Mechanism of Tumour Dissemination in Hepatobiliary and Pancreatic Tumours
In cancer patients, relapse rate and prognosis depend on the extent of cancer dissemination within the body. Furthermore, preoperative knowledge of tumour spread is the most important decision parameter in treatment planning. For the aforementioned reasons, it is important to understand cancer dissemination routes and the mechanisms that determine cancer spread. Tumour can spread locally or migrate to lymph nodes or travel to distant organs through the blood stream. A fourth, less known but nevertheless clinically important route of tumour spread is perineural invasion. The metastatic process is extremely selective and consists in a series of steps, all of which must be accomplished for metastases to develop. When the primary tumour grows to more than 1–2 mm of diameter, new vessels and lymphatics develop from the existing network to provide nutrients and oxygen to cancer tissue. Neovessels also allow cancer cells to escape into the blood stream. The few cancer cells that survive in the circulation and are caught in the capillaries of distant organs enter the surrounding tissue where they grow and eventually develop new blood vessels. This chapter will discuss the mechanisms of local and distant tumour spread and the organ-specific nature of distant metastases, with a focus on hepatobiliary and pancreatic tumours.
1.1 Local Spread
Local spread is defined as the diffusion of a tumour within an organ or throughout adjacent structures by contiguity. It represents the ability of cancer cells to thrust aside adjacent tissues to actively invade them and/or destroy them . Tumours may either expand into adjacent tissues, spread locally through direct infiltration or disseminate along blood and lymphatic vessels, nerves, and excretory biliary ducts.
1.1.1 Expansive Growth
Expansive growth is common also in intrahepatic cholangiocarcinoma (iCC) where malignant cells penetrate the bile duct wall and spread between the hepatocyte layers infiltrating the hepatic sinusoidal spaces . Surrounded by liver parenchyma, iCC grows three-dimensionally, presenting itself as an irregularly but well-defined shaped solid mass [7, 8], not invading a major branch of the portal triad and with a peripheral fibrous component [7, 9]. iCC is rarely symptomatic in the early stages  and can achieve a large dimension before being diagnosed. Patients may develop symptoms when the large mass causes compression and dilatation of a large bile duct or when liver capsular invasion and retraction is present.
Pancreatic mucinous cystic neoplasms (MCN) are frequently characterized by an expansive growth. Typically, they appear as thick walled, unilocular/multilocular cystic tumours  surrounded by a capsule composed of an inner epithelial layer secreting mucin and by an external layer of dense cellular ovarian-type stroma . In a late phase, invasive carcinoma cells may infiltrate the capsule and reach its outer layer (intracapsular invasion) or extend into the surrounding pancreatic tissue and/or thrust within the peritoneal cavity (extracapsular invasion) . Spread of tumour through vascular, lymphatic, or neural structures is however a rare occurrence and this partly explains the favourable prognosis of MCN in comparison to the more aggressive pancreatic adenocarcinoma .
1.1.2 Infiltrative Growth
Most pancreatic and biliary tumours have an infiltrative behaviour, which partly explains their dismal prognosis. From a pathological standpoint two different intramural infiltration growth-types have been observed in gallbladder cancer: the infiltrative growth-type, with muscle preservation, and the destructive growth-type where the muscle layer is destroyed [16, 17, 18]. The latter generally presents a more aggressive behaviour, also leading to a higher probability of lympho-vascular spread [16, 17, 18].
Infiltrative spread is influenced by the anatomy of the organ where the tumour originates. The gallbladder wall, for example, is composed of only four layers: mucosa, lamina propria, an irregular muscle layer, and externally by a thin stratum of connective tissue [18, 27, 28]. The absence of the submucosal layer and the irregular presence of the muscular layer make it easy for the tumour to cross the GB wall and to invade the adjacent structures by contiguity [27, 28, 29, 30]. Moreover, since the hepatic surface of the GB lacks of the serosa layer, the connective tissue of the GB is continuous with the interlobular connective tissue of the liver. These unique anatomical characteristics explain why the direct infiltration of the liver and of the sub-hepatic space are the most common routes of local dissemination in GB cancer originating from the fundus or body [18, 28]. Conversely, tumours arising from the GB neck spread along the cystic duct and reach the extrahepatic bile duct, resulting in biliary obstruction [28, 31].
