Joseph Zenga, Patrik Pipkorn, and Brian Nussenbaum Abstract
The biological mechanisms of lymphatic spread are intricate, highly regulated active processes involving the concert of trophic factors, lymphatic growth, and tumor chemotaxis. Dissemination of head and neck cancer beyond its primary site has a significant detrimental impact on treatment outcomes with clinical neck disease decreasing survival by 50%. For years, tumor spread was considered a passive process dependent on tumor volume and interstitial pressure leading to tumor shedding into regional lymphatics. During the last few decades, however, the discovery of specific lymphatic immunohistochemical markers has enabled researchers to study the mechanisms in lymphangiogenesis and lymphatic spread in greater detail. These investigations have revealed that lymphatic spread instead involves highly active and regulated mechanisms. Although much remains to be elucidated, several of the most critical steps have been identified. The process begins with tumor lymphangiogenesis and the epithelial-to-mes- enchymal transition of malignant cells. Through specific chemo- tactic signaling and extracellular matrix degradation, tumor cells intravasate into peritumoral lymphatics to reach the draining nodal basin. Although multiple trophic factors influence this sequence, the vascular endothelial growth factor C (VEGF-C)/VEGF- D/VEGFR-3 signaling axis has been shown to play a central role in the regulation of lymphatic spread. Intervention through downre- gulation of this axis has been identified as an attractive target for pharmacotherapy, preventing or limiting lymphatic dissemination. Although many agents have held early promise in preclinical animal experiments, human studies are just beginning. The exact role of these agents in the future remains to be seen.
Keywords: lymphangiogenesis, lymphatic system, metastasis, vascular endothelial growth factor, epithelial-to-mesenchymal transition, chemotaxis
For most cancer patients who succumb to their disease, it is not the result of an uncontrolled local tumor but from dissemination of cancer cells through lymphatic or hematogenous spread to regional nodes and distant organ systems. When these metastases have progressed far enough, they become resistant to conventional therapies. For certain tumors, including many sarcomas, renal cell carcinoma, and follicular thyroid carcinoma, early dissemination is typically hematogenous. For many others, however, including upper aerodigestive tract squamous cell carcinoma (SCC), initial spread is primarily to the regional lymphatics. While the reasons for these differences remain to be fully elucidated, the prognostic value of lymph node metastasis in head and neck cancer is substantial, decreasing survival probability by 50%.1 Although distant metastasis is an even more ominous prognostic sign, less than 10% of patients with head and neck cancer demonstrate overt distant metastatic disease at initial presentation.
Given the propensity of head and neck cancer to develop lymphatic metastasis and the associated decrement in survival, a great deal of work has focused on the diagnosis and treatment of regional nodal disease. Patients who present with clinical regional metastasis are treated with conventional surgical or nonsurgical management paradigms. For patients who present with clinically negative regional nodes, however, management of the neck becomes more complicated. Neck dissection with pathological assessment of the resected nodal levels is the current “gold” standard staging tool for regional metastases in a clinically negative neck. This invasive procedure comes with risk, however, and is beneficial only if the probability of detecting occult regional disease is high enough. A significant proportion of clinically negative patients are also pathologically negative and will, therefore, undergo an unnecessary invasive staging procedure. Nonetheless, in the population of patients at approximately a 20% or greater risk of harboring occult metastasis, regional control and survival are overall improved in those undergoing lymphatic staging by neck dissection as compared to observation.2
There are no current widely accepted minimally invasive techniques to achieve accurate regional staging. Although sentinel lymph node biopsy has been used, it is limited in general to SCC of oral cavity subsites and is performed at specialized centers. It is still an invasive procedure, nonetheless, and comes with its own risks and challenges. Imaging techniques are limited in resolution, and positron emission tomography scanning can identify nodal involvement as low as 7 mm in diameter but cannot reliably detect occult micrometastatic disease.3
A predictive genetic or morphologic signature of a resected primary tumor would be ideal to determine the risk of occult metastasis and help better select patients for elective neck treatment or observation. Although molecular profiling of primary head and neck tumors has been investigated to identify genetic markers of lymphatic spread, none have been widely adopted or validated in large cohorts.4 Examining the biological mechanism of lymphatic spread, including trophic factors, signaling pathways, and morphological changes, provides another possible method to identify primary tumors at high risk for regional metastasis. This requires a detailed knowledge of how tumors spread to the regional lymphatics and may provide an opportunity to develop therapeutic agents to block regional dissemination of disease. This, in turn, necessitates an understanding of the structure and function of the lymphatic system, methods for microscopic assessment, and an overview of the major molecular mechanisms driving lymphatic spread.
