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Carcinogenesis vol.27 no.9 pp.1729–1738, 2006

doi:10.1093/carcin/bgl031

Advance Access publication April 5, 2006

REVIEW

Lymphatic vessels in cancer metastasis: bridging the gaps

Ramin Shayan, Marc G.Achen and Steven A.Stacker

Angiogenesis Laboratory, Ludwig Institute for Cancer Research, PO Box 2008, Royal Melbourne Hospital, Victoria 3050, Australia

To whom correspondence should be addressed Email: steven.stacker@ludwig.edu.au

Distant organ metastasis is the most important factor in determining patient survival in cancer. This is thought to occur via the body’s own systems for transporting fluid and cells, the blood vascular and lymphatic systems. Cancer cells may exploit these vascular systems by expressing growth factors, which alter the normal pattern of angiogen- esis and lymphatic vessel growth (lymphangiogenesis), thus creating conduits for tumour metastasis. With respect to lymphatic metastasis, techniques which allow the mapping of a tumour’s lymphatic drainage and sampling of the ‘sentinel node’ from the regional lymph node group provide crucial prognostic information, determine further treat- ment and offer a window into tumour–host immune inter- actions. Aberrant drainage patterns so identified are both clinically significant, and highlight important anatomical and molecular complexities not explained by existing models of lymphatic development or anatomy. The mole- cular controls of tumour lymphangiogenesis and factors determining which lymphatic vessel subtypes are induced may be targets for novel therapeutics designed to restrict cancer metastasis. Furthermore, analyses of these control mechanisms will enhance our understanding of the inter- actions between the tumour cells and the lymphatic vascu- lature. For many years, disparate groups of clinical researchers and basic scientists have been working to unravel the mysteries of the lymphatic system. This review aims to summarize these contributions, in terms of the history, identification, structure and function of lymphatic vessels in cancer and the role they play in tumour meta- stasis. Current ideas about the roles of lymphangiogenic growth factors, their signalling pathways in lymphatic metastasis and therapeutic opportunities to restrict this spread will also be explored.

Abbreviations: E, embryonic day; ECM, extracellular matrix; LEC, lymphatic endothelial cell; MAPK, mitogen activated protein kinase; PC, proprotein convertase; PTK, protein tyrosine kinase; RTK, receptor tyrosine kinase; SMC, smooth muscle cell; VEGF vascular endothelial growth factor; VEGFR vascular endothelial cell growth factor receptor.

Brief history of the lymphatic system in cancer

The French surgeon Le Dran (1) first noted in the sixteenth century that cancers of the breast which spread to axillary lymph nodes had significantly worse survival outcomes than those that were localized only to the primary tumour. Some 200 years later, Halsted (2) set about performing sometimes disfiguring radical excisions of both the primary breast cancer and the metastatic lesions in the axillary lymph nodes (3). The next major clinical development regarding the lymphatics in cancer occurred in the 1950s when clinicians began to use radioisotope injections to better understand which regional lymph node groups drain different parts of the body (4), essential information for identifying potential routes of cancer metastasis via the lymphatic vasculature. The 1990s saw the adaptation of lymphatic mapping for the prediction of each patient’s lymphatic drainage from a specific tumour, and for identification of the ‘sentinel lymph node(s)’ (i.e. the lymph node(s) most likely to contain spreading cancer cells) within the lymphatic drainage basin (5). The same dec- ade also yielded the discovery of lymphatic-specific molecular markers to identify lymphatic vessels, which were hitherto histologically indistinguishable from blood vessels (6). The advent of these techniques led to the realization that lymphatics are fundamental to cancer metastasis and many other patho- logical processes, and has generated intense clinical and scientific interest in the lymphatic vasculature as a potentially valuable therapeutic target (7).

Structure, function and development of the lymphatic system The lymphatic vessels are integral for interstitial fluid volume regulation and absorption of dietary fat. In addition, the lymph- atics are important for immune function and in the metastatic spread of cancer (8). The intricate network of thin-walled ves- sels which constitutes the lymphatic vasculature commences within the superficial dermis of the skin as highly permeable blind-ending sacs, and are referred to as ‘lymphatic capillaries’ or ‘initial lymphatics’ (Figure 1) (8,9). Fibrillin filament anchors between lymphatic endothelial cells (LECs) and the extracellular matrix (ECM) translate interstitial fluid volume expansion into lateral displacement, to create temporary inter- cellular fenestrations (10). The lack of a continuous basement membrane surrounding the initial lymphatics facilitates entry of fluid into these vessels and restoration of normal interstitial volume, resulting in a slackening of the fibrillin filaments, and eventual return of the LECs to their overlapping resting posi- tion. Studies by Weber et al. (11) suggest that ECM signalling stimulates additional cytoskeletal structural alterations, which further aid fluid influx.

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Downloaded from R.Shayan, M.G.Achen and S.A.Stacker Fig. 1. Schematic representation and molecular

Fig. 1. Schematic representation and molecular characteristics of lymphatic vessel subtypes found in the dermal and subcutaneous layers of normal mammalian skin. The initial lymphatic capillaries are blind-ending vessels within the superficial layers of the dermis, which collect fluid and cells for transportation to the pre-collecting vessels. The pre-collecting vessels are located in the deeper layers of the dermis and are characterized by the appearance of valves, a basement membrane and SMCs within their structure. Once vessels enter the subcutaneous fat, they exhibit more uniform valves, circumferential SMC coverage, a thicker more complex wall and a continuous basement membrane. These so-called ‘collecting ducts’ constitute the major route for return of fluid to the cardiovascular system. Molecular markers for LECs are useful to detect and differentiate these vessel subtypes, and to characterize the functional role they play in lymphangiogenesis and cancer metastasis. Note that not all molecular markers are shown on the LECs.

Pre-collecting lymphatic vessels, located in the deep dermis, drain fluid from the initial lymphatics (Figure 1). They have segments which contain valves and are surrounded by a base- ment membrane and smooth muscle cells (SMCs) (12). These segments alternate with other regions that are morphologically akin to the initial lymphatic capillaries. The pre-collecting lymphatics, in turn, drain into the ‘collecting lymphatics’, which are located in the subcutaneous tissue. These vessels have circumferential SMCs and regular intraluminal valves (13). Those collecting lymphatics which are > 200 m m in diameter also have arterial-type mural intima, media and adventitia (13); however, do not normally possess pericytes (14). They propel lymph at an average of 10 m m/s by a com- bination of intrinsic wall motion (generated by specialized pacemaker-SMCs), and compression by adjacent arterial pulsation and skeletal muscle contraction (15–17), eventually returning lymph to the central veins. The collecting lymphatics coalesce into lymphatic trunks, then intrathoracic ducts. Unaided by ‘lymphatic hearts’, found in animals such as toads (18), the larger mammalian lymphatic vessels generate surges of up to 100 m m/s several times each minute to drain fluid from most tissues to a lymph node within 20 min (19–21).

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Alterations to flow may be mediated by nitric oxide synthase in LECs, acting via nitric oxide to produce SMC dilatation (22,23) and may be under autonomic control, as illustrated by splanchnic stimulation influencing thoracic duct flow (24). Recent work by He et al . (25) indicates that these lymph- atic vessels may also dilate in response to tumour-derived growth factors, hence increasing the lymph flow and capacity to transport tumour cells to lymph nodes. Lymph fluid passes through a series of lymph nodes or other secondary lymphoid organs (gastrorespiratory submucosal lymphoid aggregations such as Peyer’s patches and tonsils) (26). Afferent lymph node ducts divide before passing beneath the capsule of the node into cortical sinuses then pass through a reticuloendothelial cell filter (26). Antigen-presenting cells dis- play antigen epitopes to lymphocytes within these organs, trig- gering them to clonally expand and tailor an individualized ‘antigen-specific’ immune response (27). Whilst lymph conti- nues through the medullary sinus to the hilar region of the lymph node and into efferent ducts, tumour cells may become trapped and proliferate here or spread further to distal organs (21). The first lymphatic vessels emerge in the mouse at about embryonic day (E) 10.5, when endothelial cells sprout from the

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anterior cardinal vein to form the primary jugular lymph sacs (28). These endothelial cells express the lymphatic transcrip- tion factor Prox-1, which is essential for lymphatic develop- ment and is thought to be a major determinant of LEC fate (28). These cells also express the receptor tyrosine kinase (RTK) vascular endothelial growth factor receptor-3 (VEGFR-3), the cell surface receptor that binds the lymphangiogenic growth factors VEGF-C and VEGF-D. It is the nearby expression of VEGF-C which initiates the first sprouting of lymphatics from the anterior cardinal vein, via VEGFR-3 stimulation (29). The primary jugular lymph sacs subsequently also sprout to form a primitive lymphatic plexus, which spreads throughout the head and neck, thorax and forelimbs (8). The lymphatics continue to evolve from E12.5 onwards, via a series of posterior lymphatic sacs which are derived from local veins, and progress to pene- trate interstitial tissues nearby (28). They form a primitive dermal plexus by E15.5 (28), and continue to develop after birth into a deep dermal pre-collecting layer, from which sprout the beginnings of a subepidermal lymphatic capillary plexus (30). The LECs of the latter lymphatic vessels differ from those of the deep dermal lymphatics both morphologic- ally and in terms of the cell surface molecular markers expressed (Figure 1) (30). The timing of the formation of the initial lymphatic capillary layer also varies depending on the location of the skin, but occurs after the second post- natal day in the mouse (30). The endothelium of mature lymph- atic vessels expresses the cell surface markers podoplanin and LYVE-1, although LYVE-1 appears to be more abundant in lymphatic capillaries than in larger vessels (Figure 1) (30). In contrast, EphrinB2, a cell surface tyrosine kinase, is expressed on LECs in pre-collecting lymphatic vessels, where it is essen- tial for the maturation of these vessels after birth, and on collecting lymphatic vessels, but is not expressed on LECs in lymphatic capillaries (30) . Relatively little is known about development of lymph nodes or other lymphoid tissues. These structures arise from connective tissue protruding into embryonic lymph sacs, which later forms the first lymph node anlagen tissue at the pre- determined site of future nodes (26,27). This involves differ- entiation of mesenchymal cells into stromal organizer cells and aggregates of CD45 + CD4 + CD3 + lymphoid ‘tissue inducer cells’ (27). The two cell types interact within the anlagen to induce expression of adhesion molecules on stromal organizer cells and release homeostatic chemokines such as CCL21, CCL19 and CXCL13. These in turn attract further lymphoid ‘tissue inducer cells’ and other hemopoietic cells (27,31). Interestingly, studies in mutant mice have demonstrated that lymph node development is genetically distinct from that of the lymphatic vasculature (32).