Intravasation refers to the process in which cancer cells enter the lumen of blood vessels or lymphatics . As aforementioned, the uncontrolled growth of cancer cells amplifies the metabolic activity of newly formed tissues which in turn increases the need for nutrients and oxygen. To provide nutrients and oxygen, neoplastic cells secrete growth factors that attract endothelial cells to the tumour; the latter secretes enzymes to degrade the basement line of capillaries and promote the formation of new blood vessels and lymphatics [32, 33, 34, 35].
The process by which tumour neovessels sprout from existing blood vessels is referred to as tumour angiogenesis . Tumour vessels are malformed and highly permeable and favour the migration of neoplastic cell into the body blood stream. Characteristically, epithelial cells that give rise to most pancreatic and hepatobiliary cancers are characterized by the absence of motility due to tight cell–cell adhesion and the anchoring to the ECM by the basement membrane . Reorganization of matrix constituents during tumour growth results in the disruption of the architecture of the ECM, promoting cell transformation, tumour cell motility, and migration . Once near a vessel, the proteases produced by the malignant cells degrade the endothelium of the vessel enabling the access of cells into the blood stream .
Macroscopic invasion takes place when the tumour thrombus is visible at gross pathologic examination and/or at imaging . HCC patients with macrovascular invasion have a poor prognosis and surgical resection is usually not a treatment option . It follows that identification of venous invasion at imaging is of paramount importance to plan treatment of these patients and is included in the scoring systems that determine treatment strategies in HCC patients [43, 44, 45]. While vascular invasion is exceptional in other types of liver tumours, it may occasionally be observed in pancreatic endocrine tumours .
Lymphatic vessels compass a unidirectional fluid recycling system and play a pivotal role in tumour dissemination . In normal conditions, fluid and cells are uptaken by lymphatic capillaries and conveyed down the lymphatic ducts to the regional lymph nodes and ultimately through the thoracic duct into the venous system . Tumour cells can bolster lymphangiogenesis by altering the surrounding microenvironment and by promoting the secretion of soluble molecules that stimulate the enlargement of tumour lymphatic vessels . Differently from angiogenesis, lymphatic capillaries grow mainly around the tumour (peri-tumoural lymphatic vessels; PTV) while intra-tumoural lymphatic vessels are small, collapsed, and non-functional. As a consequence, only the PTV have the capacity to absorb fluid and cells and act as routes for cancer spread to regional lymph nodes . Cancer cells are transported from the tumour to the lymphatic capillaries where the interstitial fluid pressure is lower and invade into their lumen through the open interendothelial gaps . Lymphatic endothelial cells can support the process by secreting chemotactic factors that attract malignant tumour cells within the lymphatic lumen .
Cancer cells that enter the lumen of vessels or lymphatics migrate to distant tissues and form metastases in other organs. This is an extremely inefficient process as only <0.01% of tumour cells eventually form distant metastases [55, 56]. Typically, a 1-g tumour is composed of billions of cells that can shed 1–4 million cancer cells and most of these will die in the first 24 h by either attrition or direct cytotoxicity and lysis by Natural Killer (NK) cells on immunosurveillance . Liver and lung are very efficient in arresting cancer cells, essentially by size restriction. Liver capillaries are small (3–8 μm in diameter) and are designed to allow transit of red blood cells (7 μm in diameter and deformable), whereas cancer cells are quite large and usually stop in the pre-capillary vessels (20 μm in diameter) .
1.2.3 Extravasation and Secondary Tumour Formation
Cancer cells that survive and get stuck in small vessels extravasate into tissues by penetrating the endothelium. Extravasation can take place by two different mechanisms. In the first, cancer cells start proliferating within the vessel lumen . With growth, the cell walls are destroyed paving the access to the host tissue. In the second, cancer cells degrade the endothelium and basement membrane through proteolysis .
