Tumor Invasion and Metastasis

Tumor Dissemination

• Local tumors spread to other sites, aided by genetic and epigenetic changes that promote metastasis.
• Tumor cells must break free of the primary mass via loss of adhesion and acquire migratory capabilities.
• Immune evasion is crucial for tumor cells.
• Cells must exit vessels and integrate into new tissue.
• Metastasis accounts for much cancer-related mortality.
• Can occur through lymphatic vessels, blood vessels, or direct dissemination.

Mechanisms of Tumor Invasion and Metastasis

• A series of events is required for tumor invasion and metastasis.
• These events include adhesion, migration, stromal invasion, intravasation, tumor emboli formation and extravasation.

Step 1: Adhesion

• Detachment of tumor cells occurs with penetration of the basement membrane, and entering of the ECM.
• The dismantling of intercellular adhesion structures requires requires loss of cadherins and catenins.
• Contacts are established with ECM elements within the tumor stroma.
• Integrins and other specific receptors on tumor cell membranes recognize and bind to fibronectin, laminin, collagen, and vitronectin.
• Carcinoma cells often express increased numbers of these receptors during invasion and metastasis.

Step 2: Migration

• Tumor cells migrate actively.
• Alterations in the cytoskeleton and the cellular adhesion structures mediate migration.
• Autocrine growth factors (hepatocyte growth factor or scatter factor) stimulate migration.
• Cleavage products of ECM components, including fragments of collagen also stimulate migration.

Step 3: Stromal Invasion

• Involves penetrating the basement membrane and invading surrounding tissue.
• Proteases and antiproteases produced/activated by the tumor cells play a role.
• Tumor promoting proteases include matrix metalloproteinases (type IV collagenases) and urokinase (a serine protease).

Step 4: Intravasation

• The invasion of blood or lymphatic vessels by cancer cells.
• Involves the penetration of endothelial basement membranes, cells pass between or through endothelial cells into the vessel lumen.
• Chemotactic factors produced by multiple cell types attract vessels.

Step 5: Tumor Emboli

• Once inside a lymphatic or blood vessel, tumor cells clump together forming small emboli held together by shared adhesion molecules.
• Host lymphocytes attack tumor cells or are surrounded by platelets.
• Platelets may protect the tumor embolus from immune destruction and increase metastasis potential.

Step 6: Extravasation

• Tumor cells exit from blood or lymphatic vessels.
• Site is determined by tumor cells' ability to interact with adhesion molecules on endothelial surface.
• Tumor cells pass between or through endothelial cells following attachment to vascular endothelium
• Penetration of the basement membrane establishes a metastatic site and enters the ECM.
• Metastatic sites must provide a suitable microenvironment for tumor cell growth.
• Some tumors metastasize preferentially to specific sites.
• An example of tumors that preferentially metastasize, prostate carcinomas spread to bone in both humans and dogs.

Pathways of Tumor Metastasis: Lymphatic Spread

• Most carcinomas and some sarcomas metastasize via the lymphatic system.
• Lymph nodes closest to the tumor are usually affected first, developing the largest metastatic tumor masses.
• Lymphatic spread does not always occur in an orderly manner, regional lymph nodes do not act as a barrier.
• Adenocarcinomas of the intestine usually metastasize first to the mesenteric lymph nodes first.

Pathways of Tumor Metastasis: Hematogenous Spread

• Sarcomas typically use the hematogenous route.
• Tumors usually invade veins over arteries because venous walls are thinner and easier to penetrate.
• Tumor cells entering veins ultimately reach the vena cava, pass through the heart, and lodge in capillary beds (typically in the lungs).

Pathways of Tumor Metastasis: Transcoelomic Spread

• Cancers arising on the surface of abdominal or thoracic structures encounter few anatomic barriers to spread.
• Mesotheliomas are confined to the peritoneal, pericardial, or pleural cavities, but tumor cells readily spread to cover visceral and parietal surfaces.
• Ovarian and pancreatic adenocarcinomas preferentially spread transcoelomically in humans and dogs, resulting in carcinomatosis, which is multiple tumor masses throughout the abdomen.

Transmissible Tumors

• Spread beyond the original host by physical transplantation following direct physical contact between animals of the same species.
• Devil facial tumor disease (DFTD) affects Tasmanian devils.
• Tumors isolated from multiple affected animals have essentially identical cytologic and genetic characteristics that differ from those of the hosts.
• All DFTD tumors arose from a single tumor later disseminated to multiple animal hosts.
• Canine Transmissible Venereal Tumor is also a transmissible tumor.

