Clinical relevance of novel imaging technologies for sentinel lymph node identification and staging.

Biotechnology Advances 32 (2014) 269–279

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Biotechnology Advances journal homepage: www.elsevier.com/locate/biotechadv

Research review paper

Clinical relevance of novel imaging technologies for sentinel lymph node identification and staging Aidan Cousins a, Sarah K. Thompson b, A. Bruce Wedding c, Benjamin Thierry a,⁎ a b c

Ian Wark Research Institute, University of South Australia, Mawson Lakes Campus, Mawson Lakes, SA 5095, Australia Discipline of Surgery, University of Adelaide, Royal Adelaide Hospital, Adelaide, SA 5000, Australia School of Engineering, University of South Australia, Mawson Lakes Campus, Mawson Lakes, SA 5095, Australia

a r t i c l e

i n f o

Article history: Received 7 June 2013 Received in revised form 12 October 2013 Accepted 27 October 2013 Available online 1 November 2013 Keywords: Cancer diagnostic Prognostic Imaging Nanotechnology Metastasis MRI PET Nuclear medicine

a b s t r a c t The sentinel lymph node (SLN) concept has become a standard of care for patients with breast cancer and melanoma, yet its clinical application to other cancer types has been somewhat limited. This is mainly due to the reduced accuracy of conventional SLN mapping techniques (using blue dye and/or radiocolloids as lymphatic tracers) in cancer types where lymphatic drainage is more complex, and SLNs are within close proximity to other nodes or the tumour site. In recent years, many novel techniques for SLN mapping have been developed including fluorescence, x-ray, and magnetic resonant detection. Whilst each technique has its own advantages/disadvantages, the role of targeted contrast agents (for enhanced retention in the SLN, or for immunostaging) is increasing, and may represent the new standard for mapping the SLN in many solid organ tumours. This review article discusses current limitations of conventional techniques, limiting factors of nanoparticulate based contrast agents, and efforts to circumvent these limitations with modern tracer architecture. © 2013 Elsevier Inc. All rights reserved.

Contents 1. 2. 3. 4. 5. 6. 7.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Controversies and challenges associated with the SLN concept . . . . . . . . . . . . . . Lymphotropic contrast agents—The importance of physicochemical properties . . . . . . . Conventional lymphotropic contrast agents: Blue dyes and lymphoscintigraphy . . . . . . Optical lymphotropic imaging agents . . . . . . . . . . . . . . . . . . . . . . . . . . Computed tomography lymphotropic tracers . . . . . . . . . . . . . . . . . . . . . . Magnetic lymphotropic tracers . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1. Magnetic resonance imaging . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2. Magnetic field sensing probes . . . . . . . . . . . . . . . . . . . . . . . . . 8. Targeted SLN contrast agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9. Beyond the sentinel node concept: Lymph node staging with imaging contrast agents . . . 9.1. F-18-FDG PET lymphatic staging . . . . . . . . . . . . . . . . . . . . . . . . 9.2. Lymphatic staging with lymphotropic MRI contrast agents . . . . . . . . . . . . 9.3. The elusive quest for systemic immuno-imaging of metastatic spread . . . . . . . . 9.4. Immuno-staging of the lymphatics with interstitial agents: A necessary compromise? 10. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction

⁎ Corresponding author. E-mail address: [email protected] (B. Thierry). 0734-9750/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.biotechadv.2013.10.011

The metastatic status of regional lymph nodes is the most significant prognostic factor in breast cancer, melanoma and other solid organ tumours with lymphatic spread (Gershenwald and Ross, 2011; Krag

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A. Cousins et al. / Biotechnology Advances 32 (2014) 269–279

et al., 2010; Salhab et al., 2011). Since the initial demonstration of the sentinel lymph node concept by Morton et al. (Morton et al., 1992), there has been much focus in clinical oncology around the role the lymphatic system plays in cancer prognosis and treatment. More specifically, the sentinel lymph node (SLN) concept describes the preferential lymphatic drainage of a primary tumour to a regional lymph node(s). Studies have shown that the status of the SLN is an accurate indicator of the status of the second and subsequent tier nodes (Ball et al., 2010; Hayashi et al., 2006; Mansel et al., 2000). The SLN concept has become the standard of care for melanoma and breast cancer patients with non-clinically detectable metastases and has improved patient management, morbidity, and – although this has yet to be confirmed – mortality (Leong et al., 2011a). Current identification of the SLNs is typically performed using lymphoscintigraphy; where, after peritumoral injection of radiolabelled nanoparticulate agents, lymphatic drainage from the primary tumour is imaged preoperatively using nuclear imaging, and/or detected intraoperatively with a gamma probe. This technique can also be performed intraoperatively with blue dyes, which enable direct visual identification of the SLNs at the time of the surgery. The uptake of these lymphotropic contrast agents by the lymphatic system is monitored intraoperatively by surgeons, until the first nodes along the drainage pathway have been identified (Fig. 1A). These nodes are then surgically extracted, and the presence of cancer cells is determined through pathological examination. The presence of metastases within SLNs has been correlated with poor prognostic outcomes for a range of diseases (Carter et al., 1989; Ferrone

A

et al., 2002; Muller et al., 2001) and accurate staging of the SLNs is therefore of high clinical importance. The prognostic significance of the detection of small metastatic deposits however remains controversial. In addition, aside from being the current standard of care for breast cancer and melanoma, the clinical application of the SLN concept to other type of cancers, for instance gastrointestinal ones, remains limited and its clinical relevance controversial. One of the main factors preventing its application to other cancer types are the technological challenges yet to be solved in correctly identifying the SLNs. Imaging modalities currently used for the detection of radiolabeled agents are indeed limited by their poor spatial resolution in solid tumour types in which the SLNs are in close proximity to the primary tumour. In addition, gamma probe based detection of the SLN can be compromised when the SLN is close to the injection site because of shine-through radioactivity (Fig. 1B). Further, when blue dyes are used visualisation is not always possible – as is the case for some cancer types located in internal organs (e.g. lung and oesophageal) where tissue penetration is poor and lymph nodes often anthracotic. There is therefore, much need for improvement in the ability to detect the SLNs using lymphotropic contrast agents. An ‘ideal’ contrast agent would be one that imposes little risk to patients' health; has good specificity to the SLN; lymphatic uptake and migration speed is optimised; creates a high signal to noise ratio (e.g. good retention and distinguishable from background signals); can be detected with high resolution, and in low quantities (sensitivity); and is relatively easy and economical to use. Although no such contrast agent currently exists, the quest to find