The involvement of the lymphatics in cancer

The initial spread of cancer cells is seen histologically as tumour escape beyond a defined boundary and may include the invasion of local lymphatic vessels (33). Micrometastasis to lymph nodes, thought to occur before most primary tumours are clinically detectable, often heralds distant organ metastasis at the time of diagnosis or after some period of delay, and is therefore the most significant prognostic indicator in many human cancers (34). Hence, lymph node staging may alter the type and timing of treatment offered to the patient, and it is critical that all nodes potentially containing metastatic

Lymphatic vessels in cancer metastasis

tumour cells are sampled surgically for histological analysis (34). Failure to detect occult metastasis may be responsible for around one-third of early stage colorectal cancer patients who were originally classed as lymph node-negative on histopatho- logy, later developing systemic or recurrent disease (35,36). ‘Sentinel lymph node biopsy’ is one method of reliably sampling lymph nodes to which tumour cells have metasta- sized. Originally developed for melanoma (37), it has since been adapted for application to several other malignancies (38,39), in particular, breast carcinoma (40). It is a procedure in which a pre-operative map of lymph node(s) draining a primary cancer is obtained by injection of radiolabelled colloid (41). The sentinel node(s) is then located intraoperatively with a hand held gamma probe (42), in conjunction with visualiza- tion of blue dye injected at the commencement of the proced- ure (43). This enables accurate identification and surgical excision of the sentinel lymph node(s), deemed to be those node(s) most likely to harbour metastatic cancer cells (5,37). If histological analysis of the sentinel lymph node(s) demon- strates the presence of metastatic cells, surgical clearance of

the remaining lymph nodes within the same anatomical area is required for further histological staging and prognostication. Furthermore, histologically positive sentinel lymph nodes usu- ally pre-date distant metastasis and are, therefore, an indicator of poor prognosis (5,37), even if they occur outside the regional lymph node group (44,45). Detection of positive sen- tinel lymph node(s) results in reclassification of a patient to a group with higher risk of recurrence, which may make them eligible for systemic adjuvant anti-metastasis treatment and thus potentially result in a survival benefit (36,46–48). In con- trast, the absence of metastatic cancer cells within the sentinel node is taken to indicate disease-free status in the remaining lymph nodes in the nodal group (49), and the patient is con- sequently spared the morbidity of further lymphablation sur- gery, which could lead to undesirable side effects such as lymphoedema (50). Ablation of metastatic lymph nodes and focussed external beam radiation therapy also aid local disease control, as misdiagnosis or inadequate clearance of metastatic nodes may result in bulky, unresectable disease (51). The precise mechanisms by which tumour cells move pref- erentially towards particular lymph node(s) remain poorly defined. It was originally envisaged that lymphatic invasion took place passively as an advancing tumour front eroded the walls of any vessels in its path and that metastasis occurred by passive drainage (52); however, recent evidence indicates that

a more complex interaction between tumour cells and the

lymphatic endothelium may take place (53). The availability of markers with specificity for LECs has enabled the identi- fication and quantification of previously undiscernible lymph- atic vessels in human cancer (54), and other pathological states

(55,56).

The main ‘workhorse’ reagent for identification of lymph- atic vessels, since the late 1990s, has been anti-LYVE-1 anti- body (6,57,58). LYVE-1 is a CD44 homologue protein which

is involved in hyaluronan and immune cell transport (6,57),

and may itself be implicated in tumour cell trafficking to lymph

nodes (59). Heterogeneous LYVE-1 expression on cultured and tumour-induced LECs (59), and differential expression between normal lymphatic vessel subtypes (Figure 1) (30), means that a combination of LEC molecular markers are required to reliably identify different lymphatic vessels (9). Podoplanin is a mucin-type transmembrane glycoprotein, which despite being expressed on kidney podocytes,

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osteoblasts and Type I alveolar cells is expressed uniformly across the lymphatic vessel subtypes and has thus proven to be another useful LEC marker (60–62). Whilst its specific func- tion remains elusive, mutant mice lacking podoplanin display lymphatic defects which contribute to respiratory compromise and death, but do not interfere with normal blood vasculature (63). Podoplanin expression is regulated by the homeobox transcription factor Prox-1, which, whilst not widely used to identify cancer lymphatics, has been important for understand- ing lymphatic development during embryogenesis (64,65). VEGFR-3 was amongst the earliest markers used to charac- terize lymphatic vessels (66); however, the observation of VEGFR-3 expression on blood vessels in tumours and wound granulation tissue has meant that it is less appropriate for specific identification of lymphatics in these conditions (67,68). Finally, molecules recently identified on lymphatic vessels, such as EphrinB2 and EphB4, which are responsible for blood vessel territorial demarcation, are differentially expressed between lymphatic vessel subtypes (Figure 1) (30), and may therefore become useful in distinguishing dif- ferent lymphatic vessels in cancer. Whilst immunohistological studies using lymphatic-specific markers have demonstrated the existence of proliferating intratumoral lymphatic vessels in several types of human tumour, the functional significance of intratumoral lymphatic vessels remains controversial (69). It has been proposed that intratumoral lymphatics are not able to transport tumour cells because the elevated hydrostatic pressure within a tumour may compress these vessels (69,70). Certain studies have reported intratumoral lymphatic density as an accurate independent predicted of poor disease-free survival, and of increased meta- static propensity in such important human tumours as melan- oma, and carcinoma of the breast, endometrium, colon, lung, prostate, ovary, pancreas and in the head and neck (71–79). Peritumoral lymphatics are the lymphatic vessels immedi- ately surrounding a tumour. It has been suggested that these may represent pre-existing vessels compressed into a peri- tumoral rim by the expanding tumour mass (80). However, LECs in peritumoral lymphatics have been observed to be pro- liferating around some human cancers, suggesting that these vessels can also arise due to lymphangiogenesis (71–74). Addi- tionally, peritumorallymphangiogenesis was significantly asso- ciated with regional metastasis and poor disease-free and overall survival in malignant melanoma (81), and detection of lymph- angiogenic growth factors was also shown to be of prognostic significance in human carcinoma of the cervix, ovary, breast and gastrointestinal tract (74,75,77,78,82–85). Although the rela- tive importance of intratumoral versus peritumoral lymphatics for metastatic spread remains a subject of debate, it is clear that the lymphatic vessels associated with a tumour can be important for metastasis and patient outcome (86). Further, experimental and clinopathological studies indicate that lymphangiogenesis can contribute to the formation of these lymphatic vessels (86), therefore, an understanding of the molecular control of this process may identify novel therapeutic targets for restricting the spread of cancer (7).

Molecular regulation of lymphangiogenesis in cancer

VEGF-C and VEGF-D constitute the lymphangiogenic ‘sub- family’ of the VEGF growth factors, and have become import- ant predictors of tumour growth, lymphangiogenesis and

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metastatic behaviour (85). All of the members of the VEGF family (the others being VEGF-A, placenta-derived growth factor and VEGF-B) are related by a common VEGF- homology domain (VHD), which contains a 30% conserved amino acid sequence and a characteristic cystine-knot motif (87). Unique to the two-member lymphangiogenic ‘sub- family’, N-terminal (N-pro) and C-terminal (C-pro) propep- tides (87,88) are proteolytically cleaved from the VHD by enzymes including plasmin (89) and ‘proprotein convertases’ (PCs) (88,90). This process generates mature, bioactive forms of VEGF-C and VEGF-D, which consist of VHD dimers and display enhanced affinity for VEGFR-3 (88,91), and thus make this group of enzymes important to the tumorigenicity of cancers which secrete these growth factors (92,93). Devel- opmentally, VEGF-C is vital for normal lymphatic embryo- genesis (87), a finding supported by transgenic and gene deletion animal models (55,94). Additionally, exogenous administration of both VEGF-C and VEGF-D were sufficient to rescue lymphatic vessel sprouting (29), and transgenic expression of either ligand alone induced lymphangiogenesis independent of angiogenesis (95,96). VEGFR-3 is a major transducer of lymphangiogenic signal- ling, acting predominantly through phosphorylation of Akt, a PI-3 kinase-dependent serine-threonine kinase, and a PKC- dependent p42/p44 mitogen activating protein kinase (MAPK) (94,96,97). VEGFR-3 activation not only prevents LEC apoptosis in culture but also stimulates proliferation, migration and cell survival under serum deprivation conditions via this pathway (98).

