POM B Oncology

Tumour Biology

• Differentiation: process in which precursor stem cells change into specialize cellular types with different properties.
- Controlled by the selective and sequential inactivation of genes which cause cell proliferation and activation of genes which produce specific cell product, e.g. for red blood cells, genes for haemoglobin production is active but replication and mitotic ones are inactive (nucleus is extruded)
- Differentiation of tissue: arrangement and specialization of groups of cells (tissue) into organs that occurs during embryonic stage, e.g. migration of dermatome cells to form skin
- Differentiation of cells: specialization of individual stem cells into specialized cells within tissues. Occurs throughout life, e.g. regeneration of skin cells
• Agenesis: total congenital failure of a part or organ to develop, e.g. unilateral renal agenesis. This is usually compensated by the hypertrophy of the complimentary solitary kidney.
• Hypoplasia: partial failure of an organ to develop (congenital reduction in size)
• Atrophy: acquired reduction in the size of organs due to factors such as ageing (heart), failure of endocrine stimulation (endomentrium after menopause), or disuse (muscular degeneration).
• Hypertrophy: increase in size of organ due to increase number or size of cells OR increased size of individual cells (applies to permanent cells such as cardiac).
- Hypertrophy is usually an adaptive process that can become pathological , e.g. ventricular hypertrophy
• Hyperplasia: increase in tissue mass due to an increased number of cells as an result of imbalance between cell death and proliferation.
- Physiological: calluses on hand due to heavy manual work
- Pathological: hyperplasia of thyroid gland due to abnormal stimulation of immune system.
- Can be premalignant as the higher rate of cell proliferation allows any carcinogenic stimulus (mutated cells) to be amplified.
• Metaplasia: changing from one mature tissue type to another. This is usually an adaptive process such as Barrett’s oesophgus in which regurgitation of gastric contents into oesophagus causes it to change from squamous to columnar epithelium.
- Epithelial metaplias carry a slightly increased risk of malignant transformation due to genetic abnormalities acquired in these dysplastic changes, e.g. bronchial carcinoma is squamous cell carcinoma arising in metaplastic squamous epithelium
• Dysplasia: genetic alteration in the cells that gives rise to premalignant formations through loss of TS gene or acquisition of oncogene. This is commonly seen in epithelial metaplasia (e.g. cervix) and possible to be eradicated to prevent development of cancer
• Carcinoma in situ: benign carcinoma at the most extreme end of the dysplastic spectrum before it becomes fully malignant.
• Regenerative atypia: cell injury and death associated with inflammation or ulceration results in a regenerative response in remaining cells that show dysplastic characteristics such as nuclear enlargement, hyperchromasia (more chromatin stained), pleomorphism and mitotic activity.
- Nuclear change is not permanent and reverts if injury agent is removed. Distinguish of regenerative atypia from true dysplasia may required repeated biopsy but is made difficult due to the occurrence of true dysplasia and regeneration together during inflammation.
• Neoplasia: cells undergoing uncontrolled autonomous growth independent from stimulus.
- Major feature is monoclonality in which all cells have originated from single parent cell.
• Benign neoplasia:
- Do no metastasize
- Cells are differentiated
- Grow slowly in an expansile non-infltrative fashion
- Rarely fatal
• Malignant neoplasia
- Potential to metastasize
- Cells are less differentiated and mature
- Usually grow rapidly or high cells turnover
- Invade host tissue in an infiltrative fashion (ulcerative)
- fatal
• Structure of tumours: benign and malignant tumours are composed of both neoplastic cells and connective tissue framework from the host term tumour stroma.
• Tumour stroma: stroma is formed in response to signal molecules (growth factors) from neoplastic cells which stimulate fibroblasts etc to produce collagen fibers, and matrix material.
- Some tumour can produce their own tissue matrix such as osteosarcomma producing bone.
- Blood vessels are formed as result of tumour angiogenic factors
- Lymphatics are gained at the edge of the infiltrate and not at centre due to interstitial pressure
• Tumour vasculature: continual process of angiogenesis as the tumour grows and occludes other vessels. Resultant structure is highly disorganized with mixture of poor and rich blood flow areas.
- Areas of poor flow will suffer necrosis and this is an important characteristic of malignancy as necrosis is absent in benign tumour but more frequent in high grade tumours
- As tumour expands and infiltrate tissues, pre-existing arteries and veins are incorporated into the tumour mass, becoming enlarged to cope with flow.
• Hypoxic cells: tumour cells that reside near necrotic centres and are deprived of oxygen but viable. These develop as a result of occluded blood flow from interstitial pressure of tumours.
- Resistant to radiation
- Resistant to chemotherapy as some therapeutic agent needs oxygen for activity
- Non-proliferative so resistant to agents that inhibit division
- Further from vessels so resistant against slow diffusing drugs

