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Optical imaging using near-infrared (NIR) fluorescence provides new prospects for general and oncologic surgery. ICG is currently utilised in NIR fluorescence cancer-related surgery for three indications: sentinel lymph node (SLN) mapping, intraoperative identification of solid tumours, and angiography during reconstructive surgery. Therefore, understanding its advantages and limitations is of significant importance. Although non-targeted and non-conjugatable, ICG appears to be laying the foundation for more widespread use of NIR fluorescence-guided surgery.
The identification of structures that need to be resected (e.g. tumour tissue, lymph nodes) and structures that need to be spared (e.g. nerves, ureters, bile ducts) is of paramount importance in oncologic surgery. In daily surgical practice, surgeons mainly rely on palpation and visual inspection. However, tumour-positive resection margins and surgical morbidity as a result of damage to vital structures are not uncommon. Thus, there is a need for new intraoperative imaging modalities that can provide real-time assessment of tumour borders and affected lymph nodes, while eliminating the risk of damage to vital structures.
Optical imaging using near-infrared (NIR) fluorescence is a new technique that can be used to visualise structures in real-time during surgery. Advantages of NIR fluorescent light (700–900 nm) include high tissue penetration (millimetres to centimetres deep) and low autofluorescence, thereby providing sufficient contrast . Because the human eye is insensitive to NIR wavelengths, the use of NIR light does not alter the surgical field. Recently developed intraoperative imaging systems are able to provide simultaneous acquisition of surgical anatomy (white light, colour video) and NIR fluorescence signal [2–4]. Therefore, the use of NIR fluorescence imaging could potentially be of great value in the intraoperative detection of critical anatomical structures and oncologic targets.
In addition to NIR fluorescence imaging systems, exogenous NIR fluorescent contrast agents are necessary to visualise specific tissues. Ideally, tumour cells are labelled by targeted contrast agents. However, the only fluorescent contrast agents currently registered by the FDA and EMA for clinical applications are indocyanine green (ICG; peak emission ≈ 820 nm), methylene blue (peak emission ≈ 700 nm), and fluorescein (peak emission ≈ 520 nm, below NIR spectrum). This review is focused on the clinical use of ICG, due to its preferable fluorescent characteristics and widespread use in clinical research. ICG provides a higher signal-to-background ratio because of lower autofluorescence and increased tissue penetration at 820 nm compared to lower wavelengths and has a greater “brightness” (i.e., quantum yield) compared to methylene blue .
ICG is currently utilised in NIR fluorescence image-guided oncologic surgery for multiple indications. NIR fluorescence imaging has the potential to improve sentinel lymph node (SLN) mapping in multiple types of cancer, by real-time transcutaneous and intraoperative visualisation of lymphatic channels and subsequent detection of the SLN [3,4,6–29]. Additionally, ICG NIR fluorescence is used for endoscopic marking of colorectal tumours and intraoperative identification of certain solid tumours after intravenous injection [30–32]. Moreover, NIR fluorescence angiography using ICG can be used in intraoperative assessment of tissue perfusion in reconstructive surgery for ablative defects following oncologic surgery .
The aim of this paper is to review the available clinical studies using ICG in NIR fluorescence-guided cancer surgery in order to understand current applications, limitations, and future prospects.
Several NIR fluorescence imaging systems have been described for intraoperative clinical use (reviewed in Gioux et al. ). Although differing in their technical specifications, all of these systems provide the surgeon with an image of the NIR fluorescence signal that would otherwise be invisible to the human eye (Table I). The majority of clinical studies published to date use the commercially available Photodynamic Eye (PDE, Hamamatsu Photonics, Hamamatsu, Japan) imaging camera system . Other commercially available systems are the SPY system (Novadaq Technologies, Concord, ON, Canada) and the Fluobeam (Fluoptics, Grenoble, France). Several others imaging systems have been used in clinical studies but are not commercially available: HyperEye  (Kochi Medical School, Kochi, Japan), the FLARE and Mini-FLARE  (Beth Israel Deaconess Hospital, Boston, MA, USA), the FDPM imager  (Texas Medical Center, Houston, TX, USA), and a prototype camera system from Munich  (Technical University Munich, Munich, Germany and SurgOptix Inc., Redwood Shores, CA, USA) .
