AND BREAST CANCER
EDWIN C. GLASS
V.A. Greater Los Angeles Healthcare System, Los Angeles, California, USA John Wayne Cancer Institute, Santa Monica, California, USA
INTRODUCTION
Background of Lymphatic Mapping
The metastatic spread of breast cancer to regional lymph nodes has been recognized for centuries (1), and the propensity of melanoma for lymphatic spread was docu- mented in the nineteenth century (2).The basis for the sentinel node hypothesis is that tumors with a propensity for lymphatic invasion initially spread preferentially to nodes located on their draining lymphatic pathways. Hence these lymph nodes are the ones most likely to contain metastatic cells (3, 4), and they can be regarded as sentinel for the lymphatic spread of the cancer. Knowledge of the tumor status of these sentinel lymph nodes is now recognized as essential for staging and treat- ing malignancies with a propensity for lymphatic invasion. Before discussing the mechanics and details of sentinel node localization, it is worthwhile to review relevant lymphatic physiology.
LYMPHATIC CONTENT AND FUNCTION
The lymphatic system plays important, interrelated roles in fluid homeostasis,
removal of toxins, and immune function.The system is particularly well developed
and extensive in the skin, oropharynx, and intestinal tract where potentially offend-
ing organisms and substances are ever present and abundant.
The distal ends of lymphatic channels are lined with clefts that can be forced open by external pressure from surrounding interstitial fluid, under the influ- ences of oncotic and hydrostatic pressures. Cellular breakdown products, bacteria, proteins, and other materials gain entrance to the lymphatics through these clefts. Malignant cells can also enter through these clefts or by directly invading and penetrating the walls of lymphatic vessels. A continuous flow of interstitial fluid from these distal lymphatic origins carries materials to lymph nodes down- stream.
Molecular expressions on many substances are recognized innately by macrophages, and the materials may be engulfed peripherally or in draining nodes by macrophages or dendritic cells. Examples include the mannose on cell walls of mycobacteria and other pathogens, lipotechoic acid in the cell walls of gram-positive bacteria, or lipopolysaccharides on the cell walls of gram-negative organisms (5). These substances can be suspended in lymph, can be bound to albumin, immunoglobulins, or other proteins, or can be engulfed within macrophages. Intact cancer cells may similarly be transported to draining lymph nodes, although presumably their rate of transit would be slower due to their relatively larger size.
Upon arrival to a lymph node, foreign substances and cells are further processed, in the matrix and sinuses of the node. Importantly, antigens can be processed and presented to B- and T-lymphocytes present in the node, to initiate adaptive immune responses that allow these antigens to be recognized subsequently by anti- bodies, lymphocytes, and macrophages.
IMAGING THE LYMPHATIC SYSTEM
Strategies for imaging the lymphatic system have been developed based on the functional and mechanical properties of the system outlined above. Substances with properties that allow uptake into, transport within, processing within, or binding in the lymphatic system can be employed for imaging the system, provided that they can be monitored or visualized. Hence fluorescent materials, radiolabeled com- pounds, or magnetic molecules have been employed to study the lymphatic system using wavelength controlled light sources, radiosensitive probes or cameras, or mag- netic resonance imaging devices, respectively.
Examples of schemes employed for fluorescent lymphatic imaging include the
use fluorescent proteins or dyes, and quantum dots that exhibit near-infrared fluo-
rescence at invisible near-infrared frequencies (6). Some of the most widely
employed tracking schemes have utilized radioactive reporter molecules, including
radioactive colloidal materials and radiolabeled mannosyl compounds (7) that
are bound to innately recognized mannose receptors. Iron ferrumoxtran is an
example of a colloid material developed for imaging lymph nodes based on
trapping of the iron-containing colloidal particles within lymph nodes, with
external detection using magnetic resonance imaging devices (8) or magnetically
sensitive probes.