4.2 The Lymphatic System
4.2.1 Lymphatic Function
Nutrients in the bloodstream reach tissues by extravasation from capillary vessels under high pressures from the arterial system. This milieu of extravasated fluid and macromolecules makes up the interstitial fluid, allowing cells to take up these nutrients and dispose of waste products. Through both active and passive processes, this fluid and its components are recycled and returned to the circulation through the lymphatic system. Initial lymphatics are blind-ended channels composed of a single layer of endothelial cells with minimal basement membrane and large gap junctions to allow uptake of interstitial fluid. Anchoring filaments tether the initial lymphatics to the extracellular matrix to prevent vessels collapse under interstitial pressures. Lymph then proceeds into precollecting and larger collecting lymphatic vessels propelled by perivascular smooth muscle contraction along with extrinsic skeletal muscle pressure. These larger lymphatic vessels are less permeable, limiting extravasation, and one-way valves prevent lymphatic backflow. Lymph is first filtered through draining regional lymphatic basins, centers for antigen recognition, and initiation of any requisite immune response. From these lymphatics, lymph is ultimately returned to the venous circulation through the thoracic or right lymphatic ducts.
4.2.2 Lymphatic Origins and Development
An appreciation of lymphatic embryology can aid in understanding how tumor trophic factors may lead to lymphangiogene- sis or invasion of existing host lymphatics. Due to difficulties with visualization and tracking of nascent lymphatics, development of the lymphatic system has been a subject of controversy for over 100 years. Although the exact origins of the lymphatic and venous systems have been debated, lymphatic development appears to begin through the formation of lymphatic sacs that sprout from the embryonic venous system as early as the fifth week of fetal development. These paired lymphatic sacs, which ultimately give rise to the thoracic and the right lymphatic ducts, expand and ramify into a lymphatic plexus. Lymph node formation follows as specialized myofibroblasts proliferate and form a scaffold for lymphoid tissue, through which immune cells are recruited. Although aspects of this process remain unclear, several key factors contributing to lymphatic development and proliferation have recently been identified.
An early and critical developmental step, determining the fate of lymphatic endothelial cells, is expression of the transcription factor, Prospero-related homeobox protein 1 (Prox1). Prox1 knockout mice are devoid of lymphatics and deletion at any point during development results in lymphatic regression. Although the exact mechanisms that leads to induction of Prox1 expression remain unclear, bone morphogenic protein and Notch signaling pathways have been identified as inhibitors of Prox1-induced lymphangiogenesis and drive venous cell differentiation, while Wnt signaling appears to increase Prox1 expression in lymphatic progenitor cells. Certain transcription factors, as well, have been associated with induction of Prox1 signaling, including Sox18 and Coup-TF2. Ultimately, the Prox1 molecular switch results in expression of requisite proteins for lymphatic endothelial cell growth and migration.5
A significant advance in understanding lymphangiogenesis came with the discovery that vascular endothelial growth factors (VEGF) played a central role in stimulating lymphatic proliferation. These growth factors are composed of a family of subtypes, with five described members in mammals, including VEGF-A through VEGF-D and placental growth factor (PlGF). Each exerts its effects through specific membrane receptors coupled to downstream intracellular pathways. VEGF-A, VEGF-B, and PlGF are primarily involved in vasculogenesis and angiogenesis. VEGF-A was the first-described growth factor of this group and represents the dominant trophic signal for blood vessel endothelial cell proliferation and migration. VEGF-A binds to vascular endothelial growth factor receptors 1 and 2 (VEGFR-1 and VEGFR-2), although the majority of angiogenic effects are mediated through VEGFR-2.
