Central nervous system disorders

VI./2.4.: Issues of tumor metastizing

VI./2.4.1.: The significance and relations of metastatization

As it was already mentioned above, it is considered as a true sign of malignancy, if a tumor is capable of creating metastases with growing and surviving potential, in distant tissues. All the other, formerly mentioned histiologic and cellular tumor-characteristic, known by traditional pathology, are less important with regard to the biological behavior that eventually determines the fate of the organism. A relating feature, that is not perfectly known in all of its details, determines that individual tumors prefer to metastize in certain target organs. For this, the first possible explanation was the so called seed and soil theory. According to the second theory, location of the metastases are determinde by the direction of circulation. According to the stiffness-theory,metastasis develops in locations where the mechanical features of the given microscopical part of both the primary tumor and the target organ are almost identical.

VI./2.4.2.: Lymphogenic metastatization

Numerous factors serve the invasion of the lymphoid system by forming tumor cells that tend to disconnect from the original tumor clon and are about to wander further. Namely, in the lymphoid capillaries there is no membrana basalis (BM), their endothelium is fenestrated, their pericyta-cover is incomplete, and thus, the severing tumor cells are assisted simply by the pressure and flow of the interstitial fluid to get into the lumen of the lymphoid vessels, and to be carried to the regional lymphnodes. In the lymphatic system, tumor cells have to avoid immune-effector cells and their effect; the detailed mechanism of which is still unclear.

Tumor cells first get into the regional lymphoid vessel and lymphnodes corresponding to the location of the primary tumor, mainly – if there is such – through the sentinel lymphnode. Although, the therapeutic significance of this lymphnode is limited by the ability of tumor cells to avoid it (skipping metastasis) – for example with the help of lymphovenous anastomoses –, in clinical practice – mainly in the oncosurgery of breast cancer and mealnoma malignum –, pathohistologic analysis of the sentinel lymphnode is effectively applied in avoiding unnecessary block-dissections of lymphnodes and thus, preventing the patients from the complications of such interventions, like lymph stasis (for example, in case of breast cancer, lymph stasis or elephantiasis in the ipsilateral arm).

VI./2.4.3.: Haematogenic metastatization

The first issue of haematogenic metastatisation is the connection between the primary tumor and the blood vessels. There are multiple ways of haematogenic metastatization:

• (i) tumor cells that are already in the lymphatic system get into the blood vessels secondarily;

• (ii) tumor cells can directly invade the blood vessels of the tissue of origin;

• (iii) the tumor can create its own blood vessel system (neovascularisation), into which tumor cells can invade directly.

The first step of the process is practically the problem of local infiltration: how does the severing tumor cell gets to the first capillary from its own, original micro-milieu.

After this, the wandering tumor cell has to overcome the obstacle of pericytes around the capillary, then has to destruct the basal lamina under the endothelium, and finally, it has to „fenestrate” the lining of the endothelium. This latter is done by bioactive lipids produced by the tumor cell. These lipids make the endothelial cells transiently contracted and the tumor cell may pass through the space between the contracted endothelial cells, and get into the lumen of the vessel. There are several sources of danger for the tumor cells that got into the vascular system. Most importantly, the physical shock – mechanical destroying effect – resulting from the difference between the intravascular hydrostatic pressure and the hydrostatic pressure in the extracellular matrix. The sudden, increased hydrostatic pressure causes the immediate destruction of 95-99% of the tumor cells that got into the intravascular space.

The effect of shear forces evolving from the turbulent flow is part of this physical pressure, and the poor capacity of tumor cells for changing their shape might also play a role (although the latter is already questioned recently). For their survival, the platelet binding capacity of tumor cells is fundamental, because the platelet-cover on the tumor cell or cell group defends the cells from both the above mentioned, destructing, mechanic effects and the attack of immune-effector cells. The expression of integrins and other molecules for cell-adhesion that are partners with the cell-adhesion molecules on the surface of the platelet (e.g. the αIIβ3, αVβ3 integrins), by the tumor cell, is a necessary condition for creating a physical cell-cell connection with the platelets. Often, not only singular tumor cells, but bigger or smaller groups of tumor cells, even with proliferating forms inside, are invading the vessels (intravasation).