DEFINITION OF A SENTINEL NODE
Notwithstanding the principles on which sentinel node detection is based, as dis- cussed above, surgeons need a practical definition for routine clinical use. Many working definitions of a “sentinel” node have been proposed. Examples include the first lymph node to appear after injection of a tracer, the hottest node, all nodes with radioactive countrates exceeding a given background level, the nodes that are first encountered on all independent lymphatic drainage pathways, and other defi- nitions. These disparate definitions are usually pragmatic in orientation, and are developed to provide surgeons with a practical means for the intraoperative deter- mination of which lymph node or nodes to remove. At a more fundamental level however, the intent of sentinel node procedures is to identify, remove, and analyze the node or nodes that are most likely to contain metastatic cancer cells.
In practice, procedures are needed that will, in a period of minutes, to at most hours, define he most likely routes and locations for spread of cancer cells in lym- phatic channels. The spread of malignant cells, however, evolves over many hours to months. Since many factors may influence the biological spread of cells, a rela- tively brief lymphatic mapping procedure using a foreign tracer substance may not always reproduce the path of spread of malignant cells from a primary tumor.
Procedures for localizing sentinel nodes are therefore pragmatic, and their accuracy should be assiduously pursued, given that at best they can only approximate the complex process of cancer spread through lymphatics.
COLLOIDS IN LYMPHATICS
Among the various tools developed for lymphatic mapping for sentinel node local- ization, radiolabeled colloids have received the widest application, and have been in use for half a century (9, 10). Interstitially injected proteins and colloids are absorbed into, and migrate within, regional draining lymphatics. Early applications included mapping internal mammary lymph nodes in patients with breast cancer using subcostal injections (11), intralymphatic therapy (12), and radionuclide lym- phangiography (13).
These initial techniques were extended to evaluation of specific tumors, first to identify the specific basin of drainage (14), and subsequently to determine addi- tionally the specific nodes within that basin most likely to harbor metastases (15, 16).Although lymphatic mapping has been applied to many tumor types, this man- uscript addresses their most common uses: sentinel node localization in melanoma and breast cancer.
When injected randomly into tissue, the colloidal particles of appropriate size (in
the range of approximately 2–200 nm) gain entrance to lymphatics through clefts
in the walls of lymphatics, or by endocytosis through the walls. Some particles may
be phagocytosed by peripheral macrophages. Intralymphatic particles may thus be
either intracellular, phagocytosed by macrophages, or remain suspended in lym-
phatic fluid (17). In either case they are transported to the nearest draining lymph
node, i.e., the sentinel node for the tissue into which they were injected. In general
their rate of transport varies inversely with the size of the colloid particles, with faster transit observed for colloids with smaller particles (16, 18, 19).
Once inside a lymph node, they may be phagocytosed by macrophages or den- dritic cells, which would facilitate their intranodal retention and surgical localiza- tion. In a typical injection used for sentinel node mapping however, roughly 20–100 µg, in the case of sulfur colloid, will be injected. If approximately 1%
reaches the nearest draining node however, roughly 0.2 µg or 10
10particles would be presented to the node for potential phagocytosis. This would probably over- whelm the phagocytic cells in a single lymph node. As a result, the observed reten- tion of colloidal tracers within sentinel lymph nodes probably also represents retention and trapping by mechanisms other than phagocytosis, including nonspe- cific mechanical trapping in the reticular meshwork of the node. With time
Figure 1.
Variable nodal tracer uptake as a function of time in two patients.The upper two images demon-
strate radiocolloid in inguinal nodes, at 5 min in the upper left and at 3 h in the upper right, after an intrader-
mal injection in the right leg with filtered Tc99m sulfur colloid. The lower two images illustrate cervical
nodes at 5 min (lower left) and 3 h (lower right) after an intradermal injection of the same radiopharmaceuti-
cal in the left ear of a different patient.The two upper images demonstrate progressive accumulation of tracer
in the right inguinal nodes in time, whereas the lower images demonstrate a different, and relatively constant,
retention, and uptake in left cervical and supraclavicular nodes.The nodal uptake of tracer varies in time, but
often in an unpredictable manner in different patients. Used with permission from Springer – Cancer Metast
Rev 2006; 25:185
however, such nonspecific trapping will allow release of particles for subsequent migration to other nodes (16). Krynyckyi et al. (20) demonstrated that colloidal preparations with higher specific activity demonstrated higher retention of radioactivity within sentinel nodes than those with lower specific activity, con- sistent with the concept of a limited capacity for phagocytic uptake within a single node.