ICG is a negatively charged, amphiphilic, water-soluble but relatively hydrophobic, tricarbocyanine with a molecular mass of 776 Da [36,37]. ICG has been registered for several decades to determine cardiac output, hepatic function, and ophthalmic perfusion. Rapid registration was attributable to favourable characteristics such as the confinement to the vascular compartment by binding to plasma proteins, the fast and almost exclusive excretion into the bile, and the very low toxicity of ICG [38,39]. ICG is safe to use, as the number of allergic reactions is very low (1: 10 000, as reported by manufacturer). The dose used for standard diagnostic procedures lies between 0.1 and 0.5 mg/kg. Above 0.5 mg/kg, the incidence of immediate allergic reactions increases .
In plasma, ICG has an absorption peak around 807 nm and an emission peak around 822 nm, which is within the NIR window (Fig. 1). After intravenous administration, ICG has a short half-time of 150 to 180 seconds and is cleared exclusively by the liver . Relatively hydrophobic ICG molecules bind rapidly and almost completely to serum proteins. Protein binding reduces aggregation, increases brightness (i.e., quantum yield) by over 3-fold, and increases effective hydrodynamic diameter to that of the bound proteins [38,42–44]. Hydrodynamic diameter has important implications for distribution and transport of ICG for tumour visualisation and retention in the SLN as discussed below [6,45,46].
NIR fluorescence imaging provides new opportunities to improve and extend the indications of the SLN procedure. Gamma ray-emitting radiotracers and blue dyes are currently used as the standard of care in clinical practice. However, the use of gamma ray-emitting radiotracers requires involvement of a nuclear medicine physician, and localisation of the SLN can be difficult using a handheld gamma probe. Also, preoperative access to the injection site is required. Blue dyes cannot be easily seen through the skin and fatty tissue. Additionally, the learning curve for the standard SLN procedure using these techniques is estimated to be 60 required cases for technical proficiency when working with breast cancer patients .
NIR fluorescence imaging using ICG has been shown to visualise superficial lymphatic channels transcutaneously . Thereby, it could potentially reduce time of surgery and improve localisation of the SLN so that a small incision can be made, while maintaining a high identification rate. Moreover, the NIR fluorescence signal could aid the pathologist in both preparing and analysing the tissue specimen [13,18]. It should be noted, however, that NIR fluorescence detection is in the millimetre to centimetre range, far less than radioactive tracers, which requires caution when examining thick tissues.
Studies using ICG as a NIR fluorescent lymphatic tracer in SLN procedures are summarized in Table II. A total of 25 studies have been published using ICG as lymphatic tracer in SLN procedures in breast, skin, gastrointestinal, non-small cell lung, oropharyngeal and gynaecological cancer [3,4,7–29]. Differences in imaging systems, ICG doses and injection sites prevent direct comparison of the results. In the next sections, the results will be discussed for each tumour type separately.
Eleven studies report the use of ICG as a NIR fluorescent lymphatic tracer in the SLN procedure in a total of 548 breast cancer patients [3,12–21]. Before the introduction of NIR fluorescence imaging systems, Motomura et al. used only the intrinsic green colour of ICG and identified the SLN in 73.8% of patients. After the introduction of intraoperative NIR fluorescence imaging systems, higher identification rates of 87.5% to 100% (aggregate 98.6%) were obtained and an average of 3.4 (range 1.5 to 5.4) SLNs were identified. Two studies performed an axillary dissection irrespective of the SLN status and found an aggregate false-negative rate of 7.7% in 39 patients with a negative SLN [12,14]. Additionally, as a result of the capability of NIR fluorescence light to penetrate tissue, ICG offers non-invasive imaging of lymphatic flow (Fig. 2). Upon injection of ICG, travel time to the axilla is 1 to 10 min [13,16]. The small size of the ICG particle is probably responsible for this relatively high velocity, which has logistical advantages compared to relatively larger gamma ray-emitting radiotracers. Hojo et al.  compared ICG to patent blue in 113 patients and showed that ICG had a higher identification rate (100%) than patent blue (93%). Three studies compared the method of SLN detection by ICG fluorescence (71 out of 73 nodes) and the radiotracer (70 out of 73 nodes) [12,15,17]. Both techniques are used simultaneously in all studies; therefore, both identification rates were similar. However, no comparison can be made as to whether one is superior to the other.