VI./2.4.4.: Circumstances of metastatization in the target organs

Through intravasation, tumor cells get into the anatomically determined vascular system, however, the metastatization in different organs does not depend exclusively on the anatomical relations, but on the microenvironment found or created at the site, where tumor cells get out from the vessels, as well, as how much these circumstances, under complex regulatory processes, support the tumor cells’ survival and proliferation. Tumor cells might use their own growth/anti-apoptosis factors for their settle down and creation of the conditions of their survival and proliferation, or they might induce the production of these factors in other cells, or in the matrix.

It is noteworthy, for example, that the capillary system of bones is primarily lymphoid and not blood vessel like, and thus the extravasation of the tumor cells is simple and fast. Extravasation might take different lengths of time in case of different organs, even in case of the same tumor type: in the lung for example it is fast (<1 day), in the brain (blood-brain barrier!) it is slower (2-3 days). The group of tumor-genes behind the organ-specific metastatic capacity of the primary tumor is extremely variable, based on its special characteristics. For example, for creating a lung metastasis, breast cancer cells need to express MMP1-protese, epiregulin (EGFR-ligand), COX2, and angiopoetin-L4. For the creation of cerebral metastasis, the same cancer cells need to express another EGFR-ligand (HB-EGF), and a syalil-transferase too, next to all those that have been listed above.

In case of hepatic metastases, it is most probably an important factor that HGF (hepatocyte growth factor, hepapoietin A) is one of the dominant sinusiodal cytokins of the liver. It primarily affects epithelial cells, and regulates the regeneration of hepatic cells as a multi-functional cytokin, produced by mesenchymal cells. It is already known for two types of tumors (colon cancer and melanoma of the eye) that the increased expression of the HGF receptor (c-MET) on their cells, due to the amplification of its gene, is a necessary condition for their hepatic metastatization.

Systemic factors, produced by primary tumors, may prepare the individual organs, or serve as preconditioning factors for the organs, to receive metastases, or in other words, they may help creating the „metastatic cradle”. During this process, the mobilization of a broad scale of precursor cells takes place in the bone marrow, which cells then settle in the receiveing organ as perivascular cell groups. These cells can be mesenchymal (metastatic tumor-stroma cell precursors), or endothelial precursors ( constituting elements of metastasis-vessels), creating a receptive milieu for the arriving tumor cells.

VI./2.4.5. Metastatization and dormancy

Endothelial retraction is a condition for the extravasation of tumor cells, and then follows the crossing of the basal membrane. However, tumor cells might even start proliferating under the basal membrane. It is suspected that tumor cells may stuck here and remain in a „dormant phase” for years, capable of restarting to grow and thus, being a source of late recurrence. Long term survival of tumor cells without visible growth may occur. The so called singular-cell dormancy, the angiogenic dormancy (in which angiogenesis has not yet started) and immunologic dormancy (in which case the organism’s immune system actively suppresses the growth and development of the micrometastasis to a real metastasis) can be differentiated.

VI./2.4.6.: The efficiency of metastatization

The processes above show variable efficiency, based on the characteristics of the individual tumors, and the general condition of the host. Certain types of tumors (lung cancer, melanoma) may show extreme metastatic capacity, while in certain cases of others (colon cancer), there is less metastatic capacity. The phrase oligometastasis is for expressing the latter, the limited metastatic capacity. New therapeutic tools might effectively be applied to fight against the propagation of these latter types of tumors.

VI./2.4.7.: Circulating tumor cells

An object of intensive research activity is whether tumor cells that get into the circulation carry relevant and useful informations about for example genetic lesions typical for that specific tumor, since these might even be possible therapeutic targets. This approach is promising, since it is not as difficult to retrieve these cells than retrieving sample from any tumor tissue. Recently, it has been suggested that circulating tumor cells may not only settle in other tissues but also settle back to their original, primary tumor tissue. However, this notion has not yet been clinically justified. A similar phenomenon is the issue of the significance and useability of nucleinic-acids appearing in the circulation – e.g. DNA escaping from the tumor cells.

