Many tracers have been introduced into current medical practice with the purpose of improving lymphatic mapping techniques, anatomic visualization, and organ/tissue perfusion assessment. Among them, three tracers have dominated the field: indocyanine green, technetium-99m radiocolloid (Tc99m), and blue dye. Tc99m and blue dye are used individually or in combination; however, given particular challenges with these tracers, such as the need for a preoperative procedure by nuclear medicine and cost, other options have been sought. Indocyanine green has proven to be a promising alternative for certain procedures, as it is easy to use and has quick uptake. Its use in the management of gynecologic cancers was first described for sentinel lymph node mapping in cervical cancer, and later for endometrial and vulvar cancers. This review provides an in-depth look at these mapping substances, their uses, and the potential for new discoveries.
- surgical procedures
- vulvar and vaginal cancer
- uterine cancer
- cervical cancer
- sentinel lymph node
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- surgical procedures
- vulvar and vaginal cancer
- uterine cancer
- cervical cancer
- sentinel lymph node
The landscape of lymphatic mapping substances has evolved significantly over the years, with broadened utility in the field of gynecologic oncology. Some newer substances are associated with higher detection rates, improved side effect profiles, and lower cost.
Mapping substances range in their abilities and uses given their drastically different compositions. The ideal mapping substance should have a high detection rate with the least possible cost and side effects. In the setting of sentinel lymph node (SLN) mapping, this would entail a low false-negative rate (ie, negative SLNs on pathology in the setting of positive lymph node in the remainder of the lymph node dissection) in order to minimize undertreatment of patients. For uterine and cervical cancers, the ideal lymphatic mapping approach would also have the highest bilateral pelvic mapping rate. For vulvar cancers, the goal is to achieve the highest rate of mapping of at-risk groins.
The three conventional tracers currently used in gynecologic surgery are technetium-99m radiocolloid (Tc99m), various blue dyes, and indocyanine green. Historically, Tc99m alone or in combination with blue dye has been most often used in gynecologic surgery. This is unlike the field of ophthalmology, in which indocyanine green has been used to visualize the retinal and choroid vascularization for more than 40 years.1
Several reviews have investigated the use of various mapping substances to perform different procedures; however, none has taken a broader look at their use across multiple types of procedures. This review offers an in-depth look at the various currently available mapping substances, their use in gynecologic surgery, and the potential for new discoveries.
Landscape of Available Tracers
Tracers allow for the gross observation of biological processes that are usually invisible. These tracers include but are not limited to indocyanine green, Tc99m, and blue dye.
Indocyanine green is a tricarbocyanine compound developed by Kodak Research Laboratories in the early 1950s for use in fluorescent or near-infrared photography. By 1956, the Food and Drug Administration had approved the compound for use in clinical settings.2 Physician-scientists quickly realized the potential clinical applications of indocyanine green in medical imaging, as the chemical is water-soluble yet hydrophobic, has a low toxicity profile, and can be used to image biological properties that previously could not be viewed outside of the visible light spectrum.
Indocyanine green is composed of hydrophilic sulfonyl groups, as well as a hydrophobic polymethine chain linked with two nitrogen-based, heterocyclic moieties. This organic structure allows indocyanine green to be prepared in a water-soluble solution that can be easily delivered intravenously to patients, but that also has a strong binding affinity to albumin. Through its affinity for albumin, indocyanine green mimics the migration of biochemical compounds and can be quickly absorbed and distributed throughout lymphovascular systems.3
Indocyanine green becomes fluorescent when excited by a laser beam or near-infrared light source at wavelengths of approximately 800 nm or longer.4 The photons of energy supplied by the light source are then absorbed by indocyanine green, which then reaches an excited energy state and fluoresces light at a higher wavelength.2 5 Cameras are carefully calibrated with filters in order to capture the emitted wavelength of indocyanine green photons and thus produce a fluorescence image of indocyanine green in real time. Interestingly, cancer cells have high metabolic rates. As such, tumors have a natural proclivity for absorbing indocyanine green at higher concentrations than surrounding tissue.5 Therefore, when tumors are excited with infrared light, they fluoresce with a higher intensity.