A third vascular endothelial growth factor receptor (VEGFR-3) was subsequently discovered, and its major ligands, VEGF-C and VEGF-D, were identified as a target of Prox1 activation and a key trophic axis for lymphatic proliferation. Early in embryogenesis, however, VEGFR-3 signaling is important for vascular development, and VEGFR-3 knockout mice die at an early embryonic stage of cardiovascular defects. Later in development, VEGFR-3 expression becomes restricted to lymphatics and is critical to lymphangiogenesis and lymphatic migration. VEGFR-3 heterozygous animals exhibit substantial lymphatic defects and similarly, the human condition Milroy’s disease is associated with a VEGFR-3 mutation and results in congenital lymphedema. Further, lymphatic sprouting is at least in part a VEGF-C-dependent process, as endothelial cells have been shown to migrate toward a VEGF-C gradient and VEGF-C-deficient mice develop a hypoplastic lymphatic system. Although VEGF-D is also a VEGFR-3 ligand and a known lymphangiogenic trophic factor, lesser congenital defects in lymphatic migration are seen in VEGF-D-deficient mice, including smaller caliber and less functional superficial lymphatics.5,6
A second EGF system, the angiopoietin-Tie (Ang-Tie) axis, is involved in angiogenesis and lymphangiogenesis as well. Angio- poietin 1 (Ang1) and 2 (Ang2) along with their receptors Tie1 and Tie2 are the best characterized members of this signaling pathway. Ang1 is an obligate agonist of Tie2, while the inhibitory or stimulatory effects of Ang2 are context dependent. Although the Ang- Tie system is primarily known for its role in vascular and cardiac embryology, it has been shown to be critical to the stability and remodeling of lymphatic endothelial cells later in development.
As understanding of these pathways continues, the molecular picture becomes increasingly complex (Fig. 4.1). There appears to be crossover between the function of VEGFR-2 and VEGFR-3, including both homodimer and heterodimer formation leading to specific angiogenic and lymphangiogenic effects. VEGF-C is also known to bind the receptor neuropilin2 (Nrp2). Nrp2 is seen in high levels in the developing lymphatic plexus, and Nrp2-deficient mice exhibit normal central lymphatics but absent superficial lymphatic networks.7
In addition, all these factors are closely regulated by extrinsic and intrinsic processes. VEGF-C is regulated by collagen and calcium-binding EGF domains 1 (CCBE-1), a protein necessary for normal lymphatic development in animal models, and mutations of which lead to lymphatic dysplasia in humans. CCBE-1 plays a role in proteolytic processing of VEGF-C to its most active form. Nrp-2 signaling, which results in lymphatic proliferation when binding VEGF-C, has been shown to be regulated by semaphorins, a family of signaling proteins. When binding Nrp2, class 3 semaphorins result in inhibition of lymphatic cell proliferation and induction of apoptosis. VEGFR-3 is also highly regulated by additional pathways independent of Prox1 signaling. Ephrin-B2, a transmembrane ligand for Eph receptor tyrosine kinases, promotes lymphatic sprouting through VEGFR-3 modulation. Similarly, T-box transcription factor 1 regulates VEGFR-3 expression and appears to be necessary for lymphatic endothelial cell growth and maintenance. Finally, VEGFR-3 may be activated independently of a VEGF ligand through binding of extracellular matrix components.8
Implicated in development and wound healing
Strongly expressed on all lymphatics
Not expressed on normal blood vessels
Expressed in some pathological conditions including malignancy
Transmembrane hyaluronic acid receptor
Implicated in wound healing and cell migration
Strongly expressed on initial lymphatics
Expressed on some blood vessels and malignancies
Transmembrane tyrosine kinase receptor
Involved in lymphangiogenesis and lymphatic development
Strongly expressed on normal lymphatics
Upregulated in neoendothelium of malignancy and some vascular tumors
Involved in lymphangiogenesis lymphatic development
Highly specific for lymphatics Not expressed on neoendothelium of malignancy
Expressed on some nonendo- thelial tissue
Abbreviations: LYVE-1, lymphatic vessel endothelial hyaluronan receptor 1; Prox-1, prospero-related homeobox protein 1; VEGFR-3, third vascular endothelial growth factor receptor.