VI./2.4.8.: Bone metastases

Those tumor cell-lines that are capable of metastizing to bones have to be capable of various mechanisms, apart from proteolysis. Specifically, they express „bone-specific” proteins (e.g. osteopontin, osteonectin, BMP – bone matrix protein) next to the components of their own matrix, and they are able to cooperate with the two main types of bone cells: osteoblasts and osteoclasts. Certain tumor cells – for example breast cancer cells – produce a parathormone-like protein (PTHRP), through which they can activate osteoblasts, and stimulate their differentiation to osteoclasts, via the RANK-ligand/RANK system. By degrading the bone matrix, osteoclasts, eventually, support the sedentation of tumor cells. Osteoclasts produce catepsin-K via the activity of RHO- and SRC signal transducing systems, with which they degrade type-II collagen.

A sign of this activity is the occurrence of NTX, a collagen peptide, in the serum. If this activity does not take place, osteoclasts may release growth factors (e.g. TGFβ, IGF) from the matrix, which in turn, support the proliferation of tumor cells. The inhibition of all of these processes – the activity of osteoclasts – is the target of RANKL-inhibitors (e.g. denosumab), the SRC-kinase inhibitors that are transmitting RANKL/RANK signals (e.g. dasatinib, bosutinib, saracatinib), the catepsin-K-inhibitors (e.g. balicatib, odanacatib), and all of the osteoclast-matrix connection inhibitor bisphosphonates. Next to the osteolytic metastases detailed above, there are osteoplastic metastases either, in which, the key-molecule is the endothelin-1, produced by the tumor cells, initiating the proliferation of osteoblasts, and the production of alcaline-phosphatase. Additionally, among others, the PDGF and the Plasminogen Activation System, regulated by the WNT signalling system, may also play a role. Endothelin-1is inhibited by for example atrasentan, or zibosentran.

VI./2.4.9.: Metastasis genes

During the treatment of tumor patients, the examined, evaluated and then removed primary tumor, based on which the later, systemic treatment is chosen, is not present in the patient anymore, because of the tumor-removal surgery that made possible the examination of the tumor tissue on the first place, but only the remained metastases are present, that we treat based on the informations retrieved from the primary tumor. In the rest of the cases, when the primary tumor is still present, metastases can already be present either, during the systemic, pharmacological treatment (for example in case of neoadjuvant treatment [targeting the decrease of the tumor mass before removal-surgery] initiated based on the results of tumor biopsy; or in case of the definitive chemotherapy of small cell lung cancer). For that very reason, currently, the key-question of clinical oncology is, to what extent data from the primary tumor may be applied for the metastases.

Currently it is still undecided whether

• (i) clones that make the tumor capable of metastizing already appear in the early stages of development of primary tumors, or

• (ii) these changes appear only in later stages.

A further question is that to what extent the gene-expression profile of the primary tumor and metastases are different, and what causes the metastatic phenotype? If the profile is different, what are those differences that are important with regard to functionality? Being aware of the individual steps of tumor progression, it is unlikely that this complex process is regulated by only few key-genes.

Nonetheless, in the past decades, a great number of metastasis genes and metastasis-suppressor genes have been identified. Detailed research and analyses of these genes have yielded conflicting results, showing that while these genes in certain tumors are, in fact, key-genes, in other types of tumors they only have a partial, or supporting role, or they even function oppositely. For example, a few products of genes that have already been the target of research but their therapeutic, or diagnostic significance is not yet clear, are listed below:

• - a CD44 adhesion molecule (especially variants containing exon V6 or V3);

• - transcription factors (TWIST, SNAIL, SLOUGH) regulating the epithelial-mesenchymal transformation (EMT);

• - NEDD9 regulating the activity of FAK-kinase;

• - transcription factors (WAVE3, β-katenin, and FOXF1) regulating cellular motility.

It might be suprising, but currently, a larger number of metastasis-suppressor gene families are identified than metastasis-gene families:

• - NM23 (codes the NDP-kinase, the first, identified metastasis-suppressor gene);

• - Genes playing role in chromatin remodelling (genes of the BMRS1 transcription factor, and CRSP3);

• - Regulator elements of signalling systems (most of the tumor-suppressor genes are in this group; e.g. KISS-1 [codes the metastin protein], GPR54 [the metastin protein receptor, a G-protein-binded receptor], Rh0-GD12, Rh0-MKK4, PEBP1 (RKIP – inhibits the NfkappaB and the RAS-MEK-ERK pathway), SSECK;

• - CTGF (regulates the half-life of matrix proteins, and integrin binding);

• - DRIP130 (transriptional coactivator);

• - E-cadherin, CD-82 receptor (KIA1 – inhibits migration and invasion) (receptors of the cell membrane);

• - TIMP family (MMP-inhibitors) and RECK, which regulates them.