Indocyanine green has an excellent safety profile. Its high affinity for albumin means there is almost no leakage of the compound into surrounding tissue. The liver absorbs and excretes indocyanine green but does not metabolize it into toxic byproducts.2 Additionally, the concentrations used for fluorescence imaging in medical studies are well below the dose toxicity threshold established in animal models.2 6 Taken together, these unique properties of indocyanine green make it an ideal compound for intraoperative perfusion assessment as well as lymphatic mapping.
Technetium derives its name from the Latin word for “artificial”, as it was the first artificially formed element. While Tc99m can be found in nature, the majority of the world’s supply is manufactured.7 Technetium was first described in 1937 during an analysis of radioactive molybdenum, which was used in particle accelerators. Scientists correctly assumed that the radioactive molybdenum was a byproduct of radioactive decay of a different element that was emitting energy, that is, technetium.7
Scientists specifically isolated the “metastable” isotope of technetium-99 and named the product techenetium-99m.8 9 Unlike other technetium isotopes, this isotope is used in nuclear medicine as a tracer, since its metastable properties give it a much shorter half-life. In turn, this shorter half-life allows for the capture of an increased number of emissions over a short time period, creating an emission density that can form high-resolution images.8 Additionally, Tc99m emits gamma rays at 140 keV, an energy level strong enough for good tissue penetration but manageable enough to organize into an image without too much noise.8–10 Most importantly, this radionucleotide is capable of producing a discernable image while minimizing patient exposure to doses of harmful radiation, as the half-life is relatively short.
Imaging technologies that use technetium radiocolloids rely on the fundamentals of gamma ray decay.10 11 Technetium radiocolloid tracers emit specific energy waveforms through the process of gamma ray decay,11 which is captured via scintigraphy. Scintigraphic cameras use crystals constructed of sodium iodide. The patient is given a dose of radioactive Tc99m, which emits gamma photons as it decays. These emitted photons in turn collide with the crystals inside the cameras and excite the iodide atoms within the crystals. In an excited state, these iodide atoms are freed from the crystal’s surface and release a flash of light that is captured by the camera. This flash of light is similar to the fluorescence flashes captured in infrared cameras used for indocyanine green.9 11
Tc99m products have different biological absorptive properties depending on their oxidative states. In its most raw form, Tc99m forms “pertechnetate” when initially produced by artificial generators. This product is in a high oxidative state and is only absorbed by osteoblasts, making it exclusively suitable for bone scans.10
Over the years, physician-scientists have manipulated the formulation of “raw” Tc99m by adding reducing agents, and more recently, by adding ligands. Each formulation preserves the radioactive properties of Tc99m while altering tissue-specific binding and absorptions sites.11 One of the initial formulations of Tc99m was a sulfur colloid. In the 1960s, imaging using this formulation was the gold standard for evaluating the liver and spleen, before the development of CT. The spleen actively scavenges sulfur, which increases uptake in this organ.11 This formulation was restrictive, as sulfur colloids are too large to distribute freely into smaller vascular and lymphatic channels. As a result, Tc99m was later formulated with human serum albumin as well as human colloid albumin.11 12 These protein ligands, which are smaller, are more capable of penetrating the lymphovascular system. This development enabled increased applications of nuclear imaging involving more organs and more biological compartment.11 12 The ability to develop biotracers using the fundamentals and technologies of Tc99m is limitless, especially as the field of synthetic biology has exploded with the advent of high-throughput screening.
