproducible parenchymal injections were performed by one surgeon after refinement of the technique. For colocalization studies QDs were injected 1 minute before isosulfan blue because the latter quenches the QD-fluorescence of the lymphatic vessels and SLN. Preinjection images were obtained to document tissue autofluorescence. Lymphatic flow was visualized in real-time using the imaging system. The first lymph node encountered on the path delineated by the fluorescent lymphatic vessels was defined as the SLN. Time from injection to first appearance in the SLN was recorded.
NIR Fluorescence-Guided Nodal Dissection
After the identification of lymphatic vessels and the SLN, real-time imaging was used to guide careful dissection of the SLN. Adjacent nodes, which did not exhibit fluorescence, were also excised as negative controls. Postresection NIR fluorescence imaging of the surgical field was performed to confirm that all sentinel node tissue had been excised.
Nodal Migration
In a subset of injections the identified SLN was not immediately dissected, but allowed to remain in situ for 4 hours to determine if QDs migrated beyond the single SLN as well as to determine migratory time course. Lymphatic drainage of the porcine lung is known to advance superiorly, traversing subsequent mediastinal nodes [19].
Histologic Analysis
Nodal tissue was embedded in optimal cutting temperature compound (OCT) (Miles Laboratories, Elkhart, IN), snap-frozen, and cryosectioned at 6 µm onto Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA). Sections were stained with hematoxylin and eosin (H+E) and then examined and photographed to confirm nodal tissue. Consecutive unstained sections were photographed on a NIR fluorescence microscope [20].
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Results |
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Comment |
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TopAbstractIntroductionMaterial and MethodsResultsCommentDISCUSSIONAcknowledgmentsReferences |
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We successfully identified a SLN in 100% of the injections, with most being solitary mediastinal nodes. This distribution is not unusual because pigs are reported to exhibit no pulmonary nodes located along the segmental and lobar bronchi. In studies of porcine cadavers it was determined that lymph from the lungs drains directly into mediastinal nodes and that there are no histologically identifiable lymph nodes proximal to these [19]. Our results concur with this report, except in 1 animal where both the left upper and lower lobe injections terminated in an intraparenchymal SLN in the left hilum. All other animals sustained direct drainage to a mediastinal node. In humans the lymphatic drainage of the lungs is primarily to the bronchopulmonary nodes. Lymph drains directly into mediastinal nodes approximately 25% of the time and might be important with regard to the development of "skip" metastases [2, 21, 22].
Several studies have documented the feasibility and usefulness of SLN mapping with regard to NSCLC. Current practice in thoracic oncology supports a complete anatomic mediastinal lymph node dissection for patients with NSCLC to accurately stage the cancer. Although usually straightforward mediastinal lymph node dissection can be associated with considerable morbidity [2]. By identifying a specific lymph node for histopathologic staging, pulmonary SLN biopsy could eliminate the need for a nontherapeutic mediastinal lymph node dissection and its associated complications. Intraoperative SLN biopsy may be especially useful in smaller sized tumors [23]. By allowing the pathologist to focus on one node, or even one area of one node, for analysis, SLN biopsy can potentially uncover more micrometastatic disease [24].
Despite the worldwide validation of the SLN hypothesis and its growing use in many forms of cancer the current technique exhibits considerable limitations when applied to NSCLC. The use of blue dye cannot adequately identify the SLN in thoracic malignancies because of poor tissue penetration and the presence of anthracosis. Recent reports of SLN mapping with regard to NSCLC have thus abandoned this tracer in favor of various radioactive lymphotrophic probes. A radiocolloid is injected around the tumor either preoperatively or at the time of thoracotomy, and a hand-held gamma probe is used to detect a "hot" node. In contrast to lymphatic mapping with the blue dye method, where success is defined as the identification of a blue lymphatic channel leading to a node, radiocolloid lymphatic mapping indicates no standard definition of success. Criteria defining a positive SLN have been largely arbitrary and empiric, usually based on ratios of background to SLN radioactivity. Detection of a true SLN with the radioisotope method can also be confounded by varying amounts of "shine though" radioactivity from the primary injection site essentially causing increased background radioactivity and decreasing ratio results.
Tracer particle size exhibits an important impact on migration time in SLN mapping. Particles less than 5 nm partition into blood and those between 5–10 nm can migrate through nodal tissue often resulting in false-positives, whereas those >1000 nm largely remain at the injection site [25]. Blue dye contains a range of less than 5 nm particle size and thus can pass through the SLN and traverse multiple nodes in addition to becoming intravascular. Because radioisotopes exhibit a more uniform particle size, various preparations of technetium-99m have been used with regard to pulmonary SLN mapping. Liptay and colleagues intraoperatively injected technetium-99m sulfur colloid and used a hand-held gamma probe to identify highly radioactive nodes [8, 9]. Migration time varied considerably, but on average radiocolloid migration was detectable after 10–15 minutes. In most instances only 1 SLN was identified. Technetium-99m sulfur colloid possesses a 40 nm particle diameter and thus requires anywhere from 10–60 minutes for migration to the SLN. Technetium-99m tin colloid, with a particle diameter of 1000 nm, has also been used [10]. Because migration time of this large tracer particle exceeds 6 hours, preoperative injection was necessary. The need for CT-guided tracer injection 12–24 hours before surgery, however, severely limits the usefulness of this agent. To avoid the use of a radioactive tracer, Nakagawa and associates used ferumoxides, a 100 nm diameter nonradioactive superparamagnetic tracer particle, to perform SLN mapping in NSCLC patients [26]. Because of technical limitations regarding their magnetometer, they could only perform ex vivo SLN identification after complete nodal dissection.