Experimental models of lymphangiogenesis in cancer The VEGF-C expressing MCF-7 human breast cancer cell line demonstrates increased rates of lymph node metastasis in the presence of increased intratumoral, and particularly peri- tumoral lymphatics (99). Furthermore, soluble antibody to VEGFR-3 was shown to inhibit lymphangiogenesis and sub- sequent metastasis (99). A similar phenomenon of increased intratumoral and peritumoral lymphatic vessels was seen in an alternative VEGF-C expressing breast cancer line, MBA- MD-435 (100). The enhanced access to lymphatics was also associated with an increase in regional lymph node spread, and particularly pulmonary visceral metastasis (100). Consistent with Kukk et al.’s work (101), however, which showed the unprocessed predominantly VEGFR-3-specific 31 kD form of VEGF-C (as produced by these breast cancer models) to be insufficiently processed to stimulate VEGFR-2, significant angiogenesis did not occur in either setting (99,100). In con- trast, the VEGF-C overexpressing MeWo human melanoma- derived cell line caused increased proliferation of both blood and lymphatic vessels, both intratumorally and peritumorally, due to increased proteolytic processing of VEGF-C (102). Enlarged neolymphatics in and around the VEGF-C expressing AZ521 human gastric cancer model resulted in lymph node metastasis in 95% of AZ521-VEGF-C mice, compared with 29% of controls (103), and similar findings were demonstrated in the normally benign pancreatic b-cell tumours, when implanted in VEGF-C overexpressing transgenic mice (104). In the case of VEGF-D, growth factor expressing, stably transfected 293 EBNA tumours xenografted into SCID-NOD mice demonstrated active roles in both tumour growth and lymphangiogenesis, with a concomitant increase in regional lymph node metastasis, compared with their VEGF-A express- ing counterparts (105). Whilst VEGF-A promoted growth and

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angiogenesis, neither lymphangiogenesis nor nodal dissemina- tion were enhanced, and empty vector controls produced well contained,indolent tumours (105). Lymph node metastasis rates occurred in a VEGF-D dose-related fashion, and monoclonal antibodies raised to the bioactive region of VEGF-D competi- tively antagonised mature VEGF-D binding of VEGFR-2 and VEGFR-3, and inhibited tumour metastasis (106). Detmar et al. (107) compared a VEGF-A producing, GFP-expressing, K14 promoter-driven transgenic mouse model with a non-VEGF-A expressing control, in which squam- ous cell carcinoma was generated by multistep, topical chemical induction. Interestingly, not only did VEGF-A appear to play a role in tumour growth but also stimulated both angiogenesis and lymphangiogenesis, apparently signalling via an upregu- lated VEGFR-2 on LECs (107). Overall, these animal models correlate with findings in sev- eral human tumours. Significant tumour-induced lymphangio- genesis has been noted in human melanoma samples, and importantly, were directly related to the risk of lymph node metastasis and patient survival (46,71). Of note, several authors have found that human breast carcinoma samples seem to demonstrate metastasis in the absence of intratumoral lymphangiogenesis (69,86,108). Human breast cancer speci- mens in which intratumoral vessels were often collapsed and poorly staining with proliferation markers, but in which peri- tumoral lymphatics were increased and contained tumour emboli (86), may indicate particular significance of the peri- tumoral microenvironment in tumour lymphatic proliferation and metastatic activity. They may also illustrate the technical limitations of using single lymphatic markers, such as LYVE-1, alone (69), as they may fail to stain relevant vessels if downregulated in the particular lymphatics subtypes formed (30). Consistent with the regression to the primitive embry- ological state often seen in pathological contexts, blood vessel VEGFR-3 expression is observed on immunohistochemical studies of both benign and malignant vascular tumours (Kaposi sarcoma, 80% of spindle cell hemangiomas, 80% of angio- sarcomas), non-vascular tumours, (including melanomas, car- cinomas, other sarcomas) and perivascular tumours (109). These findings may implicate VEGFR-3 in maintaining the endothelial integrity during tumour angiogenesis, which gen- erates vessels more akin to primitive, rather than mature blood vessels (68).

Targeting lymphatics vessels for clinical benefit

Therapeutics The VEGF-C/VEGF-D–VEGFR-3 pathway. Therapeutic approaches for the inhibition of RTKs such as VEGFR-3, include monoclonal antibodies, small molecule inhibitors, pep- tide drugs and antisense techniques (7,110,111). Folkman’s vision of anti-angiogenesis as a cancer treatment has been realized with the release of an anti-VEGF agent for the treatment of metastatic colorectal carcinoma (112,113). Ana- logous to VEGF-A blockade to reduce tumour angiogenesis (114,115), an approach to block VEGFR-3 ligands VEGF-C/ VEGF-D is promising for the inhibition of tumour lymphan- giogensis and lymphogenous metastasis (105,106,116). Hence, Achen et al. (106) raised monoclonal antibodies to the VHD of human VEGF-D which bind both unprocessed and fully pro- cessed VHD with high affinity, and inhibit their binding to both VEGFR-2 and VEGFR-3.

Lymphatic vessels in cancer metastasis

Overall, experimental models demonstrating suppression of VEGFR-3 signalling have shown inhibition of both peritumoral and intratumoral lymphangiogenesis, angiogenesis and meta- static spread (116,117). Administration of soluble VEGFR- 3-immunoglobulin fusion protein, which binds VEGF-C and blocks VEGFR-3 signalling, and intravenous recombinant adenoviruses expressing VEGFR-3-immunoglobulin, both induce regression of tumour-induced lymphatic vessels (116). In addition, the interference with ligand–receptor interactions and the resulting inhibitory effect on lymphangiogenesis and metastasis produced by adeno-associated virus-delivered sol- uble VEGFR-3 decoy receptor was dose-related in VEGF-C secreting PC-3 and A375 tumour models (117). Further evid- ence of the therapeutic potential of soluble VEGFR-3 was provided by transgenic expression of soluble VEGFR-3 in mouse skin, which inhibited fetal lymphangiogenesis, and induced regression of already formed lymphatic vessels, though the blood vasculature was unaffected (93). These transgenic mice develop a lymphoedematous phenotype, characterized by foot oedema and dermal fibrosis. They survive the neonatal period despite almost complete absence of lymphatic vessels in several tissues, which later regenerate (94). Similarly, missense mutations interfere with VEGFR-3 signal transduction to cause primary (congenital) lymphoedema (56).

Proteolytic processing of VEGF-C/VEGF-D VEGF-C expressing tumours which induced angiogenesis and lymphangiogenesis, but were inhibited by mutating the pro- VEGF-C cleavage site, demonstrated that processing of pro- VEGF-C is crucial to receptor activation, and thus generation of its biological effects (93,118). The enzymes responsible include the PCs and the serine protease, plasmin, which has been shown to cleave both C-propeptides and N-propeptides from the human VEGF-C and VEGF-D VHD, to promote lymphangiogenesis and cancer metastasis (89). As a potent proteolytic enzyme which is active at cleaving both propep- tides of the lymphangiogenic growth factors, the parallel role that plasmin plays in fibrinolysis, a process which is crucial in regulating blood clotting, may help coordinate lymphangio- genesis in the later stages of tissue repair. Promisingly, alter- ations to specific PC growth factor binding sites have been shown to inhibit PDGF-A precursor processing by furin, the phosphorylation of its cognate RTK, and the resulting cellular proliferation (119). Furthermore, specific PC blockade in several stably transfected, growth factor secreting tumour models has been found to reduce tumour growth, malignant phenotype and migration (93,118). Thus, inhibitors of the PCs and plasmin, which restrict the cleavage of VEGF-C and/or VEGF-D, may also constitute novel agents for the treatment of human tumours and/or in adjuvant therapy to prevent tumour growth, metastasis or recurrence (93,118,119).

Intracellular signalling cascades The majority of work in this area has focussed on inhibiting tumour angiogenesis (110,120,121); however, blockade of intracellular signalling cascades may also prove to be useful for suppression of downstream VEGFR-3 signalling, as many of the VEGF family of RTKs employ common signalling mechanisms (97,98,120,122,123). In particular, PI-3 kinase- Akt and PKC-p42/p44 MAPK signalling cascades are the path- ways activated by growth factor stimulation of VEGFR-3 (94,96–98), and may represent potential therapeutic targets for inhibiting lymphangiogenesis induced by tumour-secreted

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growth factors (110,121). Several small molecular agents tar- geting cytoplasmic protein tyrosine kinase (PTK) in preclinical models inhibit angiogenesis and tumour progression, and have reached clinical trials (124). One example is SU11248 (sunitinib or Sutent), which has reached Phase III clinical trials, and offers targeted treatment in select tumours such as carcinoma of the lung, renal cell carcinoma, and gastrointestinal tumours (125–127). Other PTK inhibitors which target VEGFR-2 and VEGFR-3 pathways, also cur- rently involved in clinical cancer treatment trials include CEP-7055 (128), PTK 787/ZK 222584 (129,130) and BAY 43-9006 (131). The latter is a Raf-1 kinase inhibitor, which demonstrates anti-angiogenesis and anti-tumour activity via MAPK signalling and VEGFR tyrosine kinase blockade in preclinical tumour xenograft models, and is being trialed for use in a range of human tumour settings, including such common cancers as carcinomas of the breast, prostate, lung, pancreas, kidney, thyroid, pancreas and ovary (131,132).