The two substances most used as blue dyes are methylene blue and isosulfan blue (sometimes referred to as patent blue). The substances are functionally interchangeable when used as biotracers, with equal efficacy; however, their different chemical structures explain key differences between the substances.13 14 Methylene blue is cheaper, more available, and has a better side effect profile. Isosulfan blue has been associated with higher rates of anaphylaxis.15
Methylene blue has several other medical applications, extending beyond its function as a tracer. The dye was invented in the 19th century, and its first application was as an anti-malaria drug. Methylene blue has become a first-line treatment for cyanide poisoning, as well as for methemoglobinemia due to its strongly reductive properties. The reductive properties of methylene blue and isosulfan blue give these dyes an increased affinity for acids such as nucleic acids.16 This property is readily exploited in its use as a biologic tracer and dye, as it stains cancer cells a darker blue due to their increased production of DNA and RNA.
The specific structures of methylene blue and isosulfan blue are different, but they share important overall structural characteristics. Much like indocyanine green, the structure of both blue dyes contains both hydrophobic and hydrophilic regions. The hydrophilic regions allow for intravenous administration, as the substances dissolve in water and blood. The hydrophobic regions serve as high-affinity binding sites for oncotic proteins that circulate throughout the lymphovascular system. One of the differences, however, is that the blue dyes can only be seen in the visible light spectrum. This difference contributes to the overall accessibility and low cost of blue dye technology.
Use of Tracers in Gynecologic Cancer Treatment and Care
SLN Mapping in Early-Stage Vulvar Cancer
SLN mapping became an accepted standard of care for the treatment of vulvar cancer with the findings of the Gynecologic Oncology Group (GOG)−173 and the GROningen INternational Study on Sentinel nodes in Vulvar cancer (GROINSS V) studies, which demonstrated low false-negative predictive values with the use of SLN mapping compared with complete inguinal femoral lymphadenectomy.17 18 Given that SLN assessments are increasingly central to the care of our patients, it is vital to determine the best technique for mapping.
It is well established that lymph node involvement is the most important prognostic factor for survival in vulvar cancer. SLN mapping has been proposed as an alternative to inguinofemoral lymph node dissection in early-stage vulvar cancer.17 19 SLN mapping for vulvar cancer has been traditionally performed using a dual-modality approach: (1) preoperative Tc99m injection with lymphoscintigraphy intraoperative gamma probe detection, and (2) intraoperative peritumoral/vulvar injection with blue dye.
A meta-analysis performed by the Cancer Care Ontario group reported a 63% overall detection rate with the use of blue dye alone compared with 85% with the use of radiocolloid alone.20 The overall detection rate with the combination of blue dye and radiocolloid was 86.9%. The false-negative rates with blue dye alone and radiocolloid alone were 9.3% and 10.4%, respectively. The use of a combination technique lowered this rate to 6.6%.20 Given these findings, the group recommended the use of radiocolloid tracers alone or in combination with blue dye for SLN mapping of early-stage vulvar cancer.
The use of these radiotracers requires complex logistics, with the involvement of nuclear medicine, which is not readily available in all centers. As such, alternate techniques have been considered, including near-infrared fluorescence imaging with indocyanine green, for which small retrospective data are available. Crane et al reported on the successful use of indocyanine green with near-infrared fluorescence for SLN mapping in 10 patients with vulvar cancer,21 demonstrating a 90% detection rate (26 of 29 SLNs). Huttman and colleagues demonstrated a 100% detection rate with indocyanine green; however, the high detection rate was attributed to their use of indocyanine green with human serum albumin to increase the fluorescence intensity and hydrodynamic diameter of the indocyanine green.22 23 A randomized controlled trial, however, did not demonstrate a difference between indocyanine green and indocyanine green adsorbed to human serum albumin.24 Leitao et al reviewed 15 patients who underwent Tc99m injection and lymphoscintigraphy the day before surgery and indocyanine green administration the day of surgery.25 Tc99m resulted in successful mapping in 19 (83%) of 23 groins. Indocyanine green resulted in successful mapping in 25 (89%) of 28 groins. This group recently performed a larger review and reported a 96.3% detection rate (26 of 27 groins) with the use of indocyanine green alone in early-stage vulvar cancer.26 Radiocolloid tracers alone or in combination with blue dye remain the standard of care for the detection of SLNs in vulvar cancer. The use of indocyanine green warrants further investigation, as it offers the opportunity to eliminate the high cost and inconvenience of technetium radiocolloid while maintaining high detection rates.