Several factors influence the success of the SLN procedure using ICG. In the reported clinical trials, various doses of ICG have been used ranging from 0.01 mM to 6.4 mM. Sevick-Muraca et al.  found that a minimal dose of 0.01 mM ICG is required for successful SLN mapping. Mieog et al.  allocated patients in groups of escalating ICG concentrations from 0.05 mM to 1.0 mM diluted in albumin and obtained the highest brightness of the SLN using a concentration between 0.4 mM to 0.8 mM ICG (1.6 ml injection volume). Additionally, because of its relatively small hydrodynamic diameter, ICG is able to pass through the sentinel node to second-tier nodes and eventually spread through the subcutaneous tissue . To surpass this effect, imaging should to be performed shortly after ICG administration. Furthermore, as a result of the limited tissue penetration of the fluorescent signal, visualisation is limited once ICG has reached the axillary fossa, particularly in patients with a high body mass index [13,19,49].
Four studies reported the use of ICG as an NIR fluorescent lymphatic tracer in the SLN procedure in a total of 42 skin cancer patients [22–25]. The NIR fluorescence-guided SLN procedure resulted in identification of at least one SLN in 41 of the 42 patients (total number of SLNs identified was not reported). This is concordant with recent trials using conventional techniques, which showed a 93% to 100% identification rate [50,51]. Upon intradermal injection, ICG enables easy visualisation of the subcutaneous lymphatic drainage, which takes approximately 15 min after injection to reach the SLN and stays visible for at least three hours . The results of these studies are promising. However, larger trials are needed to assess patient benefit.
Nodal status is one of the most important prognostic factors in gastric and colorectal cancer. It is hypothesised that the SLN procedure in gastro-intestinal cancer patients can improve nodal staging . Currently, prophylactic lymphadenectomy is considered the standard of care for these patients. Several studies have assessed the use of the SLN procedure using radiotracers or blue dye, or both [52–54]. However, these studies show varying lymphatic drainage patterns and report high rates of skip metastases, preventing the introduction of the SLN procedure in general clinical practice.
In early gastric cancer, four studies reported the use of ICG as NIR fluorescent lymphatic tracer in the SLN procedure in a total of 158 patients [26–29]. ICG was injected during surgery or at one to three days before surgery. After both preoperative subserosal and preoperative submucosal injection of ICG, lymphatic vessels draining the tumour could be visualised [26–29]. The identification rates ranged from 90.9% to 96.4% (aggregate 94.9%) with an average number of SLNs identified of 3.0 to 7.5 [26–29]. The false-negative rates reported in these studies ranged from 14.3% to 33.3% in T1 tumours, which increased with tumour stage up to 75% in T3 gastric tumours [26,28,29]. However, the number of patients in these tumour stages with tumour-positive lymph nodes is small (range 3–10).
Several factors influence the success of SLN procedure in gastric cancer. Frequent leakage was observed from lacerated lymphatic vessels during the SLN mapping in patients with intraoperative ICG injection . Preoperative endoscopic ICG injection results in a higher number of fluorescent lymph nodes and lower false negative rate compared to intraoperative injection . Due to the longer interval between injection and imaging, it is expected that ICG passes through the SLN to the higher-tier nodes. In addition to fluorescence imaging, ICG absorption imaging has been used for SLN detection in early gastric cancer during endoscopy [27,54–57] However, as shown by Miyashiro et al. , fluorescence provides much higher contrast than absorption and is therefore preferred.
In colorectal cancer, two studies reported the use of ICG as an NIR fluorescent lymphatic tracer in a total of 51 patients with an identification rate of 88.5% and 92%, respectively, and an average number of identified SLNs of 2.1 and 2.6, respectively [7,26]. The false negative rate was 4 out of 9 (44%) patients with tumour-positive lymph nodes.
When the SLN procedure is used for nodal staging to determine prognosis and possible adjuvant therapy, as has been suggested for colorectal cancer, an ex vivo approach can be considered . Using an ex vivo approach, more optimised NIR fluorescent dyes can be used, which are not yet approved for in vivo administration, such as IRDye 800CW (LI-COR, Lincoln, NE, USA). Such dyes can also be conjugated covalently to albumin or nanocolloid to increase lymph node retention [42,59]. This ex vivo strategy was successfully applied in a recent clinical study .