B SLN

SLN

Tumour Site

Contrast Agent

Tumour Site

Injection

C

Fig. 1. Sentinel node detection: (A) Demonstration of the SLN concept: a tracer is injected at the tumour site, accumulating in the interstitial tissue. It is not until the complicated network of lymphatic vessels drains the tracer and highlights the first lymph node that the SLN can be identified (arrow), and uptake from the tumour site can be mapped in vivo. (B) In some instances proximity of the SLN to the tumour site is such that the hot spot from the injection site can mask the hot spot of the SLN (represented as shaded circles, top panel). In such instances, the sensitivity of the technique is overshadowed by its poor spatial resolution, and it may be difficult to visualise all nodes of interest (bottom panel). Reprinted from Aarsvold and Alazraki (2005) with permission from Elsevier. (C) Use of blue dyes to intraoperatively highlight the SLN. After injection, the surgical team may have less than 10 min to locate the stained sentinel node (arrow) before efferent flow stains subsequent, lower tier nodes too. Reprinted from Kitayama et al. (2007), with permission from Elsevier.


the ideal agent has encouraged researchers to explore alternatives outside of conventional lymphotropic contrast agents currently in clinical use and to also consider the use of different imaging modalities. The aim of this article is to review the key issues associated with current lymphotropic contrast agents in the pre- and intraoperative detection of the SLNs and, in view of the characteristics of these agents and their detection approaches, to discuss the clinical relevance of the SLN concept beyond its current application in breast and melanoma staging. To conclude, we discuss the emergence of novel imaging approaches designed to provide more accurate preoperative staging of lymphatic tumour spread. 2. Controversies and challenges associated with the SLN concept For breast cancer and melanoma, it is now well established that the status of the SLN is a reliable indicator of the status of non-SLN regional nodes, and ultimately is a strong independent prognostic marker for the patient (Motomura, 2012; Viale et al., 2001). However, controversy still surrounds the prognostic significance of very small metastases in SLNs and, more generally, the clinical relevance of the SLN concept in other types of cancer. A micrometastatic (MM) lesion is defined as an occult tumour deposit less than 2.0 mm in size, but greater than 0.2 mm (Salhab et al., 2011), with ‘macrometastases’ and ‘isolated tumour cells’ (ITC) representing the classifications above and below this window, respectively. The current, 7th edition of the TNM classification of malignant tumours (Sobin et al., 2009) states that (e.g. for breast cancer), ITC deposits detected during pathological examination of the regional nodes should be declared node negative (pN0(i+)), yet MM deposits are granted node positive status (pN1(mi)); the same is true for deposits detected in the SLN (denoted by the addition of suffix ‘(sn)’) (Sobin et al., 2009). Contrary to this, melanoma patients with confirmed presence of ITC in the SLNs should be upstaged to stage III according to the 2010 AJCC Cancer Staging manual (Balch et al., 2010). With the refinement of pathological examination afforded by the application of the SLN concept, the detection of such occult deposits in lymph nodes is more likely. The currently unknown significance of the detection of such occult tumour deposits in the SLNs presents a difficult problem for oncologists since the status of the SLNs currently guides treatment decisions. To answer this pressing question, a number of studies have been recently conducted, but results to date have been conflicting. In a cohort of 856 with favourable early stage breast cancer who had not received systemic adjuvant therapy, isolated tumour cells or micrometastases in regional lymph nodes were associated with a reduced 5 year rate of disease free survival (de Boer et al., 2009). Similarly, very small (b0.1 mm) deposits of melanoma in the SLNs were found to be associated with adverse clinical outcomes, including death (Murali et al., 2012). The latter finding could be related to, at least in some instance, an underestimation of the SLN tumour burden during the pathological examination. In another study, 119 node-negative oesophageal cancer patients were reanalysed and it was found that 26% had occult tumour deposits in their lymph nodes (Thompson et al., 2010). Importantly, these patients had a significantly reduced 5 year survival rate compared to those patients who remained node-negative and may have benefited from adjuvant therapy. In contrast, some studies show no significant association between survival and occult tumour deposits. For instance, the detection of SLN or non-SLN occult metastases, as detected by serial step sections at 85 μm intervals, was found to have no significant prognostic implications in 109 patients with early-stage breast cancer (Takeshita et al., 2012). Interestingly such occult metastases in SLNs and non-SLNs were detected in 25 (23%) and 10 (16%) patients, respectively, confirming the relatively high occurrence of such limited metastatic lymph node involvement even in early stage breast cancer patients. Currently, with confusion surrounding the inconsistencies between AJCC and UICC guidelines, consistent diagnosis amongst pathologists is