SLN Mapping in Early-Stage Endometrial Cancer
Surgical staging in women with early-stage endometrial cancer entails a hysterectomy, bilateral salpingo-oophorectomy, and lymph node assessment. The extent of lymph node assessment in early-stage endometrial cancer, however, continues to be highly debatable. The necessity of a full lymph node dissection was questioned after two randomized trials demonstrated no survival benefit.27 28 However, lymph node assessment remains an important part of surgical staging, as it provides valuable prognostic information and can guide treatment decisions.29 SLN mapping for endometrial cancer staging was introduced as an alternative approach to complete lymphadenectomy.
The National Comprehensive Cancer Network incorporated the use of SLN mapping in the management of endometrial cancer into their guidelines in 2014. Multiple injection methods and tracers have been studied to improve SLN detection. SLN mapping initially included lymphoscintigraphy with radiocolloid and intraoperative detection with a gamma probe, as well as an intraoperative injection of blue dye. Recently, indocyanine green with near-infrared imaging has become increasingly used, as multiple single-institution studies have reported superior detection rates with indocyanine green compared with blue dye and radiocolloid in detecting bilateral SLNs after a cervical injection.30 31
The use of indocyanine green as a standard technique has been further validated by the results of the FILM trial. In the FILM trial, which was a randomized, international, multicenter controlled trial, near-infrared imaging with indocyanine green was compared with isosulfan blue dye in detecting SLNs in both uterine and cervical cancers.32 The study reported a 74% rate of detection of at least one SLN with isosulfan blue dye compared with 96% with indocyanine green. Bilateral mapping rates were 31% and 80%, respectively. Given these results, indocyanine green offers a new, validated approach to SLN mapping in endometrial cancer.
SLN Mapping in Cervical Cancer
SLN mapping was first established as a safe and feasible approach in cervical cancer by the AGO study group trial, and more recently by the SENTICOL I and II trials.33 34 Similar to endometrial cancer, Tc99m in combination with blue dye was considered the primary technique for SLN mapping before the introduction of indocyanine green. The AGO study group trial and the SENTICOL I trial used technetium radiocolloid and blue dye. Many recent studies have demonstrated the promising potential of indocyanine green.30 32 35
Buda et al reported overall detection rates of 97%, 89%, and 100% for radiocolloid with blue dye, blue dye alone, and indocyanine green, respectively.30 As mentioned above, the FILM trial results not only support the use of indocyanine green in endometrial cancer but also in the detection of SLNs in cervical cancer.32 As with endometrial cancer, the use of indocyanine green is increasingly becoming part of standard of care for these procedures given its high detection rate, higher signal-to-background ratio, improved cost effectiveness, and decreased adverse effects and toxicity.36
Although the use of indocyanine green is gaining favor, there remains a lack of consensus regarding the optimal concentration and volume of the tracer. Different centers use different concentrations ranging from 0.39–5.0 mg/mL and volumes ranging from 0.2–4.0 mL. There is limited evidence at this time to indicate optimal dosage; however, there is some retrospective evidence suggesting that the use of larger amounts does not correlate with an improved bilateral detection rate.37 However, when comparing across multiple studies, a concentration of 1.25 mg/mL with a total of 4.0 mL administered appears to be the most commonly used dosage.38
Vascular Perfusion at Time of Radical Trachelectomy
One of the main concerns when performing a radical trachelectomy is whether the uterine artery should be preserved to maintain adequate perfusion to the uterus and not compromise future fertility. With the use of indocyanine green and near-infrared light, real-time observation of uterine perfusion after completion of trachelectomy is possible39(Figure 1). Although not routinely used in the surgical setting, this substance allows surgeons to investigate uterine perfusion patterns and offers surgeons an additional tool to evaluate uterine perfusion.