With regard to metastases, genes and genetic lesions can be divided in two main groups:

• - Metastasis initiator genes are responsible for the capability of tumor cells to get to remote organs and tissues from the primary tumor. The other group,

• - the so called metastasis-maintaining genes are responsible for the survival of the tumor cell in the foreign tissue environment, and thus, to be able to create a metastatic tissue.

From a pathologic point of view, metastasis-initiator genes, and their genetic lesions, are the ones which have prognostic significance if expressed in a primary tumor.

Metastasis-maintaining genes are not necessarily expressed in the primary tumor, often, they occur only in the metastases, but anyway they can be therapeutic targets. The success of Oncotype DX, the prognostic genetic test of breast cancer, is one of the proofs that identifying these gene families have real clinical utility. However, the whole picture will be further complicated, since it is already known for example that certain micro-RNAs (metastomirs) play a significant role in influencing the behavior of tumors, including metastizing too, although their specific role is still not clear and is the subject of research.

VI./2.4.10.: Metastatic brain tumors

Majority of the metastatic tumors originate from the lung (Macropictures 1A-C), from the breast, from melanoma malignum (Macropictures 2A-C), from the colon (Macropicture 19P-3), and recently, growing numbers of metastases are from the kidneys (Macropicture 19P-4), while approximately 10-12% is from unkown origin. In the meninges, leukemic and lymphomatic infiltration are relatively frequent, the diffuse infiltration of carcinomas can lead to meningeosis carcinomatosa.

Macropicture 1A-C: Cerebral (A.) and cerebellar (B.) metastases of epithelial lung cancer. Metastases are round, with sharp borders, on the cut- surface, granularity refers to tumorous tissue. C.: Metastasis from a pulmonary adenocarcinoma, and accordingly, the cut-surface here is rather mucosus, there is a circumstantial tumor necrosis in the middle. (A favor of Péter Molnár; University of Debrecen, Medical and Health Sciences Center, Department of Pathology)

Macropicture 2A-C: Cerebral metastases of melanoma malignum. The blueblack color of the metastases subcortically (A.), and in the brain stem (B.), is pathognomic. Sometimes the original pigment color is washed out by extensive haemorrhage (C.), in these cases, haemorrhage and cerebral metastasis can hardly be differentiated. (A favor of Péter Molnár; University of Debrecen, Medical and Health Sciences Center, Department of Pathology)

A special feature of cerebral metastatization is that the structure of cerebral vessels is specific, due to the blood-brain barrier, and they are in specific relation with astrocytes and other glia cells, and according to certain oppinions, only those cells are able to adhere in theses vessels and extravasate which are extensively expressing αVβ3-integrin. For a breast cancer cell line to be able to metastize in the brain, the cellular expression of MMP1-protease, epiregulin (an EGFR-ligand), COX2, and angiopoetin-L4, HB-EGF (another EGFR-ligand), and syalil-transferase is needed.

Metastases are more unusual to form in more caudal regions of the nervous system: certain prostate cancers for example can metastize, although rarely, to the spinal cord (Macropicture 5).

Macropicture 3: Cerebral metastasis of adenocarcinoma of the colon. The cut-surface is according to the tumor type, it is mucosus, shining. (A favor of Péter Molnár; University of Debrecen, Medical and Health Sciences Center, Department of Pathology)

Macropicture 4: Cerebral metastasis of renal cell carcinoma. (A favor of Péter Molnár; University of Debrecen, Medical and Health Sciences Center, Department of Pathology)

Macropicture 5: Metastases of prostate cancer in the spinal cord. The metastatic nodes developed among the fibers of the cauda equina. (A favor of Péter Molnár; University of Debrecen, Medical and Health Sciences Center, Department of Pathology)