NIR fluorescence SLN mapping has shown excellent results to date in breast and skin cancer. Therefore, the use of ICG should be particularly attractive to hospitals unable to work with radioactive isotopes as an adjunct or possible replacement to the use of blue dye alone [15,17]. Direct comparison between ICG fluorescence and radiotracers in adequately powered clinical trials, however, has not yet been performed. In gastrointestinal cancer, SLN procedures using ICG obtain high identification rates, although the high false negative rates in the small patient samples require further assessment. Additionally, the feasibility of NIR fluorescence SLN mapping using ICG has also been assessed in single studies in cervical, vulvar, anal, oropharyngeal and non-small cell lung cancer [4,8–11].
As the available data on ICG fluorescence in the sentinel lymph node procedure is relatively limited, conclusion on direct patient benefit and clinical outcome can not yet be drawn. Currently, several groups are performing clinical trials using NIR fluorescence imaging and ICG in the SLN procedure in multiple malignancies (JPRN-UMIN000003035, NCT00264602, NTR1981, NTR1983, NTR2003, NTR2084, NTR2479, NTR2480, NTR2481, NTR2482, as retrieved from http://apps.who.int/trialsearch/ on Jan 3, 2011).
The main goal of cancer surgery is the complete and “en-bloc” excision of tumours with adequate tumour-free margins while minimising surgical morbidity. Presently, though, intraoperative assessment of tumour margins relies on palpation and visual inspection. NIR fluorescence imaging is a promising technique for intraoperative tumour identification. NIR fluorescent probes that specifically target tumour cells could aid the surgeon in determining resection margins and possibly reduce the risk of locoregional recurrence [61,62]. Although ICG is a non-targeted probe, it can provide NIR fluorescence tumour localisation in a limited number of hepatobiliary cancer patients [31,63–66], either due to physiological uptake in well-differentiated tumours or rim uptake as a result of leakage and retention in poorly-differentiated tumours and colorectal metastases [32,67].
Liver resection is the only curative option in the treatment of hepatobiliary cancer. Intrahepatic recurrence rates after resection of colorectal cancer metastases range from 11% to 37.5% and the majority of these recurrences appear within two years after resection [68–72]. A possible explanation for this high intrahepatic recurrence rate is that these hepatic metastases were present at time of resection of the liver metastases but were undetected by preoperative imaging and intraoperative ultrasound. NIR fluorescence detection is a new technique to intraoperatively visualise hepatobiliary cancer.
ICG is excreted exclusively into the bile, which allows real-time NIR fluorescence cholangiography of biliary anatomy during cholecystectomy and other hepatobiliary surgery [73–75]. This technique provides a reliable roadmap of the biliary tree, which enables the surgeon to avoid injuring the bile duct . In hepatobiliary cancer, it is hypothesised that the NIR fluorescent signal in or around the tumour is caused by passive accumulation due to hampered biliary excretion, which, in the case of a colorectal liver metastasis, results in a fluorescent rim around the tumour (Fig. 3) . To date, five Japanese studies have reported the use of ICG in NIR fluorescence imaging of hepatobiliary cancer including colorectal metastasis, hepatocellular carcinoma and cholangiocarcinoma [31,63–66]. To identify liver tumours, the best time window is beyond 24 hours after injection, when most ICG is washed out of the healthy liver parenchyma and is still present in and around the tumour tissue .
In patients with hepatocellular carcinoma or colorectal liver metastases, 98.1% to 100% of the lesions were detected using NIR fluorescence in the resection tissue specimen [31,64,66]. However, due to limited penetration of the NIR fluorescent signal, intraoperative detection of deeper located tumours was not possible (Ishizawa et al.  reported a maximal detection depth of 8 mm). Tumours located at the liver surface provide a bright fluorescent signal and are easily detected, which is especially useful for colorectal liver metastases as these are mostly located on the surface of the liver parenchyma. In these studies this resulted in detection of new small superficial lesions by NIR fluorescence imaging that could not be detected by intraoperative ultrasonography or by visual inspection [31,66].