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difficult to achieve, and hence distinctions of MM/ITC in SLNs suffer poor reproducibility (Cserni et al., 2004; Turner et al., 2008). This has led to calls for more disciplined, standardised methodology and terminology to aid pathology of dissected lymph nodes (Cserni et al., 2004, 2008; Hermanek et al., 1999; Jamieson and Thompson, 2009; Turner et al., 2008). By adopting a series of standardised criteria and definitions for measuring and classifying MM and ITC in the SLN, an improvement was seen in agreement between pathologists from 76% (pre training) to 97% (post training) (Turner et al., 2008). Whilst this marked improvement demonstrates the need for uniform pathological criteria, agreement between the classification of ITCs close to the 0.2 mm upper threshold remained problematic; these new criteria suggest that although in such instances the lower stage category should be selected, it was found that pathologists were often reluctant to classify relatively large collections of ITCs, given that this would result in node-negative status [pNO(i+)] (Turner et al., 2008). Aside from the well demonstrated clinical significance in breast cancer and melanoma, having an understanding of the mechanisms and significance of lymphatic spread is also highly important in other cancer types (such as GI cancer) where the lymphatic pathways are more complicated and hence, less predictable (Kitagawa et al., 2005). There is no doubt that resolution of these controversies, along with more accurate lymph node staging will lead to better (and perhaps less invasive) treatment, and will ultimately lead to individualised treatment protocols. 3. Lymphotropic contrast agents—The importance of physicochemical properties The main aim when implementing the SLN concept is to map the lymphatic drainage of exogenous contrast agents, highlight the nodes of interest, and remove them for further analysis. This holds true for all imaging methods (be it visually, using a gamma probe, x-ray/CT, MRI, ultrasound, or fluorescence), and therefore, for all types of contrast agent used. Lymphotropic contrast agents range from low molecular weight organic and inorganic molecules and chelates, to large, solid nanoparticles (often referred to as radiocolloids or nanocolloids in the field) and other nanostructures able to provide contrast in a specific imaging modality. Regardless of the chemistry or physical structure of the contrast agent, the dominating factor that controls lymphatic transport is the size (usually reported as the hydrodynamic diameter) (Hiraiwa et al., 2010; Oussoren and Storm, 2001; Tsopelas, 2001). More specifically, the size of the contrast agent greatly affects uptake into the lymphatic system, speed of transport through the system, and retention inside draining lymph nodes (Fig. 2). As a consequence, contrast agent size should be optimised towards specific intended applications such as intraoperative versus pre-/postoperative imaging, reduction of background noise, and achieving sufficient migration to highlight the SLNs and any lower tier nodes of interest. As a basic rule of thumb, small sized contrast agents (less than 5– 10 nm) are taken up and transported quickly throughout the lymphatic vessels (Rao et al., 2010). Whilst efficient migration enables quick intraoperative mapping of lymphatic drainage, it also results in a short imaging window and may increase difficulty in discriminating first tier SLNs from second and third tier nodes (Fig. 1C). Small nanoparticles and low molecular weight contrast agents can also diffuse from the lymphatic vessels, reducing local concentrations and contributing to the background signal (Kobayashi et al., 2006; Tsopelas, 2001). The near infrared dye indocyanine green (ICG), which is currently being validated clinically, provides a good illustration of the potential benefits and drawbacks associated with such low molecular weight contrast agents (Polom et al., 2011). Medium sized contrast agents (in the range of 50–200 nm), conversely, have slower transport rates through the lymphatic vessels (Cong et al., 2010; Johnson et al., 2010). Although this provides a longer imaging window, such contrast agents may not migrate beyond the injection site or may skip first tier nodes if they contain metastatic tumour cells (Hiraiwa et al., 2010; Tsopelas, 2001).

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Dyes ICG

AuNP IONP

Radiocolloids (e.g. antimony trisulphide and rhenium sulphide-based)

500nm Size

Macromolecules Nanoparticles

Slow migration High Retention

Microparticles

Fig. 2. Tracer size: The main factor affecting the transport of lymphotropic contrast agents is the hydrodynamic diameter. Although there will be some dependency on the chemistry and structure of the particle, a general rule is that for agents less than approximately 10 nm, there will be high diffusion of the contrast agent from the lymphatic vessels, and fast migration through the system. For mid-range agents of about 50 nm–200 nm, the migration speed will be slower, and retention in the vessels higher as size increases. For large particles greater than approximately 500 nm, the particles will be well retained in the vessels, but the migration speed will be very slow, and the agent may not progress beyond the first node.

Large contrast agents (N 500 nm) tend to migrate in the lymphatic via macrophages and dendritic cells after phagocytosis and consequently have very slow migration rates (Cong et al., 2010; Manolova et al., 2008). Aside from the different uptake mechanisms, small nanoparticulate contrast agents are prone to aggregation in complex biological environments, which in turn can drastically affect their transport and retention in the lymphatics. The “ideal” lymphotropic contrast agent size is therefore one that is small enough to enter the lymphatics and transport swiftly to the SLNs, yet large enough to remain in the nodes long enough for imaging and SLN identification without prematurely flowing on to lower tier nodes (Cong et al., 2010). Manufacturing a contrast agent with these ideal properties often requires striking the right balance between the ‘inherent’ size of an agent, and the ‘desired’ size to achieve favourable diffusion and retention rates. A good example of this is the use of macromolecular fluorophores or dyes used as optical imaging agents; the type of molecule, its makeup, and the environment in which it resides largely dictates the hydrodynamic size of the contrast agent. These agents can be conjugated or aggregated to other molecules (such as proteins) in a controlled manner to increase their hydrodynamic size. This concept has been demonstrated with the ICG fluorophore, whereby human serum albumin (HSA) is mixed with the contrast agent to increase hydrodynamic radius (Hutteman et al., 2011). The radiocolloids currently used as SLN imaging agents in the clinic such as albumin nanocolloid (Nanocoll) and rhenium sulphide nanocolloid (Nanocis) are often obtained from commercial kits. Considering the importance of the hydrodynamic size, great care is required during their preparation (Jimenez et al., 2008). Despite well demonstrated clinical utility, some radiocolloid agents currently used have a fairly high polydispersity, which can be expected to translate into inhomogeneous transport rates inside the lymphatics and result in suboptimal mapping of the SLNs. Major advancements in the synthesis of inorganic nanoparticle contrast agents have drastically broadened the range of agents available to the biomedical community as well as provided excellent control over the size, shape and reduction in polydispersity (Goesmann and Feldmann, 2010). Progress in nanoparticle synthesis has been accompanied with progress in understanding the importance of nanoparticles biointerfaces. Functionalisation with organic (macro)molecules is often required to improve their colloidal stability (i.e. prevent aggregation) and compatibility with biological systems. One of the most commonly

used macromolecules is polyethylene glycol (PEG) which, when attached to the surface of nanoparticles, can greatly improve circulation time after systemic injection and biological stability of the nanoparticles by retarding the phagocytic process (Walkey et al., 2012). PEG can also serve as a scaffold for other ligands/labels to improve the functionality of the particles as will be discussed later in this article. 4. Conventional lymphotropic contrast agents: Blue dyes and lymphoscintigraphy The majority of clinical SLN procedures currently rely on organic dyes (to visually map the lymphatic flow intraoperatively), radioactive nanoparticulate tracers, or more often, a combination of both types. Following on from the pioneering work of Morton and colleagues in the early 90's (Morton et al., 1992), blue dyes (e.g. Evans blue, methylene blue, and Patent Blue V) have been used to visually determine lymphatic drainage after interstitial injection. Blue dyes are readily taken up in the lymphatics and provide a means for the surgeon to achieve intraoperative lymphatic mapping of the regional basin without the use of a gamma probe. Although blue dyes are easy to prepare and pose few hazards to the patient or surgeon, their efficiency in identifying SLNs is limited. The inherent low molecular weight of these dyes translates into very rapid migration in the lymphatics. In addition, the retention of dye in SLNs is poor. As a result, there is a level of skill required by the surgeon to locate and remove the SLNs before the dye spreads to other nodes (Subramanian et al., 2010). Although uncommon, dyes can also cause unwanted anaphylactic side effects in some patients (Bembenek et al., 2007). As a general rule, dyes are only useful for visualising more superficial lymph nodes (i.e. in breast cancer and melanoma) due to the presence of anthracosis in mediastinal nodes (e.g. from smoking or pollution) (Bustos et al., 2008; Rzyman et al., 2006) and the difficulty in quickly reaching SLNs that reside in deep locations, often in overweight patients (Lerman et al., 2007). Not long after the advancement of blue dyes for SLN identification, the ability to use gamma-radiation emitting tracers for the same purpose was demonstrated (Alex and Krag, 1993; Vanderveen et al., 1994). Currently the most popular gamma-emitting tracer is radioactive Technetium (Tc-99m) labelled nanoparticles (often referred to as radiocolloids). Although the chemistry of the labelling platform varies from country to country (as does the ‘standard’ size of the particles) the most common are sulphide-based nanoparticles conjugated with Tc-99m. Although