Indocyanine green is also useful for intra-urethral injections. It allows for the visualization of the ureter under near-infrared light, offering a method by which to facilitate real-time delineation of the ureter in the hope of preventing iatrogenic ureteral injury during pelvic surgery (Figure 2). Indocyanine green is administered through an open ureteral catheter that is inserted into the ureteral orifice.40 This method’s effect on ureteral injury rates has not been studied.
Tissue Perfusion During Reconstructive Surgery
There are a variety of methods by which surgeons are able to evaluate tissue perfusion. Indocyanine green angiography provides real-time feedback on perfusion, providing the surgeon with valuable information when evaluating free and pedicled flaps.41 Recent studies have demonstrated that this imaging modality is helpful in the assessment of flap harvesting and design, and quantitative and objective perfusion parameters seem to be accurate for flap perfusion assessment.41 According to a recent investigation, indocyanine green angiography is a highly valuable tool in aiding flap shaping according to optimal perfusion zones42 (Figure 3). Specifically, the arterial phase of dye distribution demonstrates a highly sensitive report of tissue vascularity and perfusion.43 This offers a promising realm for its use in vulvar flap reconstruction surgery.
Anastomotic Perfusion Assessment During Rectosigmoid Resection
Complications from intestinal surgery are devastating. Anastomotic leaks, for example, are associated with an up to 21% mortality rate.44 Poor oxygenation secondary to poor perfusion of the anastomosis is a likely culprit in failed anastomoses resulting in leakage. In the colorectal literature, indocyanine green angiography has been associated with decreased anastomotic leak rates and improved outcomes.45 46 PILLAR II was a prospective observational study that enrolled patients with colorectal pathology to undergo evaluation of colon anastomoses using intraoperative fluorescence angiography with indocyanine green.45 They demonstrated that with the use of indocyanine green, the anastomotic leak rate decreased. A more recent study investigated indocyanine green’s use in uterine and ovarian cancer debulking surgery at the time of low anterior resection and found a decrease in anastomic leaks, with significantly fewer postoperative pelvic abscesses when the substance was used.47 (Figure 4)
New and Innovative Tracing Substances
While the focus of most perfusion mapping studies in gynecologic oncology has been on technetium colloids, blue dye, and indocyanine green, alternative approaches are actively being studied in both gynecologic and non-gynecologic cancers. Some of these methodologies are meant to be used intraoperatively, where the SLNs or tissue are mapped by either visual assessment or handheld device identification. Others are radiographic imaging modalities in combination with tracer or dye. In this section, we will review the current methodologies that are being studied for perfusion mapping.
Intraoperative Identification Techniques
[99mTc]Tilmanocept is a molecule that binds receptors in reticuloendothelial cells in the nucleus and can be evaluated via an intraoperative handheld gamma detector. It is potentially advantageous over sulfur colloids, as it is better tolerated by patients and is cleared faster. It has been studied in phase 3 trials in breast cancer and melanoma and has been shown to successfully identify SLNs with 99% concordance with blue dye.48 49 Preclinical animal model trials have been successful in endometrial cancer, with SLN identification via robotic surgery, by tagging the tilmanocept to near-infrared fluorescent dye to facilitate intraoperative identification during robotic surgery, as well as with gallium-68 to allow for assessment on positron emission tomography.50 A phase 1 trial to assess the potential of fluorescent-tagged tilmanocept in SLN mapping for endometrial cancer in humans is under consideration.
Carbon nanoparticles are 150 nm particles that can be injected into the anatomical area of interest and drain through the lymphatic vessels. They are then visualized directly as a black stain in the lymphatic channels and SLN. Carbon nanoparticles are advantageous in that nuclear medicine is not required. Overall, they are well tolerated; a major side effect is skin staining when used in patients with breast cancer. They have been successfully studied in melanoma, breast, and colon cancers.51 More recently, carbon nanoparticle use was studied in endometrial cancer, demonstrating promising results in a small prospective trial of 115 patients.52 Cervical injection route (in 65 of the patients) was associated with 100% sensitivity and negative predictive value. Carbon nanoparticles were shown to have a comparable detection rate to those of currently used methods in endometrial cancer. More studies are needed to confirm these findings.