Genetics of Cancer: Heritable Alterations in Cancer

• Genetic and epigenetic abnormalities in cells results in cancer.
• Initial change may be either inherited germline or somatic mutations acquired from DNA damage (chemical, radiation, or oncogenic virus)
• Genetic changes include activating oncogenes (ras) and inactivating tumor suppressor genes (p53).

Genetics of Cancer: DNA Mutations

• Point mutations can alter gene product/level
• DNA strand breaks cause gene alterations.
• Insertions/deletions (single base pairs or thousands) can alter gene products.
• Amplifications result in more than one copy of a gene.
• Aneuploidy is abnormal numbers of chromosomes
• Chromosomal instability occurs with chromosomal translocation where pieces break off and reattach inappropriately.

Genetics of Cancer: Germline Mutations and Cancer Syndromes

• Germline mutations affecting oncogenes or tumor suppressor genes are heritable
• Transmitted to offspring and present in all cells of affected progeny
• Human families and genetically related animals that develop a specific spectrum of tumor types are said to have cancer syndrome
• Generally show an autosomal dominant pattern of inheritance

Genetics of Cancer: Acquired Somatic Mutations and Sporadic Cancers

• Somatic mutations are restricted to individual cells and their progeny
• Somatic mutations are responsible for sporadic tumors in general population.
• Somatic mutations accumulate over time, causing increased cancer risk w/ age
• Intrinsic metabolic processes and extrinsic mutagens cause somatic genetic alterations

Genetics of Cancer: Cancer Incidence By Age in Dogs

• Incidence of tumors per 100,000 years of life for male (green line) and female (red and blue lines) dogs.
• Tumor incidence in female dogs is shown both for all tumors (blue line) and for all tumors with mammary tumors excluded (red line).
• In female dogs, mammary gland tumors explain a large portion of the total tumor incidence, especially if intact.
• Tumor incidence increases with age in both sexes until the age of 11 years.

Genetics of Cancer: Epigenetic Changes in Cancer

• The term epigenetic refers to a heritable change in gene expression in somatic cells from something other than a change in the DNA sequence.
• DNA methylation involves the addition of a methyl group
• DNA methylation is essential for regulating gene expression in health.
• Hypomethylation of genes leads to gene activation and hypermethylation results in gene silencing.
• Cancer cells show overall lower levels of methylation, described as global hypomethylation.
• Histone Code is where posttranslational histone modifications play an important role in determining which genes and to what level they are expressed
• Euchromatin (open formation) is more likely to undergo transcription.
• Heterochromation (compact formation) is inaccessible to transcription.
• Histone acetylation results increased transcription of the associated gene and in a more relaxed chromatin configuration.

Molecular Determinants of Cancer

• Driver mutations: mainly responsible for tumor development
• Includes Tumor suppressor genes and oncogenes.
• Multiple changes of genetic and epigenetic nature are needed to transform a normal cell into a tumor cell.

Genetics of Cancer: Oncogenes

• Proto-oncogenes are normal cellular genes that regulate growth and differentiation.
• They encode to cell cycle regulators, DNA-binding proteins, growth factors and their receptors, protein kinases involved in signal transduction etc.
• Proto-oncogenes are termed oncogenes if activated by overexpression or mutation.
• These oncogenes drive proliferation and render cell unresponsive to normal growth inhibitory signals, causing tumor formation.

Genetics of Cancer: Model of RAS Action

• Normal cell: inactive RAS becomes activated in its active state when stimulated through a growth factor receptor.
• Activated RAS activates RAF-1 which stimulates signaling through MAPK pathway -> drives cell cycle progression.
• Cancer cells: mutant RAS proteins are continually stimulated with continual cell cycle progression because they cannot be inactivated.

Genetics of Cancer: Tumor Suppressor Genes

• Suppressor genes regulate the cell cycle, apoptosis, DNA repair, and other fundamental pathways
• When inactivated via genetic or epigenetic changes, uncontrolled cell proliferation and tumor growth occur
• Required for both alleles of a tumor suppressor gene to undergo mutation. Known as Knudson's "two-hit” hypothesis.