In the case of cholangiocarcinoma, Harada et al.  showed ICG fluorescence on the liver surface in the regions of liver with cholestasis caused by bile duct tumour invasion or thrombi. Although the tumour itself was not fluorescent, the information provided by NIR fluorescence imaging can help to estimate the extent of the bile duct tumour infiltration.
In conclusion, ICG fluorescence might be of value during hepatobiliary surgery when used as an adjunct to intraoperative ultrasound, and could be particularly useful in the intraoperative identification of small superficially located liver tumours. However, to identify deeper tumours, intraoperative ultrasound imaging is still required. Additionally, ICG fluorescence can aid in the identification of tumour lesions during pathological examination.
Endoscopic marking of intestinal lesions is essential in laparoscopic surgery or when difficulty in locating the lesion during resection is anticipated [30,67]. India ink is a frequently used dye, but is associated with complications and side effects and alters the surgical field . ICG could be a more suitable dye for tattooing, because of fewer side effects, relatively long absorption time (up to 14 days), and potential increased detection using NIR fluorescence compared to macroscopic colour perception [30,76,77].
Watanabe et al.  showed accurate and clear NIR fluorescence tumour localization after preoperative peritumoral injection of ICG. In all 10 patients, the NIR fluorescence signal was detected in the colon tumour and could be visualised clearly for at least 72 to 120 hours, whereas the marked location detection based on the intrinsic green colour of ICG was possible in only 2 patients.
It has been proposed that ICG can be used in intraoperative imaging of solid tumours other than hepatobiliary cancer. The enhanced permeability and retention (EPR) effect can potentially be used for tumour imaging. Due to newly formed, more porous blood vessels, molecules can passively accumulate in tumour tissue. Furthermore, a poorly developed tumoral lymphatic system results in increased retention [78–81].
Exploiting the EPR effect, 6 clinical studies with breast tumours used ICG for tumour identification in an outpatient, mammography-like setting [32,81–85]. These studies used optical tomography, which has higher depth penetration and potentially higher specificity, albeit with much lower resolution. During the first 10 min, ICG was retained in the breast tumour tissue and provided contrast to the surrounding healthy tissue [81,82]. Hagen et al.  and Poellinger et al.  used a prototype fluorescence mammographic imaging system and showed the ability to discriminate between malignant and benign lesions after intravenous administration of ICG. However, in an intraoperative setting a higher tumour-to-background ratio would be needed to provide sufficient tumour demarcation.
Additionally, ICG can be useful as a diagnostic tool to estimate the invasiveness of early gastric cancer during endoscopy. Four Japanese studies used NIR fluorescence endoscopy using ICG as contrast agent to differentiate between mucosal and submucosal or more invasive tumours, and obtained a diagnostic accuracy of 85% up to 93% [86–89]. The NIR fluorescence signal was visible up to 3 min in tumour tissue compared to several seconds in healthy tissue . Therefore, ICG might be useful to distinguish mucosal cancer from submucosal and deeper cancers, which is a risk factor for lymph node metastasis. However, the short duration of the signal limits its use in a surgical setting.
In conclusion, the lack of direct tumour targeting properties prevents the introduction of ICG as NIR fluorescent probe in most tumour types in an intraoperative setting.
Due to its biological characteristics following intravenous injection, ICG is a suitable contrast agent for NIR fluorescence angiography. ICG is mainly bound to plasma proteins and therefore remains predominantly in the intravascular space. Additionally, because of its rapid washout, ICG permits consecutive measurements. ICG angiography has been used in the evaluation of coronary artery bypass grafts, peripheral vascular disease, and solid organ transplantation [2,90–94].
In reconstructive surgery, intraoperative evaluation of skin flap viability is highly desirable, as loss of skin flaps is catastrophic and may produce even larger tissue deficits. Commonly used subjective methods such as tissue colour, capillary refill, dermal bleeding and more objective methods such as skin temperature, fluorescein dye perfusion, transcutaneous oxygen monitoring and Doppler ultrasound, are not optimal. NIR fluorescence angiography using ICG allows the surgeon to visualize arterial inflow, venous return and tissue perfusion, prior to harvest and after flap transfer. To date, several studies of NIR fluorescence angiography using ICG in reconstructive surgery after oncologic surgery have been performed [33,95–102].
