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Revista Română de Anatomie funcţională şi clinică, macro- şi microscopică şi de Antropologie

Vol. XIV – Nr. 2 – 2015 UPDATES

THE ANATOMY OF THE HEPATIC LYMPHATICS AND THEIR IMPLICATION IN COLORECTAL

CANCER LIVER METASTASIS

Beatrice Lintoiu-Ursut¹, A. Tulin³, B. Ursut³, O. Enciu³, S. Constantinoiu², F. Filipoiu³1. “Prof. Dr. Agrippa Ionescu” Emergency Clinical Hospital, Bucharest

2. University of Medicine and Pharmacy “Carol Davila”, Bucharest Department of General and Esophageal Surgery

“Sf. Maria” Clinical Hospital, Bucharest3. University of Medicine and Pharmacy “Carol Davila”, Bucharest

THE ANATOMY OF THE HEPATIC LYMPHATICS AND THEIR IMPLICATION IN COLORECTAL CANCER LIVER METASTASIS (Abstract): The lymphatic system is present in almost every organ of the human body. From the first report of lympatics in 1563, the research was slowly evoluating due to the lack of specific lymphatic markers. Recently, markers specific to lymphatic endothelial cells were discovered, improving the knowledge in the field. Lym-phangiogenesis plays an active role in the formation and spread of colorectal liver metastasis. The pattern of intratumoral lymphatics seems to be a predictive factor for the disease recurrence and survival. New insights into the molecular mechanisms that control lymphatic development and function may provide a better treatment for cancer patients. Key words: LYMPHATIC VESSELS, LYMPHANGIOGENESIS, COLORECTAL LIVER METASTASIS, LYMPHATIC MARKERS

INTRODUCTION The lymphatic system represents a vascular

network of thin-walled capillaries that drain protein-rich lymph from the extracellular spac-es within most organs. A continuous single-cell layer of overlapping endothelial cells lines the lymphatic capillaries, whithout a continuous basement membrane and therefore, highly per-meable. Lymph returns to venous circulation via the larger lymphatic collecting vessels, which contain a muscular and an adventitial layer. Lymphatic vessels are not normally pre-sent in avascular structures such as epidermis, hair, nails, cartilage, and cornea, nor in some vascularized organs such as brain and retina. The lymphatic system develops in parallel with the blood vascular system through a process known as lymphangiogenesis. Studies of nor-mal development and pathologic growth of the blood vascular system have thoroughly eluci-dated the molecular mechanisms that control these angiogenic processes, but studies of the

lymphatic system have slowly developed be-cause of the lack of specific lymphatic markers. The recent discovery of lymphatic endothelium-specific markers facilitates scientific advances (1,2).

HISTORICAL PERSPECTIVEBasic anatomical knowledge of the lymphat-

ic system first emerged during the Renaissance, when Eustachius (1563) observed the thoracic duct in the horse and Asellius (1627) demon-strated the mesenteric lymph vessels (“lacteae venae” or milky veins) of the dog. Lymphatic vessels on the surface of the liver were first described by Rudbeck (1653). Later, detailed studies were carried out by Mascagni (1787) and Hunter (1762) (3,4). The recently discov-ered lymphatic markers include: vascular en-dothelial growth factor (VEGF) receptor-3, LYVE-1, Prox-1, Podoplanin, macrophage man-nose receptor 1, CCL21, Desmoplakin, integrin α9 and Plakoglobin. The vascular endothelial

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The Anatomy of the Hepatic Lymphatics and Their Implication in Colorectal Cancer

growth factor receptor-3 (VEGFR-3) and the lymphatic endothelial hyaluronan receptor (LYVE-1), a specific cell surface protein of lymphatic endothelial cells and macrophages were among the first lymphatic markers to be identified (5,6,7).

LYMPHATIC REGENERATION Tissue repair requires the regrowth and re-

connection of a functional lymphatic vascular system. In full-thickness skin wounds, ingrowth of new blood vessels (angiogenesis) into the newly formed granulation tissue dominates the delayed and less pronounced formation of new lymphatic vessels, which are predominantly located surrounding the blood vessels in granu-lation tissue. Lymphangiogenesis in the adult occurs by outgrowth from preexisting lymphat-ics. Recent evidence suggests that VEGF, a major angiogenic molecule that is up-regulated during tissue, might also stimulate lymphangi-ogenesis under certain conditions. Because VEGF also induces vascular leakage and tissue edema, it remains to be established whether the lymphangiogenesis observed in tissue repair and inflammation is caused by direct activation of VEGFR-2 on lymphatic endothelium or by indirect stimulation of lymphangiogenesis by enhanced interstitial fluid accumulation. VEGF-C and VEGF-D, activating ligands of VEGFR-3, are prime candidates for molecules that control wound-associated lymphangiogenesis (8,9).