4.2.3 Lymphatic Markers
The study of the lymphatic system and its interaction with malignancy is ultimately limited by the quality of lymphatic markers. Studies evaluating tumor lymphangiogenesis and lymphatic invasion must be interpreted in light of the specificity of the technique used to identify lymphatic vessels. With traditional hematoxylin and eosin staining, lymphatic endothelium cannot be reliably detected and quantified. Immunochemical staining, therefore, must be used to target specific lymphatic markers, each with its own drawbacks and advantages (Table 4.1). There are multiple commercially available antibodies specific for the lymphatic markers examined below.9
Podoplanin is a transmembrane glycoprotein expressed in multiple tissue types including bone, lung, kidney, and choroid plexus. Although its physiological role is unclear, it is important for normal pulmonary and lymphatic development and has been implicated in wound healing and cell migration. It is strongly expressed on all lymphatics but not blood vessel endothelium. It is upregulated in certain pathological conditions, including some skin carcinomas and vascular tumors, which may limit its specificity as a lymphatic marker.10
Lymphatic Vessel Endothelial Hyaluronan Receptor 1
Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) is a transmembrane hyaluronic acid receptor also implicated in cell migration and wound healing. It is expressed on initial lymphatics
but less strongly on collecting vessels. Although it is found widely on both luminal and extraluminal surfaces of lymphatic channels, it is also found on select blood vessels including liver, spleen, and pulmonary capillaries, as well as certain activated tissue macrophages. It may also be expressed on a percentage of blood vessels in pathological conditions including some malignancies.11
Vascular Endothelial Growth Factor Receptor 3
As discussed earlier, VEGFR3 (also known as FLT4, fms-like tyrosine kinase receptor 4) is a transmembrane protein receptor involved in lymphangiogenesis and maintenance of lymphatic differentiation. Although it is a specific marker for normal lymphatics, VEGFR-3 may be upregulated in vascular endothelium when associated with malignancy. It can be seen in myoepithelial cells and is highly expressed in certain vascular tumors.
Prospero-Related Homeobox Protein 1
As discussed earlier, Prox1 is a homeobox transcription factor critical to lymphatic development. In contrast to other lymphatic markers, Prox1 immunofluorescence is nuclear rather than membrane bound. It is seen in all lymphatic endothelial cells, regardless of patient age, including lymphatic capillaries and lymphatic trunks. Prox1 is not seen in blood vessel endothelium or pericytes and does not appear to be expressed in vascular neoendothelium of carcinomas. It is seen in some nonendothelial tissue including the lens, heart, liver, pancreas, and nervous system.
4.3 Lymphatic Spread in Head and Neck Cancer
Early studies in head and neck SCC reported high rates of neck disease with low distant metastatic rates. This suggested that regional nodal involvement may be a prerequisite for distant spread and perhaps a linear process of cancer dissemination. The early prevailing view held that cancer spreads passively to the cervical nodes, which served as a filter against distant metastasis.
This theory of passive tumor shedding was challenged by emerging clinical data in the latter 20th century. First, patients with head and neck SCC were found to occasionally have clinical distant metastasis without regional nodal involvement. Second, as experience grew with unknown primary carcinoma, it became clear that a microscopic primary tumor could lead to an aggressive cervical metastasis with evidently different pathobiology. Taken together, these clinical data suggested that lymphatic and hematogenous metastases may be separate and specific events, and further that lymphatic spread was an active process with associated fundamental pathobiological changes occurring in those cancer cells that progress to lymphatic metastases (Fig. 4.2).12
Ultimately, dissemination to regional lymph nodes appears to require multiple active steps including modulation of intratumoral or peritumoral lymphatics, physical entry into lymphatic vessels, survival and transport in local and regional lymphatics, and finally deposition and proliferation in regional nodes (Fig. 4.3). Although much of the work investigating these steps has been done in other organ systems, the underlying mechanisms appear largely conserved and can be applied to understanding lymphatic spread in head and neck cancer.
4.3.1 Tumor Lymphangiogenesis
Whether tumors use preexisting host lymphatic vessels or whether tumor-induced lymphangiogenesis was critical to regional spread has been long debated. Deciphering this process, however, is vital to the development of antineoplastic therapy. An agent that blocks lymphangiogenesis will be ineffective if tumors simply employ host lymphatics for regional dissemination.
In the early 2000s, evidence emerged from animal models that tumor lymphangiogenesis leads to lymphatic metastases by upregulating VEGF-C and VEGF-D production. Experimental overproduction of these growth factors significantly increased both tumor-associated lymphatics and regional metastases. The majority of subsequent clinical and bench investigation supported these findings in many different tumor types and suggested that regional metastases were at least partly regulated by the VEGF-C/VEGF-D/VEGFR-3 signaling axis and specifically related to tumor-associated lymphangiogenesis. This pathway also appeared restricted to lymphatic proliferation and distinctly separate from angiogenesis. VEGF-C overexpression in the skin of transgenic mice resulted in hyperplasia of the dermal lymphatics without significantly affected vascular endothelium. Further, when classically nonlymphangiogenic nonmetastatic tumors were induced in transgenic mice to overexpress VEGF-C, peritu- moral lymphatic hyperplasia and regional metastases were seen. In other cancer models, when an anti-VEGFR-3 antibody was either systemically administered or secreted by tumors, decreased intratumoral lymphatics and decreased regional metastases were seen. In human studies, a correlation between VEGF-C and VEGF- D levels in primary tumors and regional metastases has been identified in multiple tumor types.13
Fig.4.2 Critical active steps in the process of lymphatic dissemination from the primary tumor to the regional nodal basin.