Chemokines and adhesion molecules Certain receptor–ligand relationships between tumour and host tissues, and soluble factors, known as chemokines, which regu- late hemopoietic cell migration, may be ‘hi-jacked’ by cancer cells to facilitate the location of and entry into lymphatic vessels, and their movement along them towards lymph nodes (133). Furthermore, tumour–endothelial interactions may be responsible for particular cancers displaying metastatic affinity towards specific tissues, and chemokine–ligand guided interactions may create a chemical gradient which predisposes circulating metastatic cells to home toward, and settle within a certain distant organ tissue (133). A prime example in lymphatic metastasis is CCL21/SLC, a ligand expressed on LECs for lymphocyte and dendritic cell signalling, and guidance towards lymph nodes via the CCR7 receptor (134–136). Human melanoma and breast cancer cell lines expressing CCR7 have demonstrated increased affinity for lymphatic endothelium and resulted in increased lymph node metastasis in animal models, a process which was inhibited by the administration of neutralizing anti-CCL21 antibodies (137). The CXCR4–CXCL12 pathway is also a well-characterized T-lymphocyte signalling mechanism, and CXCL12 is differen- tially expressed between individual colorectal carcinomas, pro- viding a prognostic factor for local recurrence, liver metastases and poor overall survival (133). Interference with CXCR4 sig- nalling may present a further target in disease-directed therapy for colorectal carcinoma (133). Expression of adhesion molecules, which normally partici- pate in immune cell traffic between interstitial and vascular compartments, may also be important in tumour entry to, or exit from the lymphovascular space during metastasis (138). Bevacqua et al . (138) demonstrated an in vitro correlation between attachment potential and metastatic propensity in malignant melanoma and breast carcinoma cell lines, using adhesion assays with human tumour cells. One particular adhe- sion molecule, L -selectin, forms a ligand–receptor pair with mannose receptor, which is expressed on lymphatic endo- thelium (139). It orchestrates the intravasation of circulating lymphocytes from interstitial tissues into lymphatic vessels, and subsequent homing to lymph node high endothelial venule addressins, a process which may be exploited by L -selectin expressing tumour cells, in order to promote lymph node meta- stasis, and which has been inhibited in animal studies by administration of anti- L -selectin monoclonal antibody (53).

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Both mannose receptor and common lymphatic endothelial and vascular endothelial receptor (CLEVER)-1 have been implicated in tumour metastasis via adhesion of malignant cells to lymphatic endothelium (140), in some human cancers, and represent further potential anti-metastatic therapeutic tar- gets (141). Proinflammatory cytokines and the resulting immune cell aggregations may also stimulate additional lymphangiogenic growth factors, and so provide an indirect method of inhibiting lymphatic vessel proliferation in the region of a tumour (142,143).

Diagnostics

Imaging localization of lymphatic drainage pathways The cutaneous lymphatic drainage pathways from the site of a primary tumour may be highly variable between patients, even within the same areas of the body (144). Up to 30% of these tumours therefore defy clinical predictability of which lymph nodes, in which regional node groups, may contain cancer cells (145), and contradict long-held anatomical ideas of drainage patterns (146). Significant examples detected usinglymphatic mappingtech- nology include drainage (and potential tumour spread) from superficial back skin to deep lymph nodes in paraaortic and retroperitoneal areas; across the midline; or from periumbilical skin through the chest wall (147,148). These routes bypass the classical drainage pathways through regional lymph nodes and may involve multiple nodal fields (149). Such cancers would be erroneously classified as lymph node-negative using conven- tional methods (147,150), in 14–37% of patients with melano- mas on their limbs, trunk, or head and neck (147–152). Future directions for lymphatic imaging will include the ongoing development of lymphatic mapping for cancers in deeper viscera such as the gastrointestinal and respiratory tracts (48). The next challenge is to develop methods of sen- tinel lymph node assessment that are non-invasive, yet as accurate as present methods. Adaptations using labelled anti- bodies or other tracers with specificity for lymphatic vessels or lymph nodes, could assist in analysing the levels at which abnormalities of lymphatic drainage occur, whether they involve unrecognized pattern variations in ‘collecting lymph- atics’, or ‘lymphaticovenous’ or ‘interlymphatic subtype’ shunts, and whether aberrant lymphatic channels may be influ- enced by tumour-derived growth factors.

Conclusion

Understanding lymphogenous tumour metastasis represents a crucial step in the treatment of human cancer. Since the devel- opment of lymphatic markers, several diverse disciplines may now converge in an attempt to define the respective roles played by lymphatic vessels and tumour cells and products in facilitating metastasis to lymph nodes and beyond. Under- standing the complexities of lymphatic development, anatomy and pathophysiology in terms of the influences of lymphan- giogenic growth factors, receptor signalling and tumour immunomodulation may yield an array of new therapeutic targets for application in the treatment of cancer.

Acknowledgements

We thank Janna Taylor and Diana Keshtiar for their assistance in generating the figure illustrations, and R.S. would also like to acknowledge the ongoing

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supervision and guidance of Prof. G. Ian Taylor, AO. S.A.S. is supported by a Senior Research Fellowships from Pfizer and M.G.A. by a Senior Research Fellowship from the National Health and Medical Research Council of Australia. The Angiogenesis Laboratory is supported by funds from the NH&MRC. R.S. is a recipient of the Raelene Boyle Sporting Chance Founda- tion and Royal Australasian College of Surgeons Cancer Research Scholarship.

Conflict of Interest Statement: S.A.S. and M.G.A. are shareholders in Lymphatix Ltd and have received funding from Imclone Systems Inc. S.A.S. also holds Pfizer Australia fellowship.

References

1. LeDran,H., Gataker,T. and Chelsden,W. (1752) Traite des operations de chirurgie (English translation Gataker). Hitch,C. and Dosley,R. London.

2. Halsted,W. (1894) The results of operations for the cure of cancer of the breast. Johns Hopkins Hosp. Rep., 4, 297–350.

3.Baum,M., Demicheli,R., Hrushesky,W. and Retsky,M. (2005) Does

surgery unfavourably perturb the ‘natural history’ of early breast cancer by accelerating the appearance of distant metastases? Eur. J. Cancer, 41, 508–515.

4. Sherman,A.I. and Ter-Pogossian,M. (1953) Lymph-node concentration of radioactive colloidal gold following interstitial injection. Cancer, 6,

1238–1240.

5.Cochran,A.J., Wen,D.R. and Morton,D.L. (1992) Management of the regional lymph nodes in patients with cutaneous malignant melanoma. World J. Surg., 16, 214–221. 6.Banerji,S., Ni,J., Wang,S.-X., Clasper,S., Su,J., Tammi,R., Jones,M. and Jackson,D.G. (1999) LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan. J. Cell Biol., 144, 789–801.

7. Stacker,S.A., Hughes,R.A. and Achen,M.G. (2004) Molecular targeting of lymphatics for therapy. Curr. Pharm. Des., 10, 65–74.

8. Oliver,G. and Harvey,N. (2002) A stepwise model of the development of lymphatic vasculature. Ann. NY Acad. Sci., 979, 159–165; discussion

188–196.

9. Oliver,G. and Detmar,M. (2002) The rediscovery of the lymphatic system:

old and new insights into the development and biological function of the lymphatic vasculature. Genes Dev. , 16, 773–783. 10.Gerli,R., Solito,R., Weber,E. and Agliano,M. (2000) Specific adhesion molecules bind anchoring filaments and endothelial cells in human skin initial lymphatics. Lymphology, 33, 148–157. 11.Weber,E., Rossi,A., Solito,R., Sacchi,G., Agliano,M. and Gerli,R. (2002)

Focal adhesion molecules expression and fibrillin deposition by lymphatic and blood vessel endothelial cells in culture. Microvasc Res., 64 , 47–55. 12.Veikkola,T., Lohela,M., Ikenberg,K., Makinen,T., Korff,T., Saaristo,A., Petrova,T., Jeltsch,M., Augustin,H.G. and Alitalo,K. (2003) Intrinsic versus microenvironmental regulation of lymphatic endothelial cell phenotype and function. FASEB J., 17, 2006–2013. 13. Scavelli,C., Weber,E., Agliano,M., Cirulli,T., Nico,B., Vacca,A. and Ribatti,D. (2004) Lymphatics at the crossroads of angiogenesis and lymphangiogenesis. J. Anat., 204 , 433–449. 14. Petrova,T.V., Karpanen,T., Norrmen,C. et al. (2004) Defective valves and abnormal mural cell recruitment underlie lymphatic vascular failure in lymphedema distichiasis. Nat. Med., 10, 974–981. 15.McGeown,J.G., McHale,N.G. and Thornbury,K.D. (1987) The role of external compression and movement in lymph propulsion in the sheep hind limb. J. Physiol., 387, 83–93. 16.McGeown,J.G., McHale,N.G. and Thornbury,K.D. (1988) Arterial pulsation and lymph formation in an isolated sheep hindlimb preparation. J. Physiol., 405, 595–604. 17.McGeown,J.G., McHale,N.G. and Thornbury,K.D. (1988) Effects of varying patterns of external compression on lymph flow in the hindlimb of the anaesthetized sheep. J. Physiol., 397 , 449–457. 18. Jones,J.M., Gamperl,A.K., Farrell,A.P. and Toews,D.P. (1997) Direct measurement of flow from the posterior lymph hearts of hydrated and dehydrated toads (Bufo marinus). J. Exp. Biol., 200, 1695–1702. 19.Thornbury,K.D., McHale,N.G. and McGeown,J.G. (1990) Contribution of lymph formation in the popliteal node to efferent lymph flow following stimulation of the sympathetic chain in the sheep. Exp. Physiol., 75,

75–80.