other radiolabels can be used (such as I-123 and Tl-201) they are not as common as Tc-99m, which is a well-established standard – owing mostly to the ease of generating Tc-99m radioisotopes, and a practical radioactive half-life of approximately 6 h. As these radiocolloids emit high energy gamma-radiation (140 keV), specialised detection/imaging equipment is needed to determine their presence. Gamma radiation is highly penetrating; hence tissue depth, density, and colouration are not an issue for Tc99m-based lymphotropic agents. When using radiocolloids, the tracer is injected much like the dyes, and the surgeon searches for ‘hot spots’: areas of high activity caused by an accumulation of the radiotracer. If given sufficient time to migrate, the hot spots should be largely localised in SLNs, although it is common for them to be a residual hot spot at the site of injection (i.e. the primary tumour). Due to the signal intensity and detection modality used (such as intraoperative handheld probes, gamma cameras, or SPECT) this method has high sensitivity (with sub-pM concentrations detectable by gamma cameras (Frangioni, 2008)), but very poor spatial resolution. As a result, if the SLNs are within close proximity to the tumour, the hot spot from the nodes can be lost in that of the primary tumour injection site (Fig. 1B). This is particularly problematic in cancers such as GI cancer where over 90% of SLNs are within 3 cm of the primary tumour site (Van de Ven et al., 1999), and lymphatic migration is particularly unpredictable due to multidirectional drainage (Kitagawa et al., 2005). By combining blue dyes with radiocolloids, the accuracy of SLN detection may be significantly improved (Aarsvold and Alazraki 2005; Tsopelas et al., 2006). This is because the lymphatic migration can be monitored visually using the dye, whilst the signal inside the nodes (which may be located too deep to be visible via staining) is intensified by the use of the radiocolloid. Table 1 shows a comparison of using a blue dye, radiocolloid, or a combination of both for detecting the SLNs in different cancer types. What is most evident here is that both methods are not without flaws, but in general there is an improvement in detection when using dyes and radiocolloids conjointly in more superficially located cancers such as breast cancer and melanoma. 5. Optical lymphotropic imaging agents Fluorescent organic molecules (fluorophores) and nanoparticles have been proposed as alternatives to conventional lymphotropic imaging agents of the SLNs (particularly blue dyes). The advantage of fluorescence-based identification of the SLNs is that these agents do not rely on colourisation of the node, but instead fluoresce under irradiation by an excitation light source, thereby providing a less ambiguous signal. Depending upon wavelength, fluorescence can penetrate through thin layers of tissue and reveal nodes otherwise hidden when using non-fluorescent dyes. In a sense, fluorescent contrast agents are a compromise between the highly penetrating signal of Tc-99m radiocolloids and poor visualisation of blue dyes. However, even in the near infrared (NIR) part of the spectrum where tissue absorption of light is minimal, fluorescence penetration remains limited in deep lymph nodes. Despite the excellent optical properties of quantum dots, the scope for clinical use remains low as most formulations are based on heavy metals such as cadmium, making them intrinsically toxic (Johnson et al., 2010). It is important to note that ICG is an FDA approved, low molecular weight organic molecule, which fluoresces in

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the NIR and has a well-established safety profile. Therefore, ICG-based imaging is considered a powerful form of optical imaging when compared to blue dyes (Jain et al., 2009). ICG was initially advanced as a visible dye marker in the detection of SLNs, and as such had comparable success to conventional dyes (for instance, 74% detection rate in breast cancer) (Motomura et al., 1999). With improvements to image capturing technology (such as sensitive infrared cameras), the use of ICG as a NIR fluorophore improved the detection rate in breast cancer to approximately 95% (Polom et al., 2011). This improvement, combined with its good safety profile, made the use of ICG to find SLNs an attractive alternative. Similar favourable detection rates have been seen in melanoma with initial studies showing 100% detection rate (although this figure is based on a very small sample size of only 10 patients) (Fujiwara et al., 2009); in gastric cancer with an average of 98% detection (Polom et al., 2011); and in lung cancer with 81% detection demonstrated (Yamashita et al., 2011). Compared to the detection rate of conventional blue dyes, there is a clear improvement when using ICG for SLN identification. This is in part due to the use of composite visible/NIR cameras in the operating theatre (Fig. 3A), which increases the sensitivity, specificity, and accuracy of SLN detection over conventional dyes (Hirche et al., 2010; Mieog et al., 2011; Parungo et al., 2005; Yamauchi et al., 2011). The main issue pertaining to the use of ICG is its low molecular weight (and therefore small hydrodynamic diameter) 0, which translate into rapid migration in the lymphatics (1–10 min to the SLNs) (Polom et al., 2011). However, the hydrodynamic radius of the ICG molecule can be increased by mixing it with HSA. Such approaches are yet to show statistically significant improvements in the detection rate (Hutteman et al., 2011; Polom et al., 2012) – although it does appear to improve the strength of the fluorescent signal (Hutteman et al., 2011). A hybrid between optical and ultrasound imaging, ‘photoacoustic imaging’, which relies on optically active contrast agents such as gold nanorods or carbon nanotubes, has recently attracted the attention of the research community for its potential to image the lymphatics and identify SLNs. Photoacoustic imaging occurs when the irradiation of the contrast agent (by a pulsed laser) creates acoustic waves in the body tissue, which can then be detected via conventional ultrasound apparatus. Whilst endogenous ultrasonic contrast – which originates from the differences in sonic absorption amongst different tissue types – can provide useful structural information, in the context of SLN imaging, exogenous contrast is required. Nanoparticles made of plasmonic metals such as gold have been used successfully to achieve photoacoustic imaging of the lymphatics, which is related to their unique optical properties, especially their ability to sustain surface plasmon resonance (Pan et al., 2010, 2012). Gold nanorods have a very high absorption coefficient in the NIR (~1.5 × 109 M−1 cm−1), which enables the photoacoustic detection of these agents in pM concentration. In addition, robust synthesis routes exist that enable the preparation of nanoparticles with well-controlled size and shape (both important characteristics for determining the absorption wavelength and sensitivity), which can in turn be used to fabricate agents with controllable lymphatic transport/clearance rates. Whilst still experimental, photoacoustic imaging often uses modified versions of existing ultrasound equipment (Agarwal et al., 2007). This not only allows real-time imaging of both structural information (endogenous) and photoacoustic signals