As a result of great variation in perforating vessels and their perfusion zones, it can be challenging to select the optimal vessel to transfer a flap. NIR fluorescence angiography using ICG is able to successfully identify perfusion zones intraoperatively in various types of flaps, including transverse rectus abdominis musculocutaneous flaps, deep inferior epigastric perforator flaps and superficial inferior epigastric artery flaps (Fig. 4) [96–98,102]. In these studies, areas with fluorescence filling defects correlated with poor clinical outcome [97,101,102]. Therefore, intraoperative measurements of the flap perfusion allows intraoperative surgical decision making based on accurate assessment of flap vascularity.
Additionally, after flap transfer, NIR fluorescence angiography can detect impaired flap blood flow and can be helpful for differentiating among poor arterial inflow, poor venous outflow, or poor perfusion [100,101]. Rapid identification of vascular compromise allows direct intervention and increased flap salvage. Komorowska-Timek et al.  reported that using ICG NIR fluorescence angiography resulted in a favourable postoperative complication rate after breast reconstruction compared to a historic cohort (4% vs. 15.1%). Furthermore, in flap re-exploration, NIR fluorescence angiography using ICG can provide excellent diagnostic accuracy for detecting microvascular thrombosis . Therefore, intraoperative assessment using ICG in NIR fluorescence angiography may be of great value in reconstructive surgery.
The recent introduction of NIR fluorescence image guidance provides new opportunities for surgery, particularly cancer surgery. Currently, ICG and methylene blue are the only clinically available NIR fluorescent probes and the reported clinical experience to date focuses on ICG. Clinical experience with ICG for intraoperative NIR fluorescence imaging is rather extensive and shows a favourable safety profile.
The current use of ICG in tumour detection and demarcation is limited because ICG cannot be conjugated to tumour specific targets, which makes ICG a non-targeted probe. Because ICG is not an ideal fluorescent probe, multiple novel fluorophores with improved optical properties, such as IRDye 800CW, have been developed. Moreover, these novel fluorophores can be conjugated to specific ligands. Various mechanisms are available to target tumour cells, for example tumour specific cell surface markers, enzymatic activity or increased glucose metabolism (reviewed in Keereweer et al. ). Multiple targeted probes are commercially available and have successfully been tested in animal studies [61,105–109]. Translating these preclinical results to clinical trials remains a major challenge. Several academic and industry groups have communicated the intention to move novel probes, such as IRDye 800CW, to the clinic and the first toxicity results for this purpose have been published .
Moreover, development of new imaging systems will further improve NIR fluorescence image-guided surgery. NIR fluorescence laparoscopic imaging systems are currently being developed, and a commercial system from Olympus is already being marketed, which will allow NIR fluorescence guided-surgery in a minimally invasive setting.[5,73,74,111] Various techniques are being evaluated to correct photon scattering and thereby enhance the depth at which NIR fluorescent signal can be detected [112–114].
When these improved NIR fluorescence imaging systems and probes become clinically available, indications for NIR fluorescence image-guided cancer surgery will be greatly expanded.
In conclusion, the availability of ICG permitted rapid clinical translation of NIR fluorescence intraoperative imaging to cancer surgery. ICG presents a new, safe and sensitive alternative or addition to the conventional SLN procedure; although, conclusions on direct patient benefit and clinical outcome cannot yet be drawn. Moreover, the use of ICG allows objective assessment of flap perfusion in reconstructive surgery and provides opportunities for intraoperative tumour imaging.
We thank Lindsey Gendall for editing.
Sources of Financial Support: This work was supported in part by NIH grant R01-CA-115296 and R01-EB-005805 and the Dutch Cancer Society grant UL2010-4732. J.S.D. Mieog is a MD-medical research trainee funded by The Netherlands Organisation for Health Research and Development (grant 92003526).
CONFLICT OF INTEREST STATEMENT: John V. Frangioni: All FLARE technology is owned by Beth Israel Deaconess Medical Center, a teaching hospital of Harvard Medical School. As inventor, Dr. Frangioni may someday receive royalties if products are commercialized. Dr. Frangioni is the founder and unpaid director of The FLARE Foundation, a non-profit organization focused on promoting the dissemination of medical imaging technology for research and clinical use.