20.Thornbury,K.D., McHale,N.G. and McGeown,J.G. (1989) Alpha-and beta-components of the popliteal efferent lymph flow response to

Lymphatic vessels in cancer metastasis

intra-arterial catecholamine infusions in the sheep. Blood Vessels, 26,

107–118.

21.McHale,N.G. (2005) Lymph circulation and lymph propulsion. In Proceedings of the First International Symposium on Cancer Metastasis and the Lymphovascular System: Basis for Rational Therapy, Springer Publishing Co., NY, p. 4. 22. Padera,T. (2005) Lymphatic pathophysiology and metastasis. In Proceedings of the First International Symposium on Cancer Metastasis and the Lymphovascular System: Basis for Rational Therapy, Springer Publishing Co., NY, p. 22. 23. Shirasawa,Y., Ikomi,F. and Ohhashi,T. (2000) Physiological roles of endogenous nitric oxide in lymphatic pump activity of rat mesentery in vivo. Am. J. Physiol. Gastrointest Liver Physiol., 278 , G551–G556. 24.Bulekbaeva,L.E. and Akhmetbaeva,N.A. (1982) Development of sympathetic influences on lymph flow in the postnatal ontogeny of dogs. Zh. Evol. Biokhim. Fiziol., 18, 140–143. 25.He,Y., Rajantie,I., Pajusola,K., Jeltsch,M., Holopainen,T., Yla- Herttuala,S., Harding,T., Jooss,K., Takahashi,T. and Alitalo,K. (2005) Vascular endothelial cell growth factor receptor 3-mediated activation of lymphatic endothelium is crucial for tumor cell entry and spread via lymphatic vessels. Cancer Res., 65, 4739–4746. 26.Cupedo,T., Kraal,G. and Mebius,R.E. (2002) The role of CD45+CD4 + CD3- cells in lymphoid organ development. Immunol. Rev., 189 , 41–50. 27.Cupedo,T. and Mebius,R.E. (2005) Cellular interactions in lymph node development. J. Immunol., 174, 21–25. 28.Wigle,J.T. and Oliver,G. (1999) Prox1 function is required for the development of the murine lymphatic system. Cell, 98, 769–778. 29.Karkkainen,M.J., Haiko,P., Sainio,K. et al. (2004) Vascular endothelial growth factor C is required for sprouting of the first lymphatic vessels from embryonic veins. Nat. Immunol., 5, 74–80. 30.Makinen,T., Adams,R.H., Bailey,J., Lu,Q., Ziemiecki,A., Alitalo,K., Klein,R. and Wilkinson,G.A. (2005) PDZ interaction site in ephrinB2 is required for the remodelling of lymphatic vasculature. Genes Dev., 19 , 397–410. 31.Cupedo,T. and Mebius,R.E. (2003) Role of chemokines in the development of secondary and tertiary lymphoid tissues. Semin. Immunol., 15, 243–248. 32. Futterer,A., Mink,K., Luz,A., Kosco-Vilbois,M.H. and Pfeffer,K. (1998) The lymphotoxin beta receptor controls organogenesis and affinity maturation in peripheral lymphoid tissues. Immunity, 9, 59–70. 33.Robbins,S. (1999) Pathologic Basis of Disease. W.B Saunders Company, Philadelphia, PA. 34. Fidler,I.J. (2003) The pathogenesis of cancer metastasis: the ‘seed and soil’ hypothesis revisited. Nat. Rev. Cancer, 3, 453–458. 35.Bilchik,A.J., Nora,D.T., Saha,S., Turner,R., Wiese,D., Kuo,C., Ye,X., Morton,D.L. and Hoon,D.S. (2002) The use of molecular profiling of early colorectal cancer to predict micrometastases. Arch. Surg., 137,

1377–1383.

36.Bilchik,A.J., Saha,S., Wiese,D., Stonecypher,J.A., Wood,T.F., Sostrin,S., Turner,R.R., Wang,H.J., Morton,D.L. and Hoon,D.S. (2001) Molecular staging of early colon cancer on the basis of sentinel node analysis: a multicenter phase II trial. J. Clin. Oncol., 19, 1128–1136. 37.Morton,D.L., Wen,D.R., Wong,J.H., Economou,J.S., Cagle,L.A., Storm,F.K., Foshag,L.J. and Cochran,A.J. (1992) Technical details of intraoperative lymphatic mapping for early stage melanoma. Arch. Surg., 127, 392–399. 38. Ferris,R.L., Xi,L., Raja,S., Hunt,J.L., Wang,J., Gooding,W.E., Kelly,L., Ching,J., Luketich,J.D. and Godfrey,T.E. (2005) Molecular staging of cervical lymph nodes in squamous cell carcinoma of the head and neck. Cancer Res., 65, 2147–2156. 39.Bilchik,A.J., Giuliano,A., Essner,R., Bostick,P., Kelemen,P., Foshag,L.J., Sostrin,S., Turner,R.R. and Morton,D.L. (1998) Universal application of intraoperative lymphatic mapping and sentinel lymphadenectomy in solid neoplasms. Cancer J. Sci. Am., 4, 351–358. 40. Park,C., Seid,P., Morita,E., Iwanaga,K., Weinberg,V., Quivey,J., Hwang,E.S., Esserman,L.J. and Leong,S.P. (2005) Internal mammary sentinel lymph node mapping for invasive breast cancer: implications for staging and treatment. Breast J. , 11, 29–33. 41.Nieweg,O.E., Tanis,P.J. and Kroon,B.B. (2001) The definition of a sentinel node. Ann. Surg. Oncol. , 8 , 538–541. 42.Leong,S.P. (2003) Selective sentinel lymphadenectomy for malignant melanoma. Surg. Clin. North Am., 83, 157–185, vii. 43.Thompson,J.F., Uren,R.F., Shaw,H.M., McCarthy,W.H., Quinn,M.J., O’Brien,C.J. and Howman-Giles,R.B. (1999) Location of sentinel lymph nodes in patients with cutaneous melanoma: new insights into lymphatic anatomy. J. Am. Coll. Surg., 189, 195–204.

1735

by guest on December 8, 2010carcin.oxfordjournals.orgDownloaded

from

R.Shayan, M.G.Achen and S.A.Stacker

44.Thelmo,M.C., Morita,E.T., Treseler,P.A., Nguyen,L.H., Allen,R.E.,Jr, Sagebiel,R.W., Kashani-Sabet,M. and Leong,S.P. (2001) Micrometastasis to in-transit lymph nodes from extremity and truncal malignant melanoma. Ann. Surg. Oncol., 8, 444–448. 45.Uren,R.F., Howman-Giles,R. and Thompson,J.F. (2003) Patterns of lymphatic drainage from the skin in patients with melanoma. J. Nucl. Med., 44, 570–582. 46.Takeuchi,H., Morton,D.L., Kuo,C., Turner,R.R., Elashoff,D., Elashoff,R., Taback,B., Fujimoto,A. and Hoon,D.S. (2004) Prognostic significance of molecular upstaging of paraffin-embedded sentinel lymph nodes in melanoma patients. J. Clin. Oncol., 22, 2671–2680. 47. Scolyer,R.A., Thompson,J.F., Li,L.X., Beavis,A., Dawson,M., Doble,P., Ka,V.S., McKinnon,J.G., Soper,R., Uren,R.F., Shaw,H.M., Stretch,J.R. and McCarthy,S.W. (2004) Failure to remove true sentinel nodes can cause failure of the sentinel node biopsy technique: evidence from antimony concentrations in false-negative sentinel nodes from melanoma patients. Ann. Surg. Oncol., 11, 174S–178S. 48.Bilchik,A.J., Saha,S., Tsioulias,G.J., Wood,T.F. and Morton,D.L. (2001) Aberrant drainage and missed micrometastases: the value of lymphatic mapping and focused analysis of sentinel lymph nodes in gastrointestinal neoplasms. Ann. Surg. Oncol. , 8, 82S–85S. 49.Thompson,J.F., Stretch,J.R., Uren,R.F., Ka,V.S. and Scolyer,R.A. (2004) Sentinel node biopsy for melanoma: where have we been and where are we going? Ann. Surg. Oncol. , 11 , 147S–151S. 50. Starritt,E.C., Joseph,D., McKinnon,J.G., Lo,S.K., de Wilt,J.H. and Thompson,J.F. (2004) Lymphedema after complete axillary node dissection for melanoma: assessment using a new, objective definition. Ann. Surg., 240, 866–874. 51. Fife,K. and Thompson,J.F. (2001) Lymph-node metastases in patients with melanoma: what is the optimum management? Lancet Oncol., 2,

614–621.