Genes and cancer

• Cancer: uncontrolled proliferation of cells due to genetic changes and mutation occurring in the genes critical for control of cell growth and death
• Causes of cancer: imbalance in the homeostasis of cell growth and death and the genetic changes can either be acquired or inherited or both.
- Environmental factors that induce cancer include diet, smoking UV radiation
- Inherited mutation may predispose to the development of cancer followed by a second somatic mutation
• Genes of cancer:
- oncogenes: genes which promote cell growth
- tumour suppressor genes: genes which inhibit cell growth
- apoptosis inhibitor: genes that block the normal pathways of cell death
- DNA repair genes: genes coding for enzymes which fixes DNA mismatches that occur after DNA synthesis and replication.
• Oncogene: dominant transforming genes termed c-onc in human genome and most are involved in signal transduction pathways of controlling cell growth and development. Oncogene normally presents in inactive proto-oncogen forms but when activated undesirably to onocogens, causes normal cells to become neoplastic cells (loss of differentiation).
• Proto-oncogene function: encodes proteins that are critical in the control of normal cell growth. Can be divided into four dominant groups
- Growth factors: sis oncogene encodes for a part of PDGF
- Growth factor receptors: a specific receptor for PDGF
- Signal transmission pathways, e.g. the ras family of oncogenes
- Nuclear transcription factors: myc
• Activation of oncogenes:
- Gene amplification: increased copy of the oncogene cause over-expression and activity of the gene, e.g. n-myc amplification in childhood neuroblastoma
- Inappropriate expression: wrong timing and excessive expression, e.g. Burkitts lymphoma
- Point mutations: change in single base of the gene, e.g. ras point mutations seen in leukaemia, colon cancer and pancreatic cancers
- Gene arrangement and translocation: structural changes unite two chromosomes to form either a new oncogene or influence one of the gene to increase expression, e.g. myeloid leukaemia
- Epigenetic mechanism: loss of methylation allowing silenced gene to become active.
• HER-2: proto-oncogene that encodes for EGF (epidermal) receptor in mammary cells. Amplification of the HER-2 gene over expresses the receptor and causes cancer with EGF stimulation.
• Burkitt’s lymphoma: myc gene on chromosome 8 is juxtaposed with immunoglobulin gene on chromosome 14 (heavy chain). Upregulation of myc gene by neighbouring promoter cause cancer
• Tumour suppressor genes: genes coding for inhibitory protein that halt and control cell proliferation. Cancer can arise from the loss of activity of these genes, usually both alleles. Functions include:
- regulation of cell cycle
- growth inhibition by cell to cell contact
- repair DNA damage
- maintain genome integrity
• Discovery of tumour suppressor genes:
- Loss of heterozygosity (loss of allele function) is common in human tumours
- Inherited cancer syndromes, e.g. retinoblastoma where inheritance of a defective tumour suppressor gene causes cancer following a second somatic hit inactivating the other allele.
- Fusion of tumour with normal cells reverse “abnormal growth”
• Mechanism of loss of TS gene: all require double hit/mutation both alleles for loss of function
- Point mutations
- Loss of heterozygosity
- Altered methylation
• Apoptosis genes: genes involves in regulating pathways of apoptosis. This will cause a slow accumulation of malignant cells. Relevant cancers include indolent lymphoma and chronic lymphocytic leukemia
• Mechanism of cancer through loss of apoptosis:
- Loss of expression of genes which mediate apoptosis, eg. Fas and p53
- Up regulation of genes that block apoptosis, e.g. up-regulation of bcl2 gene when it is translocated from chromosome 18 to chromosome 14 next to the immunoglobulin gene creating B-cell lymphoma
• DNA repair mechanism: no repair of DNA error for a critical cancer gene will predispose cancer (multi-genes are needed for cancer development). E.g. familial colorectal cancer
• Multistep Evolution in Cancer: somatic mutation gives a cell a growth advantage and its daughter cells will be genetically identical with the same proliferative advantage. With progressive mutations, changes will accumulate until enough have occurred to allow the cell to become invasive, induce angiogenesis, and metastasize.
• Chronic Myeloid leukemia: cancer due to proliferation of myeloid cells in the bone marrow. Radiation exposure is the only known environmental risk factor.
- Diagnostic features: high white cell count, splenomegaly, presence of the Philadelphia chromosome in the bone marrow
- Clinical course: median survival of 3.5 – 4 years. Has three phases: chronic, accelerated, terminal blast crisis
- Genetics: Philadelphia chromosome
• Philadelphia chromosome: acquired cytogenetic abnormality that arises in a bone marrow stem cell and is found in all the blood cells (not found in non-haemotopoietic elements). The chromosome is a reciprocal translocation between the long arms of chromosome 22 and 9
- Reciprocal translocation brings the c-abl gene on chromosome 9 to bcr gene on chromosome 22, producing the bcr-abl fusion gene which is a potent tyrosine kinase that activate cell proliferation
• Testing: analysis of the Ph chromosome can be done by cytogenetics or molecular techniques such as PCR to monitor response to therapy and identify potential target for therapy
• Treatment of CML:
- bone marrow transplantation though only 20% of patient have matched donor or at a safe age
- targeting bcr-abl fusion protein for therapy as it is only found in leukemia cells and is the key event in development
- Glivec drug (imatinib) inhibits the c-abl tyrosine kinase and suppresses the growth of leukemia cell lines. It is available orally and in phase II patients.
• Features of Glivec: tablet based therapy with mild and predictable side effects.