Fig. 4.3 Schematic of lymphatic spread with critical questions that this review will address including the role of tumor lymphangiogenesis, intratumoral and peritumoral lymphatics, and tumor cell entry into functional lymphatic vessels.
Although clinical evidence in head and neck SCC is limited, several studies have identified an association between prolymphan- giogenic factors and regional metastases. The majority of these data come from retrospective studies evaluating tissue levels of signaling molecules and their correlation with tumor stage and prognosis. Although many have found a significant association between upregulation of the VEGF-C/VEGF-D/VEGFR-3 signaling axis and regional metastases, others have found no such correlation.14.15
Other proteins involved in the lymphangiogenic cascade have also been implicated in head and neck cancer including Proxl, neuropilins, and semaphorins.14.16 Ultimately, although prolymphangio- genic signals play a role in the development of regional metastasis, the process is more complex, requiring not only growth factors, but also changes in lymphatic vasculature, tumor invasion and migration, as well as survival in regional nodal basins to achieve clinical regional dissemination.
4.3.2 Significance of Lymphatic Vessel Density
If upregulation of lymphangiogenic signaling pathways plays a role in lymphatic metastasis, it becomes critical to understand where in the tumor environment lymphatic proliferation occurs, both for informing prognosis from surgical specimens and to identify therapeutic targets. Increases in both intratumoral and peritumoral lymphatic vessel density (LVD) have been correlated with lymphatic spread in multiple tumor types. Although the association of tumoral microvessel density with survival has been recognized since the early 1990s, in head and neck cancer it appears that LVD has even greater prognostic value. A recent meta-analysis identified intratumoral LVD as a more important predictive factor than blood vessel density, with a greater than twofold increased risk of death in patients with high intratumoral lymphatic counts.17.18
Although intratumoral LVD has significant prognostic value, peritumoral lymphatics may be more functionally important for lymphatic spread. In animal models, intratumoral lymphatics have been shown to be nonfunctional in many cases, either invaded or compressed by tumor, and do not provide an effective draining pathway to regional lymphatics. Peritumoral vessels, however, are often dilated and functional, particularly in the presence of VEGF-C overexpression. Further, regional metastases have been demonstrated in the absence of intratumoral lymphatics, suggesting that a peritumoral lymphatic network is sufficient for lymphatic spread. Additionally, meta-analyses in other tumor types, including breast cancer and melanoma, have identified a stronger correlation of regional metastases with peritumoral LVD than with intratumoral LVD.14
4.3.3 Tumor Entry into Lymphatics
Although dilation and functional drainage of peritumoral lymphatics may be associated with lymphatic spread, to enter the lymphatic system, tumor cells must make an epithelial-to-mes- enchymal transition (EMT), migrate to nearby lymphatics, and intravasate. Although early theories proposed that this was a passive process, driven by changes in interstitial fluid pressure and volume, recently, EMT and intravasation have been recognized as complex and active processes involving the concert of multiple chemotactic factors and signaling pathways.
Epithelial cells are characterized by cell polarity and adhesion. Migration and metastases require cell detachment and cytoskeletal reorganization. Two fundamental cellular changes, downregulation of E-cadherin and upregulation of vimentin, have been associated with loss of cell-to-cell adhesion, increased cell motility, and an EMT phenotype. Head and neck cancers with this clinical signature have a nearly universal metastatic rate, while those without it have a metastatic rate of less than 50%. A large number of other signaling components, along with trophic stimuli from transforming growth factor-p (TGF-в) and epidermal growth factor (EGF) have been identified, all contributing to morphological and functional changes that lead to cell detachment and invasion.19
Once EMT is established, tumor cells must migrate toward functional lymphatics to achieve lymphatic spread. In nonpathological conditions, lymphatic vessels are an important source of chemotactic factors, including chemokine ligand 21 (CCL21) that binds to chemokine receptor 7 (CCR7) on immune cells, allowing them to migrate to and enter the lymphatic system to initiate a normal immune response. Upregulation of CCR7 in certain tumors has been associated with an increase in lymphatic metastases, suggesting that lymphatic chemotaxis plays an important role in the process of lymphatic spread.13 In addition, effective tumor cell migration requires degradation of the extracellular matrix, mitigated by upregulation of certain proteases, particularly the matrix metalloproteinases (MMP). This family of endopeptidases play a critical role in tumor-associated basement membrane degradation including entry into lymphatics and extracapsular spread from lymph nodes.20
4.4 Clinical Applications
4.4.1 Importance of Head and Neck Subsites
It is well known that head and neck cancers at various subsites have different propensities and patterns for nodal metastasis.