TUMOR LYMPHANGIOGENESISIn most human cancers, the lymphatic sys-

tem serves as the primary conduit for the met-astatic spread of tumor cells to regional lymph nodes and via the thoracic duct and blood cir-culation to distant organs. Tumor cell metasta-sis to lymph nodes represents a major factor for evaluating the prognosis of cancer patients and for the choice of adjuvant chemotherapy and/or radiation therapy. However, few studies are available regarding the molecular mecha-nisms by which tumor cells gain access to the lymphatic system and consequently are able to spread. There are three causes for the lack of insight into the early metastatic process: the absence of specific markers for tumor-associ-ated lymphatic vessels, the lack of knowledge about lymphangiogenesis factors and the ab-sence of experimental cancer metastasis models for the quantitative evaluation of lymph node metastasis. Based on the recent discovery of

several lymphatic-specific markers, scientific advances during the last years have provided new insights into the molecular mechanisms that control lymphatic metastasis. These ex-perimental studies have also provided convinc-ing evidence for an active role of malignant tumor cells in inducing peritumoral and intra-tumoral lymphangiogenesis, taking advantage of molecular mechanisms operative in the im-mune response (9,10,11,12).

Skobe et al. (13), using an orthotopic human MDA-435 breast cancer model in immunosup-pressed mice, showed that lymphatic vessels are present both surrounding and within malignant tumors, and that overexpression of the lymphang-iogenesis factor VEGF-C resulted in enhanced infiltration of breast cancers by proliferating lymphatic vessels that frequently contained can-cer cells.. VEGF-C-induced tumor lymphangi-ogenesis resulted in enhanced tumor metastasis to regional lymph nodes and the extent of lung metastasis was correlated with the extent of lymphangiogenesis of the primary tumor. (14). Recent studies using podoplanin as a lymphat-ic marker determined a significant correlation between the lymphatic microvascular density and the lymph node status in human breast cancer (15). On the contrary, increased lym-phatic microvessel density was associated with a favorable prognosis in early-stage cervical cancer (16).

LYMPHATICS OF THE LIVER The liver is a unique organ with overwhelm-

ing innate and adaptive immune cells, playing important roles in host defense against the inva-sion of exogenous pathogens and tumors. The lymphatic vessels in the liver function as a drainage and an immunological control system. It consists of noncontractile initial lymphatic network and collecting lymphatic vessels. The initial vessels are tubulosaccular, with unidirec-tional valves and a discontinuous or absent basement membrane. Collecting lymphatic ves-sels are endowed with smooth muscle cells. 25 to 50 % of the lymph from the thoracic duct comes from the liver. The hepatic lymphatic vessels, according to their locations are classi-fied in: portal, sublobular and superficial. 80% of hepatic lymph drains into portal lym-phatic vessels (3,4). Fluid in the space of Disse passes through channels between hepatocytes of the limiting plate and through the space along the initial segment of the hepatic sinusoids to

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enter the interstitial space of the portal tract. It can also travel through channels between hepat-ocytes to enter sublobular and superficial lym-phatic vessels. Mall (1901) showed that color gelatin injected into the portal vein first ap-peared in the perisinusoidal space (Disse), then reached the perilobular space (the space of Mall) and finally entered portal lymphatic ves-sels. Immunohistochemistry to markers specific to lymphatic vessels such as LYVE-1 shows the existance of lymphatic vessels in the portal tract (3,4,17). An experimental study demonstrated that the hepatic sinusoids and the space of Disse significantly expand when the thoracic duct is ligated. This study and several recent ones in-dicate that hepatic lymph fluid comes from the hepatic sinusoids. Fluid filtered out of the si-nusoids into the space of Disse flows through the channels traversing the limiting plate either independently of blood vessels or along blood vessels and enters the interstitial space of either the portal tract, sublobular veins or the he-patic capsule (18). It is not yet demonstrated any direct transition of prelymphatic vessels to lymphatic vessels. Sublobular lymph vessels lead into lymphatic vessels running along the inferior vena cava. Superficial lymphatics from the convex surface of the liver run through the coronary ligament, mainly the right and left triangular ligament as well as through the fal-ciform ligament. They cross the diaphragm to enter the precardiac, superior phrenic and jux-taesophageal lymph nodes or accompany the right and left inferior phrenic artery to reach the celiac nodes. From these nodes the lymph passes into the intestinal lymph trunks, which then enter the cisterna chyli and then into the thoracic duct. Some lymph vessels cross the anterior margin of the liver and communicate with the hepatic lymph nodes at the porta he-patis (3,4) Superficial lymphatics from the con-cave surface mostly run to the hepatic lymph nodes with the exception of some lateral vessels of the right lobe,which pass directly to the right lateral aortic group. From the caudate lobe lymph vessels drain into the precaval nodes. The deep lymph vessels leave the liver at the porta hepatis, where 12-15 separate vessels run together with the hepatic artery or the bile duct. These communicate with the foraminal node at the epiploic foramen and the superior pancre-atic nodes, which are connected with the lat-eral aortic group. Other lymph nodes are situ-