52. Pepper,M.S. (2001) Lymphangiogenesis and tumor metastasis: myth or reality? Clin. Cancer Res., 7, 462–468. 53.Qian,F., Hanahan,D. and Weissman,I.L. (2001) L-selectin can facilitate metastasis to lymph nodes in a transgenic mouse model of carcinogenesis. Proc. Natl Acad. Sci. USA, 98 , 3976–3981. 54.Beasley,N.J., Prevo,R., Banerji,S., Leek,R.D., Moore,J., van Trappen,P., Cox,G., Harris,A.L. and Jackson,D.G. (2002) Intratumoral lymphan- giogenesis and lymph node metastasis in head and neck cancer. Cancer Res., 62, 1315–20. 55. Jeltsch,M., Kaipainen,A., Joukov,V., Meng,X., Lakso,M., Rauvala,H., Swartz,M., Fukumura,D., Jain,R.K. and Alitalo,K. (1997) Hyperplasia of lymphatic vessels in VEGF-C transgenic mice. Science, 276 ,

1423–1425.

56.Karkkainen,M.J., Ferrell,R.E., Lawrence,E.C., Kimak,M.A., Levinson,K.L., McTigue,M.A., Alitalo,K. and Finegold,D.N. (2000) Missense mutations interfere with VEGFR-3 signalling in primary lymphoedema. Nat. Genet., 25, 153–159. 57. Prevo,R., Banerji,S., Ferguson,D.J., Clasper,S. and Jackson,D.G. (2001) Mouse LYVE-1 is an endocytic receptor for hyaluronan in lymphatic endothelium. J. Biol. Chem., 276, 19420–19430. 58.Koukourakis,M.I., Giatromanolaki,A., Sivridis,E., Simopoulos,C., Gatter,K.C., Harris,A.L. and Jackson,D.G. (2005) LYVE-1 immunohistochemical assessment of lymphangiogenesis in endometrial and lung cancer. J. Clin. Pathol., 58 , 202–206. 59. Jackson,D.G., Prevo,R., Clasper,S. and Banerji,S. (2001) LYVE-1, the lymphatic system and tumor lymphangiogenesis. Trends Immunol., 22,

317–321.

60.Kerjaschki,D. (2005) Lymphatic neoangiogenesis in human neoplasia and transplantation as experiments of nature. Kidney Int., 68, 1967–1968. 61.Evangelou,E., Kyzas,P.A. and Trikalinos,T.A. (2005) Comparison of the diagnostic accuracy of lymphatic endothelium markers: Bayesian approach. Mod. Pathol., 18, 1490–1497. 62.Breiteneder-Geleff,S., Soleiman,A., Kowalski,H. et al. (1999) Angiosarcomas express mixed endothelial phenotypes of blood and lymphatic capillaries: podoplanin as a specific marker for lymphatic endothelium. Am. J. Pathol., 154, 385–394. 63. Schacht,V., Ramirez,M.I., Hong,Y.K. et al. (2003) T1alpha/podoplanin deficiency disrupts normal lymphatic vasculature formation and causes lymphedema. EMBO J. , 22, 3546–3556. 64.Hong,Y.K., Harvey,N., Noh,Y.H., Schacht,V., Hirakawa,S., Detmar,M. and Oliver,G. (2002) Prox1 is a master control gene in the program specifying lymphatic endothelial cell fate. Dev Dyn., 225, 351–357. 65. Petrova,T.V., Makinen,T., Makela,T.P., Saarela,J., Virtanen,I., Ferrell,R.E., Finegold,D.N., Kerjaschki,D., Yla-Herttuala,S. and Alitalo,K. (2002) Lymphatic endothelial reprogramming of vascular

1736

endothelial cells by the Prox-1 homeobox transcription factor. EMBO J. , 21, 4593–4599. 66.Kaipainen,A., Korhonen,J., Mustonen,T., van Hinsbergh,V.W., Fang,G.H., Dumont,D., Breitman,M. and Alitalo,K. (1995) Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothlium during development. Proc. Natl Acad. Sci. USA, 92 , 3566–3570. 67.Valtola,R., Salven,P., Heikkila,P., Taipale,J., Joensuu,H., Rehn,M., Pihlajaniemi,T., Weich,H., deWaal,R. and Alitalo,K. (1999) VEGFR-3 and its ligand VEGF-C are associated with angiogenesis in breast cancer. Am. J. Pathol., 154, 1381–1390. 68.Kubo,H., Fujiwara,T., Jussila,L. et al. (2000) Involvement of vascular endothelial growth factor receptor-3 in maintenance of integrity of endothelial cell lining during tumor angiogenesis. Blood, 96 , 546–553. 69. Padera,T.P., Kadambi,A. and di Tomaso,E. (2002) Lymphatic metastasis in the absence of functional intratumor lymphatics. Science, 296,

1883–1886.

70.Leu,A.J., Berk,D.A., Lymboussaki,A., Alitalo,K. and Jain,R.K. (2000) Absence of functional lymphatics within a murine sarcoma: a molecular and functional evaluation. Cancer Res., 60, 4324–7. 71.Dadras,S.S., Bertoncini,P.T., Brown,L.F., Muzikansky,A., Jackson,D.G., Ellwanger,U., Garbe,C., Mihm,M.C. and Detmar,M. (2004) Tumor lymphangiogenesis: a novel prognostic indicator for cutaneous melanoma metastasis and survival. Am. J. Pathol., 162, 1951–1960. 72.Kyzas,P.A., Geleff,S., Batistatou,A., Agnantis,N.J. and Stefanou,D. (2005) Evidence for lymphangiogenesis and its prognostic implications in head and neck squamous cell carcinoma. J. Pathol., 206, 170–177.

73.Yokoyama,Y., Charnock-Jones,D.S., Licence,D. et al. (2003) Expression of vascular endothelial growth factor (VEGF)-D and its receptor, VEGF receptor 3, as a prognostic factor in endometrial carcinoma. Clin. Cancer Res., 9, 1361–1369. 74.Yokoyama,Y., Charnock-Jones,D.S., Licence,D. et al. (2003) Vascular endothelial growth factor-D is an independent prognostic factor in epithelial ovarian carcinoma. Br. J. Cancer, 88, 237–244. 75.Renyi-Vamos,F., Tovari,J., Fillinger,J., Timar,J., Paku,S., Kenessey,I., Ostoros,G., Agocs,L., Soltesz,I. and Dome,B. (2005) Lymphangiogenesis correlates with lymph node metastasis, prognosis, and angiogenic phenotype in human non-small cell lung cancer. Clin. Cancer Res., 11,

7344–7353.

76.Wang,T.B., Huang,Y.H., Lan,P., Song,X.M. and Wang,J.P. (2005) Correlation of lymphangiogenesis to progression of colorectal cancer. Ai Zheng, 24, 1276–1279. 77.Zeng,Y., Opeskin,K., Horvath,L.G., Sutherland,R.L. and Williams,E.D. (2005) Lymphatic vessel density and lymph node metastasis in prostate cancer. Prostate, 65, 222–230. 78. Schoppmann,S.F., Bayer,G., Aumayr,K. et al. (2004) Prognostic value of lymphangiogenesis and lymphovascular invasion in invasive breast cancer. Ann. Surg., 240, 306–312.

Kerjaschki,D. and

Kloppel,G. (2004) Expression of lymphangiogenic factors and evidence of intratumoral lymphangiogenesis in pancreatic endocrine tumors. Am. J. Pathol., 165, 1187–1197. 80. Stacker,S.A., Achen,M.G., Jussila,L., Baldwin,M.E. and Alitalo,K. (2002)

Lymphangiogenesis and cancer metastasis. Nat. Rev. Cancer, 2, 573–583. 81.Valencak,J., Heere-Ress,E., Kopp,T., Schoppmann,S.F., Kittler,H. and Pehamberger,H. (2004) Selective immunohistochemical staining shows significant prognostic influence of lymphatic and blood vessels in patients with malignant melanoma. Eur. J. Cancer, 40, 358–364. 82.White,J.D., Hewett,P.W., Kosuge,D., McCulloch,T., Enholm,B.C., Carmichael,J. and Murray,J.C. (2002) Vascular endothelial growth factor-D expression is an independent prognostic marker for survival in colorectal carcinoma. Cancer Res., 62, 1669–1675. 83.Kitadai,Y., Kodama,M., Cho,S., Kuroda,T., Ochiumi,T., Kimura,S., Tanaka,S., Matsumura,S., Yasui,W. and Chayama,K. (2005) Quantitative analysis of lymphangiogenic markers for predicting metastasis of human gastric carcinoma to lymph nodes. Int. J. Cancer, 115 , 388–392. 84.Rubbia-Brandt,L., Terris,B., Giostra,E., Dousset,B., Morel,P. and Pepper,M.S. (2004) Lymphatic vessel density and vascular endothelial growth factor-C expression correlate with malignant behavior in human pancreatic endocrine tumors. Clin. Cancer Res., 10 , 6919–6928. 85. Stacker,S.A., Williams,R.A. and Achen,M.G. (2004) Lymphangiogenic growth factors as markers of tumor metastasis. APMIS, 112, 539–549. 86.Williams,C.S., Leek,R.D., Robson,A.M., Banerji,S., Prevo,R., Harris,A.L. and Jackson,D.G. (2003) Absence of lymphangiogenesis and intratumoural lymph vessels in human metastatic breast cancer. J. Pathol., 200 , 195–206.