In the oral cavity, it is widely known that the incidence of lymphatic metastasis is associated with subsite, T-stage, and depth of invasion. For the larynx, early on, glottic cancers were noted to have a low risk of nodal spread, while oral and pharyngeal sites were more likely to develop regional metastases. More recently, a dramatic increase in human papillomavirus (HPV) related oropharyngeal cancers have renewed interest in elucidating the mechanisms of lymphatic spread. These HPV-driven tumors are typically small at the primary site with early and sometimes extensive spread to regional nodes.
Several hypotheses have been suggested for the discrepancies observed in nodal spread between subsites. Earlier symptoms from vocal cord tumors may lead to earlier detection and therefore less nodal disease. Even advanced glottic cancer, however, appeared to have less lymphatic spread than similar supraglottic primaries, and it was hypothesized that the density of the lymphatic system in the submucosa may account for the different rate of lymphatic invasion. Beginning in the 1980s, dyes studies performed in cadaveric specimens as well as in vivo demonstrated sparse lymphatics in the glottic submucosa, particularly anteriorly, and a somewhat more developed network in the deeper connective tissue with few anastomotic connections. These studies also suggested that there were few lymphatic connections in a vertical axis between subglottis, glottis, and supraglottis. Although initial reports using different methodology often led to different conclusions, the use of electron microscopy combined with immunohistochemical lymphatic staining provided compelling evidence that the baseline density of lymphatics is intrinsically different between laryngeal subunits. This difference may, to some extent, explain the difference in nodal spread between glottic and supraglottic cancers.21,22
In addition to differences in metastatic rate, specific head and neck subsites appeared to drain to distinct nodal stations (Fig. 4.4). Based on pathological nodal stage from large groups of patients undergoing comprehensive neck dissection for head and neck cancer, the most common drainage pathways for various head and neck subsites were determined.23 This knowledge has allowed for more targeted treatment with selective neck dissection in N0 patients to limit morbidity. The predictability of the exact pathways of lymphatic drainage may be variable between patients and sites, particularly for cutaneous primary tumors.
A visual understanding of differential drainage from different head and neck primary sites was achieved with the development of lymphoscintigraphy. Peritumoral injection of radioopaque dye revealed that distinct lymphatic channels existed specific to each patient and tumor location. This gave surgeons extensive information about the variability of the cervical lymphatic system and valuable clues to explain the existence of skip metastases and patients with involvement of multiple nodal stations. This concept was further adapted to intraoperative lymphatic mapping to identify the first echelon, or sentinel, node as a minimally invasive method to stage a clinically negative neck. This hypothesis, that lymphatic drainage from a given primary site would reliably drain to primary echelon nodes and the status of these nodes would be representative of the status of the neck basin, was confirmed and widely applied to cutaneous malignancy. The sentinel lymph node concept appears to be effective for staging N0 necks for mucosal primary sites as well.
Fig. 4.4 (a) Typical sites for regional lymph node metastases. 1, submental; 2, submandibular; 3, parotid and preauricular; 4, retroauricular; 5, jugulodigastric; 6, deep cervical chain; 7, jug- uloomohyoid; 8, pretracheal and peritracheal; 9, prelaryngeal lymph nodes. (b) Laryngeal carcinoma. (c) Carcinoma of the tongue. (d) Tonsillar carcinoma.
4.4.2 Antilymphangiogenic Therapeutic Targets
As discussed earlier, the view that regional metastasis is a passive process has largely been replaced by evidence that lymphatic spread is an intricate active process involving the coordination of several events including multiple trophic factors and signaling cascades. Tumor lymphangiogenesis is an early critical step that provides an attractive target for pharmacologic intervention. Limiting lymphatic ingrowth in early- stage primary tumors may limit the development of lymphatic spread. By decreasing intratumoral and peritumoral lymphatic channels and their associated chemotactic factors, those tumor cells that have made the EMT may be less likely to achieve regional dissemination.