It is logical to conclude that with longer time periods after injection of colloids that greater numbers of lymph nodes will be visualized using colloids. In fact there is evidence that this occurs, but there is also evidence that images acquired as long as 24 h after injection may demonstrate patterns of nodal uptake that are very sim- ilar to those observed soon after injection (21, 22). Hence there appears to be both a wash-through phenomenon as well as a mechanism for prolonged retention within nodes that receive a high flux of colloidal materials (Fig. 1).
RATIONALE OF LYMPHOSCINTIGRAPHY AND PROBE LOCALIZATION The arguments for sentinel node procedures include accurate localization, removal, and evaluation of the node(s) most likely to harbor metastases, improved staging and prognostication, shorter and simpler surgery, reduced surgical morbidity, and the potential for adjuvant therapy and improved survival. Lymphoscintigraphy, which pictorially identifies these nodes, is extremely helpful in this effort since the drainage of tumors is often unpredictable on a clinical basis, and since unusual or unexpected patterns are common (15). For example, the variable drainage patterns of melanomas near Sappey’s line, or on the head and neck, are now well recognized.
Breast cancers in any part of the breast, not just in medial quadrants, can drain to internal mammary nodes (23).
Lymphoscintigraphy also portrays the temporal sequence of nodal uptake, depicting the first node to intercept lymph drainage from a tumor, i.e., the true sentinel node, which is the lymph node most likely to harbor metastatic cells. In addition to visualizing the lymphatic channels, basins, and nodes, preoperative lymphoscintigraphy can be used to apply skin marks for additional operative guid- ance.These considerations all support the use of preoperative lymphoscintigraphy.
GOALS OF LYMPHOSCINTIGRAPHY
(a) To identify the specific channels, nodal basin(s), and nodes of drainage (b) To determine the temporal sequence of nodal uptake
(c) To mark the sentinel nodes and their afferent channels on the skin (d) To generate clear, easily interpreted images for use in operating room (e) To review findings with the surgeon preoperatively
If the above goals are met, the following clinical benefits are accrued:
(a) Shorter and simpler surgery
(b) Increased accuracy of surgery
(c) Reduced operative morbidity
RADIOPHARMACEUTICALS FOR LYMPHATIC MAPPING
The radiopharmaceuticals most commonly used for lymphoscintigraphy are col- loidal and are radiolabeled with Tc99m. Colloidal tracer remains within lymphatic vessels rather than diffusing out of lymphatic vessels into the blood. Endolymphatic particles undergo subsequent mechanical and phagocytic trapping by macrophages in sinusoids of lymph nodes. Agents now commonly employed include Tc99m albumin nanocolloid and Tc99m rhenium sulfide in Europe, Tc99m antimony trisulfide colloid in Australia, and Tc99m sulfur colloid in unfiltered form or filtered through a submicron filter in the United States. No radiopharmaceuticals are cur- rently approved by the U.S. FDA for lymphoscintigraphy.
Particle size influences colloid properties. Optimal size is in tens of nanometers, with the above agents ranging 5–250 nm. Small particles or nonparticulate agents, such Tc99m-labeled albumin (noncolloidal), migrate faster and provide better delineation of lymphatic channels (16, 18, 24), but also tend to leak into the blood pool. Small particles or nonparticulate tracers are also more likely to travel beyond the first or sentinel node, resulting in multiple hot nodes (18). This is more likely when larger numbers of particles are injected, which overwhelm the trapping capacity of macrophages in the first (sentinel) node.