Genetics of Cancer: Retinoblastoma

• Sporadic form involves two normal copies of Rb gene and "two hits" to inactivate.
• Familial form: Born with one inactivated copy of the Rb gene

Genetics of Cancer: Haploinsufficiency

• Inactivation of only one copy for certain genes (haploinsufficiency) is sufficient for tumor growth.
• Many tumor suppressor genes are key components of cell cycle
• p53 or "Guardian of the Genome”: DNA-binding protein that stops cell cycle to allow for DNA repair and signals for apoptosis.
• The p53 protein is most commonly inactivated, by mutation, in more than half of human cancers

Genetics of Cancer: Multistage Carcinogenesis

• Certain tumors follow an orderly morphologic progression through premalignant to malignant -> invasive/metastatic disease.
• Colorectal cancers show this orderly progression through premalignant stages.
• The initiating event is the loss/mutation of the APC, which leads to adenoma formation
• Activating mutations of ras oncogene and subsequent loss of tumor suppressor genes follow this initial mutation
• This leads to emergence of malignant carcinoma.

Mechanisms of Carcinogenesis

• Intrinsic Factors: by-products of ordinary cell metabolism (E.g ROS) or changes to DNA or chromosomal abnormalities resulting in decrease telemere length/activity

• Extrinsic Factors: include chemical and physical environmental agents and oncogenic viruses interact with DNA causing cancer. -Mutagens that create DNA damage and cause mutations, -Carcinogens are ultimately the agents that cause cancer.

Carcinogenesis: Chemicals

• Wide variety of chemicals can be carcinogenic
• Ptaquiloside (toxin in bracken fern) causes bladder cancer in cattle
• Direct-acting chemical carcinogens: effective in their entered form
• Indirect-acting carcingoens (or procarcinogens): Requiring metabolic activation by cellular enzymes of the liver to form ultimate carcinogens

Carcinogenesis: Radiation

• Complete carcinogens: All forms of radiation which can initiate and continue to promote tumorgenesis

• Secondary tumors: can arise at formerly treated cancer sites

• Direct radiation: the damages caused include primarily single- and double-strand breaks and base elimination.

• UV radiation: component of sunlight exposure cause cancer in nonpigmented/hairless animals, ears of white cats + conjunctiva of Hereford cattle.

Carcinogenesis: Viruses

• Viruses cause cancer when called oncogenic viruses
• Use a large array of direct and indirect mechanisms to induce these cancers

Cancer Prognosis

• Prognosis is determined by tumor type, grade, stage and completeness of surgical excision.
• Prognosis helps clinician develop the most appropriate treatment plan for the patient.

Tumor Diagnostics and Prognosis: Histopathologic Diagnosis

• A definitive diagnosis obtained by:
  • Standard histopathologic evaluation of tumor biopsy.
  • Cytologic studies of tumor aspirates. -Cells are scrutinized for these features of malignancy: -Abnormal morphologic features -High mitotic index -Presence of abnormal mitoses -High nuclear to cytoplasmic ratio -Evidence of invasion or metastasis. -Degree of differentiation -Malignant neoplasms are frequently poorly to moderately diffrentiated
• Immunohistochemistry used for specific cell markers to aid in diagnosis (e.g. determination of B-cell vs T-cell lymphoma)

Tumor Diagnostics and Prognosis: Grade

• Assigned by a pathologist
• Indication of how similar or dissimilar the neoplastic cells are to normal counterparts: provides some indication about biologic behavior
• All grading schemes assess differentiation of tumor cells.
• Well-differentiated (very similar to normal cells)
• Moderately differentiated (somewhat more similar to normal cells)
• Poorly differentiated (anaplastic) cells translates to categories: -Grade I = Low -Grade II = Medium -Grade III = High.

Tumor Diagnostics and Prognosis: Tumor Stage

• Assigned by a clinician
• Indication of extent of tumor growth and spread in the animal.
• Guides how the clincian will develop a therapeutic plan, and gives some means to estimate patient's prognosis
• TNM system is used:
  • T0-4 = size of the primary tumor
  • M0-3 = degree of lymph node involvement
  • M0-2 = extent of metastasis

TNM System Breakdown:

-Stage O - carcinoma in situ -Stage I - invaded a little into tissue of origin -Stage II - invaded into that tissue extensively -Stage III - spread to local lymph nodes -Stage IV - extensive metastasis throughout the body