Although many of the mechanisms underlying lymphatic embryology, and the analogous process of tumor lymphangio- genesis, have been elucidated, development of antilymphan- giogenic pharmacotherapy has been more limited. The majority of evidence comes from preclinical studies focusing largely on the VEGF-C/VEGF-D/VEGFR-3 signaling axis. In animal models, inhibitory antibodies or downregulation of this signaling cascade has been shown to reduce the rate of lymph node metastasis.
The first VEGF inhibitor approved for human use was bevacizumab, a humanized monoclonal antibody specific for VEGF-A. Used in several different solid tumors, including colorectal, lung, and renal cancers, its efficacy is primarily related to its antiangiogenic properties, although it has shown some antilymphangio- genic effects as well. VEGFR-2, the primary receptor for VEGF-A, has been identified on lymphatic endothelial cells and may lead directly to lymphatic proliferation. In addition, VEGF-A has been shown to be a chemotactic factor for immune cells, which in turn secrete VEGF-C and VEGF-D, leading to lymphangiogenesis. Many other multitargeted receptor tyrosine kinase inhibitors acting on VEGF pathways have been developed since, several with anti-VEGFR-3 activity, including sunitinib, pazopanib, and axiti- nib. In recent clinical trials, several of these inhibitors have shown some modest activity in head and neck SCC, particularly in the recurrent-metastatic setting. Despite the theoretical anti- lymphangiogenic activity of these inhibitors, the extent and clinical importance of their effect on lymphatic growth remains uncertain.
Along with multitargeted therapies, several agents specific to the VEGF-C/VEGF-D/VEGFR-3 axis are in development. IMC- 3C5 and VGX-100, monoclonal antibodies specific for VEGFR-3 and VEGF-C, respectively, have undergone phase I trials in patients with advanced solid tumors. Although minimal direct antitumor activity was observed, these agents are relatively well tolerated, and require testing in larger-scale trials powered to detect changes in recurrence and metastasis to determine ef- ficacy.24 In addition to direct antibody-mediated inhibition, the proteolytic processing of VEGF-C and VEGF-D may be targeted, preventing the conversion of these growth factors into their most active form. Although this has shown promise in preclinical models, its in vivo effects remain unexplored.
In addition to the VEGF-C/VEGF-D/VEGFR-3 axis, other components of lymphangiogenic signaling may be targeted including VEGF-A/VEGFR-2, Ang/Tie, neuropilins, semaphorins, and the transcriptional control of lymphatic proliferation. Although several inhibitors if the VEGF-A/VEGFR-2 and Ang/Tie systems have been developed, their primary mechanism of action is thought to be through regulation of tumor angiogenesis and the extent and efficacy of their antilymphangiogenic effects are not well understood. Inhibitors of several other molecular targets of the lymphangiogenic signaling cascade are in development and have shown promise in preclinical reports. In addition, modulation of the transcriptional control of lymphatic proliferation, including Prox1 and its transcriptional inducer Sox-18, which are critical to normal lymphatic development as well as tumor lymphangiogenesis, may provide an essential complement to downstream molecular inhibitors. In vitro and in vivo knockout studies have shown that inhibition of lym- phangiogenic transcription factors decreases lymphatic migration and proliferation.25
Lymphatic dissemination of head and neck cancer is the most common route of metastatic spread and is associated with a significantly worse prognosis along with the need for more intensive multimodality treatment. Recent molecular advances, including the development of lymphatic-specific markers, have improved understanding of the mechanisms of lymphatic dissemination. Contrary to early hypotheses, lymphatic spread is an active process with fundamental pathobiological changes in those metastatic cells enabling them to dissociate from the primary tumor mass, migrate toward intratumoral or peritumoral lymphatics, make lymphatic vessel entry and travel to the regional nodal basin while remaining viable. These steps are governed by multiple molecular pathways and chemotactic agents, with the vascular EGFs and receptors central among them. As understanding of these processes grows, the development and therapeutic role of specific antilym- phangiogenic targets is likely to expand.