Other noncolloidal lymphoscintigraphic agents specifically target lymph nodes, including radiolabeled antitumor antibodies, agents that bind to receptors in lymph nodes (7), and agents that mimic heme breakdown products, e.g.,Tc99m phthalo- cyanine tetrasulfonate (PCTS) (25). Noncolloidal dyes (e.g., isosulfan blue or meth- ylene blue) migrate much faster than colloids (24). Although usually used in nonradioactive form, radiolabeled noncolloidal dyes are also now being investigated (26, 27). Like PCTS, these labeled dyes offer the possibility of rapid visual, lym- phoscintigraphic, and probe identification of sentinel nodes.
Despite general differences observed among tracers and techniques, considerable variation is observed in tracer kinetics between patients, particularly with breast cancer. No single imaging time is optimal for all patients.The radiopharmaceutical employed is a much less significant factor in overall accuracy than the skill, atten- tion to detail, and understanding of the persons performing lymphatic mapping.
CONSIDERATIONS IN TECHNIQUE OF LYMPHATIC LOCALIZATION FOR CUTANEOUS MELANOMA
Techniques for lymphatic mapping are fairly well standardized for melanoma but not for breast cancer (28). Both radioisotopic and dye-based tracer methods are used. In general, most blue nodes are hot, but not all hot nodes are blue (29). In practice, nodal localization is optimized by using both visible dyes and radioactive tracers.The tracers are used both for lymphoscintigraphy and for probe localization. The overall repro- ducibility of cutaneous lymphoscintigraphy in melanoma has been found to be approx- imately 85% (30–32). Results can be affected, however, by previous surgical resections or radiation that alters migration patterns or by any process that affects epifascial tissues.
In experienced hands, cutaneous lymphoscintigraphy will identify a sentinel
node in almost all patients, but nodal basin recurrence rates after sentinel node
Figure 2.
Upper panels show early (upper left) and delayed (upper right) images from lymphoscintigram of melanoma in midback that demonstrates bilateral axillary migration.The patient refused any axillary sampling but underwent removal of the melanoma on his back.Two years later he returned and underwent FDG PET imaging (oblique views in lower two panels) that demonstrated not only bilateral axillary metastases but also in transit metastases that probably had already seeded prior to removal of the primary after the initial lym- phoscintigraphy. Supraclavicular nodal metastases are also demonstrated that were not predicted by the lymphoscintigram
localization and surgery are higher for melanoma than for breast cancer (33).
Melanoma is a particularly dangerous malignancy however, with no well-defined
medical adjuvant therapies. Despite the relative ease of identification of the sentinel
node in melanoma, recurrences are common, and not always fully predicted by the
results and findings from lymphatic mapping (Fig. 2). In the Multicenter
Lymphadenectomy Mapping Trial I for example, the rates of recurrence and melanoma-specific mortality were 16.8 and 9.7%, respectively, at 5 years in patients without evidence of metastases in the sentinel node (34). It is essential to pay meticulous attention to detail when performing lymphatic mapping.
Other technical considerations that may influence nodal localization in melanoma include the times of imaging after injection, location and depth of the injection, positioning of the patient including arm elevation, characteristics of the radiopharmaceutical employed, including the size of the colloid particles in the injectate, and the use of adjunctive measures that modulate the flow of lymph, such as local heat and massage.
Imaging with a scintillation camera should begin immediately after intradermal (not subcutaneous) injection, in order to identify lymph channels, and to identify the first (sentinel) node(s). Subcutaneous injections result in slower and lesser migrations of tracer (24). Immediately after the injection, the site of skin puncture should be covered with a cotton ball and tape, or similar materials, to prevent seepage of tracer at that site, with resultant artifactual contamination of the skin. The use of a small- bore needle ((27) gauge or smaller) helps minimize this problem, and is also less uncomfortable for the patient. Positioning of the patient for lymphoscintigraphy should correspond, in at least some views, to the position of the patient during sur- gery, since lymph nodes can translate with body movement and positioning.