Pulmonary SLN mapping using NIR QDs overcomes these limitations. QDs can be engineered to precise sizes to optimize migration time and SLN retention. The optimal size of 15–20 nm allows for rapid transit to, and retention in, the first node. Additionally these optical biological probes are bright in the NIR region and can easily be viewed at depths of approximately 1 cm of skin, allowing visualization of a mediastinal SLN beneath thick pleura. The intraoperative use of NIR QDs to visually map lymphatic flow and identify SLNs obviates the necessity for preoperative radiocolloid injections and the use of blue dye. Instead surgeons can track lymphatic flow in real-time and guide their nodal dissection with the aid of on-line video feedback after one simple intraoperative injection. NIR fluorescence from the injection site does not interfere with visualization of the SLN on the video monitor and the actual surgical field remains unchanged because NIR QDs are invisible to the human eye. Finally the entire NIR-imaging system could be potentially translated to a thoracoscopic platform because it is optically based and completely portable.
A limitation regarding the clinical use of NIR QDs for SLN mapping is their potential toxicity. These biological probes contain heavy metals at their cores with an amphiphilic organic coating. Cadmium, telluride, selenide, and alkyl phosphines exhibit known acute and chronic toxic profiles as isolated heavy metals, but their toxicities as precomplexed nanocrystals are unknown. In our nonsurvival animal studies we observed no evidence of acute toxicity; heart rate and rhythm, blood pressure, and oxygen saturation remained stable during prolonged operative procedures. As we reported elsewhere extrapolating known dose-effect relationships for inhalation and oral exposures of these heavy metals indicates that our dose of 200 pmol of NIR QDs corresponds to a dose fifty times lower than that defined as causing respiratory symptoms and three-hundred times lower than that required to produce renal insufficiency [14]. Of importance to this study, most of the injected dose is removed by resecting lung and nodal tissue, so toxicity may, in fact, be negligible.
In summary we report a highly sensitive technique for pulmonary SLN mapping using NIR fluorescent QDs that overcomes many of the formidable limitations of current techniques. The brilliant fluorescence and low tissue scatter of NIR QDs, along with their precisely engineered size, constitutes them as optimal lymphotropic optical probes. Thus in the future they may prove useful with regard to SLN mapping of human lung cancer.
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DISCUSSION |
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TopAbstractIntroductionMaterial and MethodsResultsCommentDISCUSSIONAcknowledgmentsReferences |
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Our group and others have achieved a greater than 80% success rate identifying sentinel nodes in early stage lung cancers. Preoperative or intraoperative injection of radioisotopes are both limited by in vivo tumor background radioactivity and tracer migration time. Dr Soltesz and his colleagues have presented an elegant study demonstrating the success of an approach that may overcome several limitations regarding the radioisotope technique.
I have three questions for the authors. First, the majority of sentinel nodes were mediastinal in your porcine model. In humans, the sentinel node is more often located deeper in the lobar and hilar areas. Will your technique exhibit more difficulty with regard to visually identifying these deeper nodes?
Second, we inject tracer directly into the tumor to avoid aerosolization of radioisotope and peribronchial background radioactivity. Was that an issue for you when injecting the fluorescence directly into the normal lung parenchyma?
Lastly, you mentioned this in your talk briefly, but how will you address the potential toxicities of the heavy metals used in your nanocrystal tracer when adapting this technique to humans?
I thank the members of the Society for the privilege to discuss this promising work and I thank the authors for providing me with an advanced draft of their manuscript. Thank you.
DR SOLTESZ: Dr Liptay, thank you for your kind remarks. In response to your questions, we do not expect our imaging system to experience difficulty with regard to visualizing any of the hilar lymph nodes that would be expected to be present in humans. In other studies, we were able to visualize a depth approaching 1.5 cm in the skin using near-infrared quantum dots with our imaging system. Importantly, when we injected the quantum dots into the medial surface of the right upper lobe at a depth of 1.5 cm, we were still able to visualize the fluorescence fairly clear through the lateral surface of the lung lobe at a depth of 4 cm. Therefore, we do not expect there to be difficulty visualizing what would amount to hilar lymph nodes in the human.
Second, in terms of difficulty injecting the quantum dots peritumorally or intratumorally, we did not experience any leakage. There was no toxicity of the quantum dots in terms of skin irritation or aerosolization risk. The amount of quantum dots that we inject, which is on the order of 200 pmol, is approximately 300 times less than what would be expected to cause any toxicity to a human.
Finally, we expect the toxicity related to the use of these quantum dots to be minimal, because we use such a small dose and resect the lung tissue along with the node. Also, because the particle size is 15–20 nm, we have conclusively demonstrated that the quantum dots are actually retained in the first node. We do not have to be concerned regarding systemic spread and removal of that remaining quantum dot load. Thank you.
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