India ink is an injectable colloid suspension of carbon particles that dyes SLNs black. A study in melanoma showed that it did not improve the identification of lymph nodes when used in combination with sulfur radiocolloid and blue dye.53 In a study in early-stage cervical cancer, India ink was used in combination with radiocolloid and blue dye,54 once again demonstrating no improvement in overall SLN detection.
Superparamagnetic iron is a tracer that can be injected preoperatively for intraoperative magnetic detection. It has been studied as an alternative to SLN mapping in breast cancer and has been shown to be non-inferior.55 56 For use in endometrial and cervical cancers, this would require the development of a new surgical device that could be used for magnetic detection during abdominal surgery. It could also be considered for further study in inguinal lymph node assessment in vulvar cancer.
Contrast-enhanced ultrasound is a technique that employs the injection of perfluorobutane microbubbles to visualize lymphatic channels on ultrasound and facilitate the identification of SLNs for subsequent biopsy or resection with skin marking or wire localization. This method has been studied in breast cancer, with varying success (70–100% detection rate).57 It has not been tested in gynecologic cancers.
Hybrid dextran-gadolinium nanoparticles were developed for potential use in SLN mapping using MRI. Current MRI contrast dyes cannot be used for SLN assessment. In an animal model study, this new particle demonstrated a good safety profile and successfully identified SLNs, which were subsequently identified via methylene blue injection to the same site.58 This model has yet to be tested in humans.
Laser speckle imaging is a non-invasive technique that has been used for assessing tissue perfusion in multiple settings, including intraoperatively during breast reconstruction and to assess gastric blood flow after esophagectomy.59 60 The technique is designed to detect movement of red blood cells based on laser light formation, and it is able to detect perfusion to the level of the dermis. Trial data with this approach, within and outside the field of gynecologic cancer, are lacking. However, it could be considered for evaluation of bowel anastomoses or vulvar flap procedures.
There are several available tracers for lymphatic mapping, anatomic visualization, and organ/tissue perfusion assessment. When pertaining to lymphatics, the current standard of care depends on the anatomical location. In vulvar cancer, the combination of technetium radiocolloid and blue dye is the most commonly employed method. However, recent retrospective trials have suggested that indocyanine green may be an appropriate alternative, as it has the potential to improve detection rates while decreasing cost and side effects. In endometrial and cervical cancers, emerging randomized controlled trial data have shown the benefit of indocyanine green in SLN mapping.
Indocyanine green offers intraoperative anatomical detection, such as detection of the ureter. Indocyanine green could be adapted to identify other structures in the pelvis that require anatomical delineation during complex dissection. Indocyanine green has also shown efficacy in evaluating bowel anastomosis in the colorectal literature and is currently being investigated in the gynecologic realm. This tracer offers a multitude of opportunities and warrants further investigation in the field of gynecologic surgery.
New tracers with the potential for implementation in gynecologic oncology are emerging. Investigation into the potential use of [99mTc]tilmanocept and carbon nanoparticles in endometrial cancer has begun. Some tracers, such as India ink, have not demonstrated promising initial findings, while more novel tracers, such as super-paramagnetic iron, require further investigation. Radiographic imaging modalities in combination with tracer or dye offer additional opportunities. The optimal technique in gynecologic oncology continues to evolve, necessitating continued investigation at each step.
Editor's note This paper will feature in a special issue on sentinel lymph node mapping in 2020.
Twitter @jackiefeinberg, @leitaomd
Contributors All authors have contributed significantly to warrant authorship.
Funding This study was funded by National Cancer Institute.
Competing interests Outside the submitted work, ML is an ad-hoc speaker for Intuitive Surgical.
Patient consent for publication Not required.
Provenance and peer review Commissioned; externally peer reviewed.