Extravasation does not necessarily result in metastasis. Most single cells that enter tissues from circulation either are destroyed by immune cells, go into apoptose or remain dormant . Some metastases can reside within the new environment without proliferating due to the absence of growth factors of the original tissue . Micro-metastases can undergo various mutations during time, giving the tumour the potential to proliferate. Once the tumour has reached a size of 1–2 mm, neoangiogenesis takes place regulating the transition between the avascular to the vascular phase . At this point, the patient faces a poor prognosis.
1.2.4 Organ Specificity of Metastases
Metastases show an organ-specific pattern of spread. For example, colorectal cancer has a propensity to metastasize to the liver while breast cancer develops brain, bone, and lung metastases. As mentioned above, other tumours metastasize to RLN and can then either enter blood circulation or keep on travelling in the lymphatic system . Stephen Paget in his 1889 paper  observed that “distribution of secondary growths (metastases) was not a matter of chance” and hypothesized the “seed and soil” theory. According to Paget’s theory, organ-specific distribution of metastases depended both on the characteristics of cancer cells (the seed) and of the secondary organ (soil). The “seed and soil” theory was later challenged by James Ewing which suggested that blood-flow patterns determine which organ tumour cells reach first . Indeed, the two mechanisms are not mutually exclusive and probably both have a role in the development of metastases [63, 64, 65]: the initial delivery of the tumour cells seems to be mechanically driven (circulatory theory) while the secondary growth depends on tumour compatibility with the host organ (seed and soil theory) . The “seed and soil” hypothesis is now widely accepted and numerous genetic and epigenetic alterations have been found that endow cancer cells with the competence to colonize distant organs . Haematogenous dissemination of pancreatic cancer cells to the liver and the development of aggressive metastases are very common and accounts for the high relapse and mortality rates of this type of cancer . Certainly, the high frequency of liver metastases from pancreatic cancer is due to anatomical reasons since blood drains to the pancreas through the portal system . It has also been hypothesized that blood entering the portal circulation follows a streamline phenomenon such that patients with carcinoma of the tail and body of the pancreas, particularly in the presence of splenic vein invasion, develop metastases preferably in the left lobe . However, the presence of a short latency in metastasis relapse and the aggressiveness of pancreatic adenocarcinoma can be explained only by genetic mechanisms . Like pancreatic adenocarcinoma, diffuse type HCC develops via intrahepatic dissemination of cancer cells through the portal vein in a short period of time .
1.3 Perineural Invasion
It was initially thought that progression along the nerves was favoured by the nerve sheath serving as a low resistance pathway for tumour cell spread . This theory was rebutted when studies with electron microscopy demonstrated that vice versa the nerve sheath is a highly resistant pathway [78, 79, 80]. Although the molecular mechanisms of PNI pathogenesis and the aptitude of certain tumours to develop a PNI are still not well understood, the main driver of nerve invasion is the symbiotic relationship between cancer and host, in which both parties facilitate the metastatic process [71, 81]. Signalling mechanisms involving tumour cells, nerve cells, and the stromal environment play a pivotal role, through the production of neurotrophins, chemokines, and proteinases, as matrix metalloproteinases, facilitating tumour invasion [82, 83].
In many hepatobiliary malignancies, PNI has emerged as a key pathological feature and a marker of poor disease outcome . PNI is a common finding in pancreatic cancer (70–100% of cases), bile duct cancer (75–85% of cases), and gallbladder carcinoma (44–72% of cases) [82, 84]. In pancreatic cancer, tumour cells extend by PNI through the plexuses from the celiac and superior mesenteric artery ganglia. In such occurrence, complete tumour removal with safe margins is difficult to achieve and is an important determinant of the patient prognosis. In a series of 72 patients with pancreatic cancer and lymph node negative disease, Ozaky et al.  reported a 5-year survival rate of 75% in patients without PNI versus 29% in those with PNI.
Mechanisms of cancer spread have not yet been fully investigated and much still remains to be unveiled. However, current knowledge on the pathways of tumour dissemination and principles of organ-specific metastatization provide imaging doctors with tools for better comprehension of findings and clinicians with useful information for treatment planning and prognostic assessment.
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