79. Sipos,B., Klapper,W., Kruse,M.L., Kalthoff,H.,

by guest on December 8, 2010carcin.oxfordjournals.orgDownloaded

from

87.Kukk,E., Lymboussaki,A., Taira,S., Kaipainen,A., Jeltsch,M., Joukov,V. and Alitalo,K. (1996) VEGF-C receptor binding and pattern of expression with VEGFR-3 suggests a role in lymphatic vascular development. Development, 122, 3829–3837. 88. Stacker,S.A., Stenvers,K., Caesar,C. et al. (1999) Biosynthesis of vascular endothelial growth factor-D involves proteolytic processing which generates non-covalent homodimers. J. Biol. Chem. , 274, 32127–32136. 89.McColl,B.K., Baldwin,M.E., Roufail,S., Freeman,C., Moritz,R.L., Simpson,R.J., Alitalo,K., Stacker,S.A. and Achen,M.G. (2003) Plasmin activates the lymphangiogenic growth factors VEGF-C and VEGF-D. J. Exp. Med., 198 , 863–868. 90. Joukov,V., Sorsa,T., Kumar,V., Jeltsch,M., Claesson-Welsh,L., Cao,Y., Saksela,O., Kalkkinen,N. and Alitalo,K. (1997) Proteolytic processing regulates receptor specificity and activity of VEGF-C. EMBO J. , 16,

3898–3911.

91. Joukov,V., Kaipainen,A., Jeltsch,M., Pajusola,K., Olofsson,B., Kumar,V., Eriksson,U. and Alitalo,K. (1997) Vascular endothelial growth factors VEGF-B and VEGF-C. J. Cell Physiol., 173, 211–215. 92. Siegfried,G., Khatib,A.M., Benjannet,S., Chretien,M. and Seidah,N.G. (2003) The proteolytic processing of pro-platelet-derived growth factor-A at RRKR(86) by members of the proprotein convertase family is functionally correlated to platelet-derived growth factor-A-induced functions and tumorigenicity. Cancer Res., 63, 1458–1463. 93. Siegfried,G., Basak,A., Cromlish,J.A., Benjannet,S., Marcinkiewicz,J., Chretien,M., Seidah,N.G. and Khatib,A.M. (2003) The secretory proprotein convertases furin, PC5, and PC7 activate VEGF-C to induce tumorigenesis. J. Clin. Invest., 111 , 1723–1732. 94.Makinen,T., Jussila,L., Veikkola,T. et al. (2001) Inhibition of lymphangiogenesis with resulting lymphedema in transgenic mice expressing soluble VEGF receptor-3. Nat. Med., 7, 199–205. 95.Veikkola,T., Jussila,L., Makinen,T. et al. (2001) Signalling via vascular endothelial growth factor receptor-3 is sufficient for lymphangiogenesis in transgenic mice. EMBO J. , 20, 1223–1231. 96.McColl,B.K., Stacker,S.A. and Achen,M.G. (2004) Molecular regulation of the VEGF family—inducers of angiogenesis and lymphangiogenesis. APMIS, 112, 463–480. 97. Schlessinger,J. (2000) Cell signaling by receptor tyrosine kinases. Cell, 103 , 211–225. 98.Makinen,T., Veikkola,T., Mustjoki,S. et al. (2001) Isolated lymphatic endothelial cells transduce growth, survival and migratory signals via the VEGF-C/D receptor VEGFR-3. EMBO J. , 20 , 4762–4773. 99.Karpanen,T., Egeblad,M., Karkkainen,M.J., Kubo,H., Ylu-Herttuala,S., Jaattela,M. and Alitalo,K. (2001) Vascular endothelial growth factor C promotes tumor lymphangiogenesis and intralymphatic tumor growth. Cancer Res., 61, 1786–1790. 100. Skobe,M., Hawighorst,T., Jackson,D.G., Prevo,R., Janes,L., Velasco,P., Riccardi,L., Alitalo,K., Claffey,K. and Detmar,M. (2001) Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis. Nat. Med., 7, 192–198. 101. Kukk,E., Lymboussaki,A., Taira,S., Kaipainen,A., Jeltsch,M., Joukov,V. and Alitalo,K. (1996) VEGF-C receptor binding and pattern of expression with VEGFR-3 suggests a role in lymphatic vascular development. Development , 122, 3829–3837. 102. Skobe,M.,Hamberg,L.M.,Hawighorst,T.,Schirner,M.,Wolf,G.L.,Alitalo,K. and Detmar,M. (2001) Concurrent induction of lymphangiogenesis, angiogenesis, and macrophage recruitment by vascular endothelial growth factor-C in melanoma. Am. J. Pathol., 159, 893–903. 103. Yanai,Y., Furuhata,T., Kimura,Y., Yamaguchi,K., Yasoshima,T., Mitaka,T., Mochizuki,Y. and Hirata,K. (2001) Vascular endothelial growth factor C promotes human gastric carcinoma lymph node metastasis in mice. J. Exp. Clin. Cancer Res., 20, 419–428. 104.Mandriota,S.J., Jussila,L., Jeltsch,M. et al. (2001) Vascular endothelial growth factor-C-mediated lymphangiogenesis promotes tumour metastasis. EMBO J., 20 , 672–682. 105. Stacker,S.A., Caesar,C., Baldwin,M.E., Thornton,G.E., Williams,R.A., Prevo,R., Jackson,D.G., Nishikawa,S., Kubo,H. and Achen,M.G. (2001) VEGF-D promotes the metastatic spread of tumor cells via the lymphatics. Nat. Med., 7, 186–191. 106. Achen,M.G., Roufail,S., Domagala,T. et al. (2000) Monoclonal anti- bodies to vascular endothelial growth factor-D block its interactions with both VEGF receptor-2 and VEGF receptor-3. Eur. J. Biochem., 267, 2505–2515. 107. Hirakawa,S., Kodama,S., Kunstfeld,R., Kajiya,K., Brown,L.F. and Detmar,M. (2005) VEGF-A induces tumor and sentinel lymph node lymphangiogenesis and promotes lymphatic metastasis. J. Exp. Med., 201, 1089–1099.

Lymphatic vessels in cancer metastasis

108. Vleugel,M.M., Bos,R., van der Groep,P., Greijer,A.E., Shvarts,A., Stel,H.V., van der Wall,E. and van Diest,P.J. (2004) Lack of lymphangiogenesis during breast carcinogenesis. J. Clin. Pathol., 57,

746–751.

109. Partanen,T.A., Alitalo,K. and Miettinen,M. (1999) Lack of lymphatic vascular specificity of vascular endothelial growth factor receptor 3 in 185 vascular tumors. Cancer, 86, 2406–2412. 110. Paul,M.K. and Mukhopadhyay,A.K. (2004) Tyrosine kinase–role and significance in cancer. Int. J. Med. Sci., 1, 101–115. 111.McColl,B.K., Loughran,S.J., Davydova,N., Stacker,S.A. and Achen,M.G. (2005) Mechanisms of lymphangiogenesis: targets for blocking the metastatic spread of cancer. Curr. Cancer Drug Targets, 5, 561–571. 112. Kabbinavar,F., Hurwitz,H.I., Fehrenbacher,L., Meropol,N.J., Novotny,W.F., Lieberman,G., Griffing,S. and Bergsland,E. (2003) Phase II, randomized trial comparing bevacizumab plus fluorouracil (FU)/leucovorin (LV) with FU/LV alone in patients with metastatic colorectal cancer. J. Clin. Oncol., 21, 60–65. 113. Kabbinavar,F.F., Hambleton,J., Mass,R.D., Hurwitz,H.I., Bergsland,E. and Sarkar,S. (2005) Combined analysis of efficacy: the addition of bevacizumab to fluorouracil/leucovorin improves survival for patients with metastatic colorectal cancer. J. Clin. Oncol., 23 , 3706–3712.

114. Folkman,J. (1971) Tumor angiogenesis: therapeutic implications. N. Engl.

J. Med., 285, 1182–1186.

115. Folkman,J. (1972) Anti-angiogenesis: new concepts of therapy of solid tumors. Ann. Surg., 175, 409–416.

116. He,Y., Kozaki,K., Karpanen,T., Koshikawa,K., Yla-Herttuala,S., Takahashi,T. and Alitalo,K. (2002) Suppression of tumor lymphan-

giogenesis and lymph node metastasis by blocking vascular endo- thelial growth factor receptor 3 signaling. J Natl Cancer Inst., 94,

819–825.

117.Lin,J., Lalani,A.S., Harding,T.C. et al. (2005) Inhibition of lymphogenous metastasis using adeno-associated virus-mediated gene transfer of a soluble VEGFR-3 decoy receptor. Cancer Res., 65, 6901–6909. 118. Khatib,A.M., Siegfried,G., Chretien,M., Metrakos,P. and Seidah,N.G. (2002) Proprotein convertases in tumor progression and malignancy:

novel targets in cancer therapy. Am. J. Pathol., 160, 1921–1935. 119. Khatib,A.M., Siegfried,G., Prat,A., Luis,J., Chretien,M., Metrakos,P. and Seidah,N.G. (2001) Inhibition of proprotein convertases is associated with loss of growth and tumorigenicity of HT-29 human colon carcinoma cells:

importance of insulin-like growth factor-1 (IGF-1) receptor processing in IGF-1-mediated functions. J. Biol. Chem., 276, 30686–30693. 120. Yoshiji,H., Kuriyama,S., Ways,D.K. et al. (1999) Protein kinase C lies on the signaling pathway for vascular endothelial growth factor-mediated tumor development and angiogenesis. Cancer Res., 59, 4413–4418. 121.Bicknell,R. and Harris,A.L. (2004) Novel angiogenic signaling pathways and vascular targets. Annu. Rev. Pharmacol. Toxicol. , 44 , 219–238. 122. Gerber,H.P., McMurtrey,A., Kowalski,J., Yan,M., Keyt,B.A., Dixit,V. and Ferrara,N. (1998) Vascular endothelial growth factor regulates endothelial cell survival through the phosphatidylinositol 3 0 -kinase/Akt signal transduction pathway. Requirement for Flk-1/KDR activation.