Table 1 Conventional approaches: A comparison of the blue dye method, radiocolloid method, and combination blue dye and radiocolloid method for detecting the SLN in 4 types of cancer. Note that it is rare for two studies to have complete agreement with the accuracy in identifying the SLN, so the numbers provided below are ‘typical’ values, and have been rounded to the nearest 5%. Cancer type

Blue dye method

Radiocolloid method

Blue dye + radiocolloid method

Breast Melanoma Lung Oesophageal

65% Giuliano et al. (1994) 70% Albertini et al. (1996a) 50% Sugi et al. (2003) 85% Grotenhuis et al. (2009)

80% Krag et al. (1993) 85% Albertini et al. (1996a) 80% Liptay et al. (2000) 95% Thompson et al. (2011)

90% Albertini et al. (1996b) 95% Landi et al. (2000) 55% Tiffet et al. (2005) –

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A

D

C

B

E

Fig. 3. Novel approaches: (A) Intraoperative use of the ICG fluorophore to identify the SLN 5 min (top panel) and 45 min post injection, where lower tier non-SLNs become evident (bottom panel). By combining the images from an NIR camera with that from a visible camera, a real-time composite image can be created to aid in the mapping of the lymphatic system, and produce more accurate detection of SLNs. Reprinted from van der Vorst et al. (2013), with permission from Elsevier. (B) The comparison of ICP-MS measured concentration of gold in the removed popliteal lymph nodes of mice injected with either CD45-targeted AuNPs or PEGylated AuNPs. Reprinted from Liu et al. (2013), with permission form Elsevier. (C) The use of iron oxide nanoparticles to identify and hence stage lymph nodes presenting metastatic growth in MR imaging. A preinjection MRI (top panel) showing the axillary lymphatics in a patient with breast cancer does not show evidence of malignancy if one adopts the conventional size-based criteria (white bar indicates 5 mm), yet when compared to a post injection MRI of the same region (below) the heterogeneous uptake of contrast agent into one of the lymph nodes reveals a 3 mm metastatic deposit (white arrow). Image reproduced from Harisinghani and Weissleder (2004). (D) Whole-body PET image shows strong uptake of F-18-FDG tracer, revealing high metabolic activity in the gallbladder (dotted arrow) and the right breast and an axillary lymph node (solid arrows) – suggesting metastasis via the lymphatics has occurred. Image adapted with kind permission from Springer Science + Business Media: Kim et al. (2009) (E) Fluorescence images showing targeted, interstitially injected agents highlighting metastatic deposits of two different cell lines in ALNs of a murine model. Such a technique demonstrates the ability for interstitially injected tracers to provide immuno-staging of occult metastases (in this case 24 h post injection). Image reproduced from Tafreshi et al. (2012).

(nanorods, etc.), but also creates a non-invasive, low cost, and flexible alternative to detecting SLNs via the use of dyes, radiocolloids, and fluorescent molecules. 6. Computed tomography lymphotropic tracers X-ray computed tomography (CT) using low molecular weight iodine-based contrast agents has been used to image the lymphatic network. However, to date, CT imaging remains limited in its ability to accurately stage the SLN, with detection rates as low as 30% (Hyung et al., 2010). This may, in part, be due to the rapid clearance rates of small sized iodinated contrast agents (such as the water-based

Iopamidol) creating very short imaging windows [peak attenuation reached within 1 min, and image intensity greater than 100 HU maintained for less than 10 min (Chung et al., 2010; Hayashi et al., 2006; Hyung et al., 2010)]. Iodinated emulsions have been advanced as substitutes to these low molecular weight water-based agents. Iodinated emulsions have more favourable pharmacokinetics due to their size, and subsequent increased retention in the lymphatics compared to water-based agents (Chung et al., 2010). A second approach involves the use of inorganic tracers such as gold nanoparticles, which provide longer imaging windows due to their larger size and increased sensitivity [absorption coefficient of gold at 100 keV is 5.16 cm2/g compared to 1.94 cm2/g for iodine (Hainfeld