 Roberts TJ, Colevas AD, Hara W, Holsinger FC, Oakley-Girvan I, Divi V. Number of positive nodes is superior to the lymph node ratio and American Joint Committee on Cancer N staging for the prognosis of surgically treated head and neck squamous cell carcinomas. Cancer. 2016; 122(9):1388-1397
 D'Cruz AK, Vaish R, Kapre N, et al. Head and Neck Disease Management Group. Elective versus therapeutic neck dissection in node-negative oral cancer. N Engl J Med. 2015; 373(6):521-529
 Erdi YE. Limits of tumor detectability in nuclear medicine and PET. Mol Imaging Radionucl Ther. 2012; 21(1):23-28
 Lallemant B, Evrard A, Chambon G, et al. Gene expression profiling in head and neck squamous cell carcinoma: clinical perspectives. Head Neck. 2010;
 Semo J, Nicenboim J, Yaniv K. Development of the lymphatic system: new questions and paradigms. Development. 2016; 143(6):924-935
 Srinivasan RS, Escobedo N, Yang Y, et al. The Prox1-Vegfr3 feedback loop maintains the identity and the number of lymphatic endothelial cell progenitors. Genes Dev. 2014; 28(19):2175-2187
 Secker GA, Harvey NL. VEGFR signaling during lymphatic vascular development: From progenitor cells to functional vessels. Dev Dyn. 2015; 244(3):323-331
 Alitalo A, Detmar M. Interaction of tumor cells and lymphatic vessels in cancer progression. Oncogene. 2012; 31(42):4499-4508
 Ordonez NG. Immunohistochemical endothelial markers: a review. Adv Anat Pathol. 2012; 19(5):281-295
 Ugorski M, Dziegiel P, Suchanski J. Podoplanin: a small glycoprotein with many faces. Am J Cancer Res. 2016; 6(2):370-386
 Baluk P, McDonald DM. Markers for microscopic imaging of lymphangiogenesis and angiogenesis. Ann N Y Acad Sci. 2008; 1131:1-12
 Allen CT, Law JH, Dunn GP, Uppaluri R. Emerging insights into head and neck cancer metastasis. Head Neck. 2013; 35(11):1669-1678
 Karaman S, Detmar M. Mechanisms of lymphatic metastasis. J Clin Invest. 2014; 124(3):922-928
 Zhang B, Gao Z, Sun M, et al. Prognostic significance of VEGF-C, semaphorin 3F, and neuropilin-2 expression in oral squamous cell carcinomas and their relationship with lymphangiogenesis. J Surg Oncol. 2015; 111(4):382-388
 de Sousa EA, Lourenço SV, de Moraes FP, et al. Head and neck squamous cell carcinoma lymphatic spread and survival: Relevance of vascular endothelial growth factor family for tumor evaluation. Head Neck. 2015; 37(10):1410-1416
 Sasahira T, Ueda N, Yamamoto K, et al. Prox1 and FOXC2 act as regulators of lymphangiogenesis and angiogenesis in oral squamous cell carcinoma. PLoS One. 2014; 9(3):e92534
 Weidner N, Semple JP, Welch WR, Folkman J. Tumor angiogenesis and metastasis: correlation in invasive breast carcinoma. N Engl J Med. 1991; 324(1):1-8
 Yu M, Liu L, Liang C, et al. Intratumoral vessel density as prognostic factors in head and neck squamous cell carcinoma: a meta-analysis of literature. Head Neck. 2014; 36(4):596-602
 Smith A, Teknos TN, Pan Q. Epithelial to mesenchymal transition in head and neck squamous cell carcinoma. Oral Oncol. 2013; 49(4):287-292
 Kim HS, Park YW. Metastasis via peritumoral lymphatic dilation in oral squamous cell carcinoma. Maxillofac Plast Reconstr Surg. 2014; 36(3):85-93
 Werner JA, Schünke M, Rudert H, Tillmann B. Description and clinical importance of the lymphatics of the vocal fold. Otolaryngol Head Neck Surg. 1990; 102(1):13-19
 Kirchner JA. Glottic-supraglottic barrier: fact or fantasy? Ann Otol Rhinol Lar- yngol. 1997; 106(8):700-704
 Shah JP. Patterns of cervical lymph node metastasis from squamous carcinomas of the upper aerodigestive tract. Am J Surg. 1990; 160(4):405-409
 Dieterich LC, Detmar M. Tumor lymphangiogenesis and new drug development. Adv Drug Deliv Rev. 2016; 99:148-160
 Rho CR, Choi JS, Seo M, Lee SK, Joo CK. Inhibition of lymphangiogenesis and hemangiogenesis in corneal inflammation by subconjunctival Prox1 siRNA injection in rats. Invest Ophthalmol Vis Sci. 2015; 56(10):5871-5879