J. Biol. Chem. , 273 , 30336–30343.

123.Thakker,G.D., Hajjar,D.P., Muller,W.A. and Rosengart,T.K. (1999) The role of phosphatidylinositol 3-kinase in vascular endothelial growth factor signaling. J. Biol. Chem., 274, 10002–10007. 124. Deplanque,G. and Harris,A.L. (2000) Anti-angiogenic agents: clinical trial design and therapies in development. Eur. J. Cancer, 36, 1713–1724. 125. Arora,A. and Scholar,E.M. (2005) Role of tyrosine kinase inhibitors in cancer therapy. J. Pharmacol. Exp. Ther. , 315, 971–979. 126. Staehler,M., Haseke,N., Schoppler,G., Stadler,T., Adam,C. and Stief,C.G.

(2006) Therapy strategies for advanced renal cell carcinoma. Urologe A, 45, 99–112. 127.Wakelee,H.A. and Schiller,J.H. (2005) Targeting angiogenesis with vascular endothelial growth factor receptor small-molecule inhibitors:

novel agents with potential in lung cancer. Clin. Lung Cancer, 7 (Suppl. 1), S31–S38. 128.Ruggeri,B., Singh,J., Gingrich,D. et al. (2003) CEP-7055: a novel, orally active pan inhibitor of vascular endothelial growth factor receptor tyrosine kinases with potent antiangiogenic activity and antitumor efficacy in preclinical models. Cancer Res., 63, 5978–5991. 129.Wood,J.M., Bold,G., Buchdunger,E. et al. (2000) PTK787/ZK 222584, a novel and potent inhibitor of vascular endothelial growth factor receptor tyrosine kinases, impairs vascular endothelial growth factor-induced responses and tumor growth after oral administration. Cancer Res., 60,

2178–2189.

130. Drevs,J., Zirrgiebel,U., Schmidt-Gersbach,C.I. et al. (2005) Soluble markers for the assessment of biological activity with PTK787/ZK

1737

by guest on December 8, 2010carcin.oxfordjournals.orgDownloaded

from

R.Shayan, M.G.Achen and S.A.Stacker

222584 (PTK/ZK), a vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitor in patients with advanced colorectal cancer from two phase I trials. Ann. Oncol., 16, 558–565. 131.Wilhelm,S.M., Carter,C., Tang,L. et al. (2004) BAY 43-9006 exhibits broad spectrum oral antitumor activity and targets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis. Cancer Res., 64 , 7099–7109. 132.Wilhelm,S. and Chien,D.S. (2002) BAY 43-9006: preclinical data. Curr. Pharm. Des., 8, 2255–2257. 133. Kim,J., Takeuchi,H., Lam,S.T., Turner,R.R., Wang,H.J., Kuo,C., Foshag,L., Bilchik,A.J. and Hoon,D.S. (2005) Chemokine receptor CXCR4 expression in colorectal cancer patients increases the risk for recurrence and for poor survival. J. Clin. Oncol., 23, 2744–2753. 134. Pachynski,R.K., Wu,S.W., Gunn,M.D. and Erle,D.J. (1998) Secondary lymphoid-tissue chemokine (SLC) stimulates integrin alpha 4 beta 7-mediated adhesion of lymphocytes to mucosal addressin cell adhesion molecule-1 (MAdCAM-1) under flow. J. Immunol., 161,

952–956.

135. Gunn,M.D., Tangemann,K., Tam,C., Cyster,J.G., Rosen,S.D. and Williams,L.T. (1998) A chemokine expressed in lymphoid high endothelial venules promotes the adhesion and chemotaxis of naive T lymphocytes. Proc. Natl Acad. Sci. USA, 95, 258–263. 136. Ohl,L., Mohaupt,M., Czeloth,N., Hintzen,G., Kiafard,Z., Zwirner,J., Blankenstein,T., Henning,G. and Forster,R. (2004) CCR7 governs skin dendritic cell migration under inflammatory and steady-state conditions. Immunity, 21, 279–288. 137.Wiley,H.E., Gonzalez,E.B., Maki,W., Wu,M.T. and Hwang,S.T. (2001) Expression of CC chemokine receptor-7 and regional lymph node metastasis of B16 murine melanoma. J. Natl Cancer Inst., 93,

1638–1643.

138.Bevacqua,S.J., Welch,D.R., Diez de Pinos,S.M., Shapiro,S.A., Johnston,M.G., Witte,M.H., Leong,S.P., Dorrance,T.L., Leibovitz,A. and Hendrix,M.J. (1990) Quantitation of human melanoma, carcinoma and sarcoma tumor cell adhesion to lymphatic endothelium. Lymphology, 23, 4–14. 139. Irjala,H., Johansson,E.L., Grenman,R., Alanen,K., Salmi,M. and Jalkanen,S. (2001) Mannose receptor is a novel ligand for L-selectin and mediates lymphocyte binding to lymphatic endothelium. J. Exp. Med., 194 , 1033–1042. 140. Farnsworth,R.H., Achen,M.G. and Stacker,S.A. (2006) Lymphatic endothelium: an important interactive surface for malignant cells. Pulm Pharmacol. Ther. , 19 , 51–60. 141. Irjala,H., Alanen,K., Grenman,R., Heikkila,P., Joensuu,H. and Jalkanen,S. (2003) Mannose receptor (MR) and common lymphatic endothelial and

1738

vascular endothelial receptor (CLEVER)-1 direct the binding of cancer cells to the lymph vessel endothelium. Cancer Res., 63, 4671–4676. 142.Ristimaki,A., Narko,K., Enholm,B., Joukov,V. and Alitalo,K. (1998) Proinflammatory cytokines regulate expression of the lymphatic endothelial mitogen vascular endothelial growth factor-C. J. Biol. Chem., 273, 8413–8418. 143. Schoppmann,S.F., Birner,P., Stockl,J., Kalt,R., Ullrich,R., Caucig,C., Kriehuber,E., Nagy,K., Alitalo,K. and Kerjaschki,D. (2002) Tumor- associated macrophages express lymphatic endothelial growth factors and are related to peritumoral lymphangiogenesis. Am. J. Pathol., 161,

947–956.

144.Leong,S.P., Morita,E.T., Sudmeyer,M., Chang,J., Shen,D., Achtem,T.A., Allen,R.E.,Jr and Kashani-Sabet,M. (2005) Heterogeneous patterns of lymphatic drainage to sentinel lymph nodes by primary melanoma from different anatomic sites. Clin. Nucl. Med., 30, 150–158. 145. de Wilt,J.H., Thompson,J.F., Uren,R.F., Ka,V.S., Scolyer,R.A., McCarthy,W.H., O’Brien,C.J., Quinn,M.J. and Shannon,K.F. (2004) Correlation between preoperative lymphoscintigraphy and metastatic nodal disease sites in 362 patients with cutaneous melanomas of the head and neck. Ann. Surg., 239, 544–552. 146. Netter,F.J. and Hansen,J.T. (2002) Atlas of Human Anatomy. Saunders, New York. 147.Thompson,J.F. and Uren,R.F. (2001) Anomalous lymphatic drainage patterns in patients with cutaneous melanoma. Tumori, 87, S54–S56. 148.Leong,S.P., Achtem,T.A., Habib,F.A., Steinmetz,I., Morita,E., Allen,R.E., Kashani-Sabet,M. and Sagebiel,R. (1999) Discordancy between clinical predictions vs lymphoscintigraphic and intraoperative mapping of sentinel lymph node drainage of primary melanoma. Arch Dermatol., 135, 1472–1476. 149. Uren,R.F. (2004) Lymphatic drainage of the skin. Ann. Surg. Oncol., 11,

179S–185S.

150. Uren,R.F., Howman-Giles,R.B., Thompson,J.F., Shaw,H.M. and McCarthy,W.H. (1995) Lymphatic drainage from peri-umbilical skin to internal mammary nodes. Clin. Nucl. Med., 20, 254–255. 151. O’Brien,C.J., Uren,R.F., Thompson,J.F., Howman-Giles,R.B., Petersen- Schaefer,K., Shaw,H.M., Quinn,M.J. and McCarthy,W.H. (1995) Prediction of potential metastatic sites in cutaneous head and neck melanoma using lymphoscintigraphy. Am. J. Surg., 170, 461–466. 152. Pathak,I., O’Brien,C.J., Petersen-Schaeffer,K., McNeil,E.B., McMahon,J., Quinn,M.J., Thompson,J.F. and McCarthy,W.H. (2001) Do nodal metastases from cutaneous melanoma of the head and neck follow a clinically predictable pattern? Head Neck, 23, 785–790.

Received January 30, 2006; accepted March 20, 2006