et al., 2006)]; they are also considered relatively safe for use in patients (Hainfeld et al., 2006). Unfortunately, with CT imaging, an efficient contrast agent calls for quantity as much as it does quality of attenuation. As CT imaging of the lymphatics requires attenuation of x-rays by the contrast agent, if gold is to be used, enough nanoparticles must be injected to effectively block portions of the x-ray beam. For gold nanoparticles this may require impractically high concentrations. For example, Boote et al. (2010) injected 85–99 mg/kg of gold nanoparticles to obtain sufficient CT contrast in swine models. In an 80 kg human, this would translate to approximately 7 to 8 g of gold injected into the body! Similar results were found in a study by Hainfeld et al. (2006) (10 mg/mL of gold; 54 mg per mouse), who found that very high concentrations of contrast were needed in order to get satisfactory images (Chien et al., 2010; Popovtzer et al., 2008). As such, use of this technique as a SLN mapping tool is impractical for in vivo imaging in humans. 7. Magnetic lymphotropic tracers 7.1. Magnetic resonance imaging MRI is characterised by its excellent spatial resolution and high sensitivity to imaging soft tissues, making it an ideal imaging technology for the lymphatics. Much like CT however, the small size of conventional MRI contrast agents such as gadolinium chelates translates to poor retention in the lymphatics, and diffusion out of the vessels only adds to the background noise (Kobayashi et al., 2006). The speed of the clearance compared to the necessary image acquisition time also means that it is very difficult to determine the direction of flow, and hence the identity of SLNs. With the aid of exogenous nanoparticulate contrast agents such as iron oxide nanoparticles, however, it has been possible to improve the mapping of lymphatic migration from the primary tumour site. These particles possess low toxicity (Anzai et al., 2003; Kou et al., 2008), high biocompatibility (Nahrendorf et al., 2010; Sun et al., 2008), prolonged retention in lymph vessels (Kobayashi et al., 2006), tuneable signal strength (Jun et al., 2005), and ease of manufacture (Pinna et al., 2005). If the size of the iron oxide nanoparticles could be increased in order to reduce the migration speed – to prevent progression to second tier LNs – then it may be possible to use MRI for noninvasive identification of the SLNs. This is an attractive option as the patient would not be exposed to ionising radiation (such as gamma radiation and x-rays) nor invasive surgery (as conventional and optical agents require). These nanoparticles also offer a stable platform for coatings or labels that gadolinium chelates cannot provide. Coatings can increase the performance and retention of the iron oxide nanoparticles (Corot et al., 2006) – for example, using dextran or biocompatible PEG and PVA coatings to enhance tracer efficiency (Amiri et al., 2011). 7.2. Magnetic field sensing probes Iron oxide nanoparticles coupled with a handheld magnetometer probe could manifest itself as a viable alternative to gamma probe detection of radiocolloids. Analogous to SLN identification using radiocolloids and a gamma probe, magnetic nanoparticles can be injected at the tumour site and taken up by the lymphatic system. A magnetic resonance (MR) based probe is then used in vivo (Minamiya et al., 2006), ex vivo (Nakagawa et al., 2003), or transdermally (Shiozawa et al., 2010) to locate the SLNs by measuring the magnetic signature of nearby nodes. Unlike a gamma probe, MR probes (such as the ‘SentiMag’ by Endomagnetics) do not measure a signal intrinsic to the particles (due to their superparamagnetic behaviour), but rather the particles' response to an externally applied magnetic field. With this technology, products such as the SentiMag are aiming to measure as few as 100 μg of nanoparticles at a distance of 30 mm (Pankhurst, 2007). If a commercial ferumoxide colloid (i.e. Endorem/ Ferridex) was used, this would then equate to the detection of approximately 10 μL of contrast agent accumulated in the SLNs, or approximately

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5% of a typical dose of 0.2 mL; thus highlighting the potential for such a probe to replace conventional intraoperative detection, especially if coupled to preoperative imaging with MRI. 8. Targeted SLN contrast agents Once a contrast agent has been injected into the interstitial tissue and uptake into the lymphatics has occurred, its retention in draining lymph nodes is largely driven via non-specific phagocytosis of the agents by macrophages or other scavenger cells (Eck et al., 2010; Sun et al., 2008). As reviewed in the previous sections, the passive migration and uptake of lymphotropic agents are mostly controlled in antagonist fashion by their hydrodynamic size. In most diseases however, an ideal lymphotropic contrast agent for highlighting the SLNs would have rapid uptake by the lymphatics and rapid clearance from the injection site, yet strong retention in the first draining lymph nodes. The difficulty in designing such an agent based solely on hydrodynamic size is illustrated by the broad range of lymphotropic agents currently used worldwide. This issue has motivated intense research in controlling retention in the lymph nodes – and more generally, the lymphatics – regardless of the size or type of the contrast agent. Enhanced retention can be achieved by taking advantage of biological receptors; i.e. attaching an active ligand to the contrast agent that will specifically bind to lymphoid cells – such as mannose binding to reticuloendothelial cells, or monoclonal antibodies (mAbs) that are targeted by macrophages, T-cells, or other scavenger cells in the lymphatics. Lymphoid cell and tissue targeting is especially suitable for agents with small hydrodynamic diameter (low molecular weight soluble agents and sub-20 nm nanoparticles), where balance must be sought between fast clearance from the injection site and prolonged retention in the SLNs. A contrast agent that can clear rapidly from the injection site, yet have prolonged retention in the SLNs, is of particular interest in gamma-imaging (using Tc99m), as this means that the injection site hot spot can be reduced significantly without adversely affecting uptake in the SLNs, thus reducing the “shine-through” effect. Such properties can be found to some extent in mannose-based radiolabeled contrast agents (such as ‘Lymphoseek’, which has recently gained FDA approval in the USA) (Wallace et al., 2007). Lymphoseek consists of a mannosylated 10 kDa dextran backbone labelled with Tc99m, using diethylene triamine pentaacetic acid (DTPA) as a chelating agent. Though such mannose-based low molecular weight soluble agents can be produced with a well-controlled, sub-20 nm hydrodynamic diameter (Leong et al., 2011b; Wallace et al., 2007), conventional radiolabelled particulates (such as antimony trisulphide or rhenium sulphide) can range anywhere from approximately 10 nm to 2000 nm, and with typically quite large size distributions (Tsopelas, 2001). Given the difference in particle size, injection site clearance half-life can vary from N20 h (radiocolloid) to approximately 2 h with mannose-based alternatives (Wallace et al., 2007). The favourable lymphotropic nature of mannose targeted soluble agents translates to improved SLN detection rates, with mannose-based radiolabelled contrast agents presenting results as high as 90% for breast cancer and 98% for melanoma without invoking the use of blue dyes (Leong et al., 2011b). Enhancing specific uptake via the conjugation of mAbs to a chosen contrast agent has also been trialled. To demonstrate the potential of small nanoparticulate lymphotropic agents with high binding affinity to lymphoid cells, 18 nm gold nanoparticles were conjugated with an antibody against the leukocyte common antigen CD45 (Liu et al., 2013). As a result of their high binding affinity to CD45+ lymphoid cells and small hydrodynamic diameter, the immunotargeted nanoparticles were retained in the popliteal and axillary lymph nodes after interstitial injection in mouse fore/hind paw whilst their non-conjugated counterparts were cleared from the lymphatics rapidly (Fig. 3B). Peak accumulations in LNs were in the range of 2.5 to 3% of the injected dose, which compare favourably with approaches based on targeting the mannose

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receptor (Nagashima et al., 2006; Sondak, 2009). Similarly, contrast agents targeted with an anti-CD4 mAb were selectively taken up by the macrophages and scavenger cells of the lymphatic system which resulted in efficient uptake and higher concentrations in the lymph nodes when compared to non-targeted controls (Eck et al., 2010). Targeting lymphatic tissues has also become a focus for designing improved lymphatic imaging agents. In 1999, the lymphatic vessel endothelial hyaluronan receptor (LYVE-1) was identified as a lymph-specific receptor (Banerji et al., 1999) and hence, with the use of an anti-LYVE-1 mAb conjugated to a contrast agent, specific lymphatic mapping is achievable (McElroy et al., 2009). As an extension of this concept, it has been proposed that imaging of tumour-induced LN lymphangiogenesis using LYVE-1 targeted contrast agents might serve as a novel target to image the very early stages of the metastatic process. Anti-LYVE-1 immuno-PET enabled the visualisation of lymphatic vessel expansion in LNs containing spread in a model of inflammation-induced LN lymphangiogenesis (Mumprecht et al., 2010). Similar work may also be possible with antibodies such as D2-40; like anti-LYVE-1 variants, this antibody is specific to lymphatic endothelial cells (Fohn et al., 2011) – although its current use is mainly limited to ex vivo staining of tissue sections.