Elevation of the arm, for example, will alter the position of axillary nodes relative to overlying skin marks.The location of the sentinel node should be determined pre- cisely, and skin marks applied directly over the node and its afferent channel. This can be greatly facilitated by using a radiosensitive probe, after and/or during imaging in nuclear medicine. Inspection for dual channels and additional nodes that are in close proximity to the obvious ones should be carefully performed and reported. Copies of the clearest and best images should be available to the surgeon for intraoperative ref- erence. Body outlines should be included on images to facilitate interpretation (e.g., in surgery). Body outlines should be routine in lymphoscintigraphy.
OTHER ISSUES IN THE DETECTION OF SENTINEL NODE FOR MELANOMA
Problems often encountered by less experienced users include (1) imaging too late after injection, when multiple nodes may be hot, (2) suboptimal positioning of patient (e.g., omitting oblique views of axilla with arm extended or elevated), (3) failure to perform lymphoscintigraphy at all, (4) poor communication between nuclear medicine physician and surgeon (e.g., poorly formatted images that cannot be interpreted by surgeon or others who were not involved in the generation of those images), (5) inaccurate or no skin marks over the sentinel node, and (6) failure to review the findings with the surgeon preoperatively.These problems can all result in erroneous identification of the sentinel node.
In experienced hands, cutaneous lymphoscintigraphy can successfully identify a
sentinel node in almost all patients. However, the identification of lymph channels
is also important in melanoma. When a separate lymph channel leading to a node
is observed, that node should be regarded as a sentinel node, regardless of whether or not it is the most intense node at some later time.Additionally, knowledge of the locations of lymph channels can facilitate surgery; it directs the surgeon in the search for a blue lymphatic channel, which is often identified intraoperatively before the sentinel node itself. The channel can then be traced intraoperatively to the sentinel node. Imaging should begin immediately after injection so that lymph channels can be identified. Inspection for additional (two or more) channels and additional nodes that are in close proximity to the obvious ones should be carefully performed and reported, since additional channels may drain to additional sentinel nodes.
CONSIDERATIONS OF TECHNIQUE FOR LYMPHATIC LOCALIZATION IN BREAST CANCER
The identification of sentinel nodes in breast cancer is often more challenging than in melanoma, and techniques for lymphatic mapping are less standardized for breast cancer. Both dye-based and radioisotopic tracer methods are used, with optimal results usually obtained with the use of both techniques in combination. As with melanoma, nodes are usually, but not always both blue and hot. In general, nodes are more often hot and not blue, than blue alone (29, 35, 36).This is usually attrib- uted to the retention of the particles by macrophages in lymph nodes, whereas blue dye is not trapped as efficiently in the node and will flow through it.Although there are usually more hot nodes than blue nodes found in the axilla, this depends on time lag after injection of either of the two tracers. Early after injection (e.g., 5–30 min), slow moving particles may not yet have arrived to the lymph basin, but the faster moving dye will have arrived, whereas later, the particles are retained, but the dye has washed out.
Techniques for breast injection differ among institutions.The best technique for injection is one of the more controversial topics in the realm of sentinel node studies.
Different types of injections include parenchymal peritumoral, intratumoral, subdermal, subareolar, and periareolar injections.The relative advantages and disad- vantages of the different methods are now reasonably well understood. Longer times for localization are required after parenchymal injections of radiocolloids than after dermal, subdermal, or periareolar injections, although parenchymal injections are more likely to identify nonaxillary sites of drainage, e.g., internal mammary nodes. Intratumoral or parenchymal peritumoral injections are conceptually logi- cal. So why not always use them? The principal disadvantage of intratumoral and peritumoral injections is a lower success rates for visualization of axillary sentinel nodes. Additionally, they require longer transit times to nodes, and are somewhat more difficult to perform.
To address the problems with these parenchymal injection methods, injections
into the skin or subcutaneous tissue overlying the tumor or in the periareolar
region have been utilized.These methods yield higher rates of visualization of axil-
lary nodes and are procedurally simpler. Additionally, injecting the day before sur-
gery (21, 22) has been employed, almost assuring the identification of hot nodes by
Figure 3.