ated along the common and proper hepatic arteries and drain into the celiac nodes. The lymphatics that accompany the hepatic veins leave the liver as five or six separate vessels that continue in the wall of the inferior vena cava. The further drainage of the hepatic lymph vessels is represented by four chains, of which the thoracic duct is the most important collect-ing vessel. The other chains consist of lymph nodes in the anterior and the posterior medi-astinum and along the internal thoracic vessels (3,4).

LYMPHATIC VESSELS IN PATHOLOGICAL CONDITIONS OF THE LIVERDiseases affecting the hepatic lymphatics

manifest as lymphedema, lymphatic masses or cystic lesions. Lymphatic disruption after trau-ma or surgery is depicted as perihepatic fluid collections of lymph (lymphocele). Abnormal distended lymphatics are seen in periportal spaces as linear hypoattenuations on CT or strong linear hyperintenities on MR imaging. In hepatic cirrhosis there are diffuse abnor-malities of the liver’s architecture and the por-tal lymph flow is highly increased (19). In patients with cancer, tumor metastasis to lymph nodes by means of the lymphatic vascular sys-tem results in poor prognosis. In hepatocellular carcinoma (HCC) and some metastasized tu-mors, LYVE-1 and Prox-1-positive lymphatic vessels are abundant in the immediate vicinity of the tumor. Poorly differentiated HCCs ex-press VEGF-C significantly stronger than well- or moderately differentiated HCCs. Thus lym-phangiogenesis is associated with enhanced metastasis (20). Almost half of the patients affected by colorectal cancer have or will de-velop hepatic metastases. The only currative treatment is the surgical removal, although 60-70% will develop recurrences of the disease. Tumor angiogenesis has been implicated as a major factor in the development and spread of these metastasis (21). Studies have suggested that lymphangiogenesis also plays an active role in the formation and spread of colorectal liver metastasis. The patterns of intratumoral lym-phatics may have potential clinical significance, as a predictive marker of disease recurrence and patient survival (21,22). Lymph node ratio is independently correlated with distant organ metastases of CR cancer being an important

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predictive factor for estimating the prognosis of CRC (23). Lupinacci at al. (21) after the histologic evaluation of CRCLM obtained from 118 patients concluded that intrahepatic lym-phatic invasion is a significant prognostic factor and lymphatic system is the main route for dissemination of colorectal liver metastasis.

For decades, angiogenesis, the formation of new blood vessels, has been the main focus of research in the pathogenesis of tumor metasta-sis. Several studies have focused on the mi-crovessel density (MVD) as a prognostic tool, elevated levels of MVD being associated with poor prognosis (24). Early studies shows that lymphatic vessel density (LVD) in the tumor periphery, centre, and adjacent liver may be used as a prognostic marker to predict the like-lihood of disease recurrence following hepatic resection (24,25,26,27). Investigating the ex-pression of VEGF-C, Matsumoto et al. report-ed that VEGF-C overexpression significantly correlated with tumor invasion, lymphatic inva-sion, and lymph node metastases (28). Clear consensus on the potential use of LVD as a prognostic marker remains to be elucidated. Determining the lymphatic development within tumors may further play a significant role in

the selective use of biological agents with the ability to target lymphatics being currently un-der development (28,29,30).

CONCLUSIONSAfter more than 300 years since the initial

description of the lymphatic vessels by Gaspa-ro Aselli, some of the mechanisms controlling the normal and pathological development of the previously neglected lymphatic vasculature are being revealed. The identification of specific markers for the lymphatic vessels has been obtained. The concept of tumor lymphangio-genesis is starting to be considered as an im-portant aspect of cancer metastasis. The future of this field of research is very promising and could eventually lead to better diagnosis and treatment of a variety of lymphatic disorders and certain types of cancer.

ACKNOWLEDGEMENTSThis paper is supported by the Sectoral

Operational Programme Human Resources Development (SOP HRD), financed from the European Social Fund and by the Romanian Go vern ment under the contract number POSDRU/159/1.5/S/137390”

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Corresponding author

Adrian Tuline-mail: dr_2lin@yahoo.com