9. Beyond the sentinel node concept: Lymph node staging with imaging contrast agents Despite the well demonstrated clinical benefits of the SLN concept, its current successful application relies ultimately on histopathological examination of the resected SLNs. There is an increasing need for the development of an imaging contrast agent that could identify whether a SLN was positive or negative without removing it. This could minimise the need for unnecessary invasive procedures, i.e. in situations where the SLNs are negative.

9.1. F-18-FDG PET lymphatic staging Perhaps the most common imaging modality for identifying the presence of metastatic spread (either in lymph nodes or other distant sites) preoperatively is metabolic mapping using positron emission tomography (PET). The glucose analogue fluorodeoxyglucose (FDG) has been commonly coupled to the positron-emitting radioactive Fluorine-18 to highlight areas of hypermetabolic activity during PET scans (Gambhir, 2002). Although not specifically used to distinguish SLNs from lower tier, non-SLNs, this technique could be used to highlight metastatic nodes or lesions due to the increased glucose uptake that occurs in diseased tissue. Unfortunately, increased metabolism is not limited to metastatic tissue, and in some patients it can be difficult to distinguish a metastasis from inflamed or infected tissues (Barrington and O'Doherty, 2003). To remediate some of the limitations associated with FDG-based PET imaging, radiolabeled choline has been developed (Hara et al., 1997). Choline is required in the synthesis of phosphatidylcholine, a key constituent of tumour cell membranes and can therefore be used as a marker of malignant tumour deposits. The main limitation of metabolic PET remains its lack of spatial resolution and specificity, which limits its relevance to accurately stage lymphatic spreading. Even when coupled with computed tomography (PET/CT), PET imaging cannot distinguish positive lymph nodes in close proximity to the primary tumour (due to the shine-through effect). F-18-FDG PET is typically not reliable for the detection of positive lymph nodes less than 7 to 8 mm in size and most certainly does not have the sensitivity required to image the presence of micrometastatic lesions in lymph nodes (Shimizu et al., 2009). Hence, using FDG-based PET, there is a “miss, mask, or mimic” effect and increased potential for false positive readings (Barrington and O'Doherty, 2003; Crippa et al., 2000).

9.2. Lymphatic staging with lymphotropic MRI contrast agents Owing to its excellent spatial resolution and sensitivity, MRI has an (albeit limited) ability to stage healthy versus tumour burdened nodes. A healthy node can be generally characterised as having a diameter less than 8 mm (or if elongated, a short-axis diameter less than 10 mm) (Harisinghani et al., 2003). Typical features of a malignant node include exceeding the above-mentioned dimensions, abnormalities of the fatty central hilum, changes in shape, and irregularities in nodal contours (Luciani et al., 2006). The main limitation of direct MRI staging is that it is almost impossible to determine the presence of small metastatic deposits (e.g. micrometastases). Using iron oxide nanoparticles injected systemically as exogenous contrast, a pioneering study by Ralph Weissleder's group revealed that the localised accumulation of these particles in lymph nodes could be used to discriminate healthy versus tumour burdened nodes (Harisinghani et al., 2003). In contrast to the case for F-18-FDG staging, lymphotropic contrast agents are taken up by lymphoid cells (such as macrophages) and hence accumulate in the lymph nodes, but not inside metastatic lesions (Harisinghani et al., 2003). If sufficient contrast is injected into the patient, then the nodal architecture of the lymph nodes is revealed as the agents concentrate in the sinuses of the nodes. If a node is negative, then the architecture of a healthy node is revealed through the homogenous uptake of the contrast agent in the node. If, however, there is a metastatic deposit, then this results in the contrast of the node appearing blotted and heterogeneous (Fig. 3C) (Guimaraes et al., 2008; Harisinghani et al., 2006; Mouli et al., 2010). Since this approach is mostly qualitative and only areas of healthy tissue are highlighted, there are limitations to the size of metastasis that can be detected. As a result, any staging must be based on the ‘apparent’ uptake of the contrast agent. This difference in uptake can sometimes be strikingly obvious, but in cases where the metastasis is in its early stages, individual interpretation can mean the difference between correct and false-negative diagnosis (Harisinghani et al., 2006). Consequently, despite its merits, the use of systemically injected lymphotropic MRI contrast agents has not become a mainstream clinical procedure. 9.3. The elusive quest for systemic immuno-imaging of metastatic spread The last decade has seen a major research effort towards the development of systemic imaging agents able to accurately detect metastatic spread (Weissleder and Pittet, 2008). Ideally, a contrast agent would be injected intravenously, it would circulate through the body, and it would accumulate at metastatic sites in concentrations compatible with reliable detection using an imaging modality. With such a system, a full body account of metastatic spread could be accurately delivered without the need for an invasive procedure (Fig. 3D). Pioneering studies with radiolabeled probes with strong binding affinity to cancer biomarkers have paved the way to modern molecular imaging; which is fast breaching the gap between fundamental biological studies and clinical oncology (Kaur et al., 2012; Weissleder and Pittet, 2008). The emergence of diagnostic nanotechnology is also rapidly remodelling this field with the promise of more efficient molecular imaging agents (McCarthy et al., 2010). Owing to their high sensitivity, it is not surprising that PET, SPECT and MRI have been the most broadly investigated molecular imaging modalities. In order to achieve malignant specificity in PET imaging, positron emitting contrast agents must be coupled with biological ligands with high and specific binding affinities to known cancer biomarkers. A number of immuno-PET and SPECT tracers have been developed and tested in animal models or clinical studies; a topic that has been reviewed recently (van Dongen et al., 2007). Whilst few clinical studies have been performed, with the use of disease-specific markers, immuno-PET was found to have increased specificity (and hence diagnostic relevance) when compared to FDG-PET (van Dongen and


Vosjan, 2010; van Dongen et al., 2007; Verel et al., 2005), demonstrating the potential of immuno-targeting PET contrast agents for the body. Importantly, improvement in PET imaging technology and increased availability of PET tracers with longer half-life (which is more suitable for molecular imaging) has renewed the interest in immuno-PET, which is likely to find further clinical use in the near future. The inherent high spatial resolution and non-ionising nature of MRI makes it an attractive modality in the field of molecular imaging in comparison to PET and SPECT. Low molecular weight MRI contrast agents as well as nanoparticulate ones have been successfully conjugated with biological ligands to target tumour markers and image their distribution in vivo (Kievit et al., 2012; Koyama et al., 2012; Lee et al., 2007; Liu et al., 2013; Rasaneh et al., 2011). With such a system, it should be possible to highlight not only large tumours, but also small metastatic deposits found in secondary organs, as well as lymphatics. Whilst the safety and high spatial resolution of MRI are certainly significant advantages over PET, the sensitivity of this technique for molecularly targeted agents requires further development if the detection of sub-mm metastases is to be realised. The result is, whilst the specificity of ‘molecular MRI’ – like immuno-PET – shows significant improvements in the detection of tumour deposits, molecular MRI does not currently possess the sensitivity to provide a reliable, realistic alternative to current SLN staging techniques (Dassler et al., 2012). 9.4. Immuno-staging of the lymphatics with interstitial agents: A necessary compromise? Whilst systemic administration routes of tumour-specific ligands radiolabeled or conjugated to nanoparticulate imaging probes represent the most attractive strategies, their successful clinical implementation remains an elusive goal. This is especially evident for small metastatic deposits, considering the numerous physiological barriers to the delivery of these imaging agents to cancerous tissues (Bertrand and Leroux, 2012; Weissleder and Pittet, 2008). In the context of lymph node staging, interstitial routes offer more direct access to the tumour deposits in lymph nodes than systemic ones and appear to have significant translation potential. In support of this hypothesis, recent studies demonstrated that mammaglobin-A or carbonic anhydrase specific mAbs conjugated to NIR fluorescent dyes could detect as few as 1000 cancer cells in the axillary lymph node after interstitial injection (Tafreshi et al., 2010, 2012). These findings suggest the ability to image specifically occult deposits well below the size of micrometastases. A specific and durable fluorescence signal was observed in target positive metastases until at least 48 h post injection, long after completely clearing from the mammary fat pad injection site (Fig. 3E). Importantly, the imaging probes were not retained in ALN metastases that did not express the target marker. Although fluorescence-based detection approaches are intrinsically limited to animal studies, at least in the current state of the imaging technologies (Ntziachristos, 2010), the application of this approach to other imaging modalities holds promise for the future and warrants further studies. 10. Conclusion The SLN concept remains a powerful prognostic approach for breast cancer and melanoma patients. In order for the SLN concept to reach its potential in other cancer types, the properties of the passive lymphatic tracers require refinement with respect to the clearance rate from the injection site, speed of migration, retention in the SLN, and sensitivity of tracer detection. The ideal passive lymphotropic contrast agent can achieve both rapid migration speed associated with small contrast agent size, yet prolonged SLN retention via the conjugation of biological receptors specific to the lymphatics. Additionally, the detection method for these contrast agents is of high importance: high sensitivity to the contrast agent reduces the doses of tracer administered to the patient, and low-invasive detection reduces complications associated with the

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procedure. Currently, there is future scope to remove the reliance on biopsy to determine the tumour burden of a patient, with the implementation of highly specific immuno-targeted contrast agents. Whilst the systemically implemented immuno-targeted agents closely represent the ideal staging methodology, current limitations to imaging resolution and sensitivity make it currently unfeasible for use in humans. Interstitial immuno-targeted agents, on the other hand, focus on detecting metastases in the nodes closest to the tumour site, and hence efforts are concentrated where they are most needed, and the demand on sensitivity is not as high. As the technology behind immuno-staging tracers is further developed, questions may be raised about the significance of ITC deposits in the lymph nodes, and the prognostic significance they hold – a topic currently considered controversial. 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Intraoperative Fluorescence Imaging for Sentinel Lymph Node Detection: Prospective Clinical Trial to Compare the Usefulness of Indocyanine Green vs Technetium Tc 99m for Identification of Sentinel Lymph Nodes.

Novel handheld magnetometer probe based on magnetic tunnelling junction sensors for intraoperative sentinel lymph node identification.

Gene expression profiling for molecular staging of cutaneous melanoma in patients undergoing sentinel lymph node biopsy.

Sentinel lymph node biopsy in breast cancer: predictors of axillary and non-sentinel lymph node involvement.

Internal Mammary Sentinel Lymph Node Biopsy With Modified Injection Technique: High Visualization Rate and Accurate Staging.

Internal mammary sentinel lymph node biopsy: minimally invasive staging and tailored internal mammary radiotherapy.

[Relevance of the sentinel lymph node biopsy in breast multifocal and multicentric cancer].

The history of sentinel lymph node biopsy.

Sentinel Lymph Node Mapping of Liver.

Imaging sentinel lymph nodes.

Indocyanine green and fluorescence lymphangiography for sentinel lymph node identification in cutaneous melanoma.

Sentinel node biopsy in vulvar cancer: Implications for staging.

Sentinel lymph node biopsy for cutaneous head and neck malignancies.

Fluorescent iodized emulsion for pre- and intraoperative sentinel lymph node imaging: validation in a preclinical model.

Low-Volume Lymph Node Metastasis Discovered During Sentinel Lymph Node Mapping for Endometrial Carcinoma.

Lymph node ratio for postoperative staging of laryngeal squamous cell carcinoma with lymph node metastasis.

Clinical outcome of patients with lymph node-positive prostate cancer following radical prostatectomy and extended sentinel lymph node dissection.

Sentinel lymph node biopsy is indicated for patients with thick clinically lymph node-negative melanoma.

Clinical Significance of Extracapsular Invasion at Sentinel Lymph Nodes in Breast Cancer Patients with Sentinel Lymph Node Involvement.

Missing sentinel lymph node in cutaneous melanoma.

99mTc-Tilmanocept: A Novel Molecular Agent for Lymphatic Mapping and Sentinel Lymph Node Localization.

Priority Claim to "Sentinel Lymph Node Transfer".

Sentinel lymph node biopsy in gastric cancer.

Utility of sentinel lymph node biopsy for solitary dermal melanomas.