Familial Cancer

• Familial cancer: inheritable cancer
- Many close relatives with cancer (at least one first degree)
- Cancers in successive generation
- Young age of onset
- Multiple cancers in same patients or within family
• Amsterdam Criteria: set of condition to assess the familiarity of the cancer, e.g. in hereditary non-polyposis colorectal cancer. Criteria includes:
- An individual has 3 or more relatives with an HNPCC related cancer
- Involves at least 2 generations
- One of the relatives must be first degree with others
- 2 or more diagnosed with cancer before 50 years of age
• Molecular basis of familial cancer: germline mutations of genes are already present in one allele i.e. oncogenes and tumour suppressor (mainly affected) and result is similar to sporadic acquired cancers,
- Somatic: germline mutations of Met (transmembrane receptor for hepatocyte growth factor) associated with hereditary papillary renal carinoma
- Tumour suppressor: inherited germline mutation inactivates one alleles with second acquired mutation eliminating activity of the second allele leading to loss of function of TS gene
- Oncogone: example RET (Rearranged during transfection), mutation of gene coding tyrosine kinase receptor is associated with MEN type 2 medullary thyroid cancer.
• Two hit theory: example with Retinoblastoma - embryonic tumour of eye
- Due to abnormalities of the TS gene, Rb. Familial forms due to inherited mutation of 1 Rb allele followed by the 2nd somatic mutation of the other allele. Sporadic forms involve two somatic acquired mutation of each Rb allele with a normal germline.
• Familial bowel cancer: autosomal dominant disease with two main subtypes
- Hereditary non-polyps colorectal cancer
- Familial adenomatous polyposis
• HNPCC: approximately 85% risk
- Aetiology: inherited abnormality of a DNA mismatch repair gene such as MLH1, MLH3, MSH2, MSH6, PMS1, PMS2. Mutation/inactivation of both allele can predispose cancer by accelerating progression from adenoma to carcinoma, and increase replication error in microsatellite regions (microsatellite instability)
- Clinical features: colorectal and endometrium cancer along with other malignancies such as ovarian, renal and GI tract
- Diagnosis: Amsterdam criteria, DNA analysis for microsatellite instability, loss of expression of mismatch repair genes (immunohistochemistry gene) and mutational analysis (i.e. gene sequencing) can all be used
- Management: regular screening and genetic testing for the gene, changing diet
• Familial adenomatous polyposis: multiple polyposis and adenoma with very early onset (2nd decade)
- Aetiology: Inherited mutation cause truncations of APC (adenomatosis polyposis coli) gene on 5q prevent function of protein to bind to and degrade beta-catenin (a transcription activator). Second hit often required such as hypomethylation
- Clinical features: present in adolescence with hundreds of colonic polyps (potential to transform to cancer)
- Management: identification of individuals and families with possible FAP and offer genetic counselling and testing, cancer screening. Colectomy at early stage before cancer develop
• Familial breast and ovarian cancer: autosomal dominant inheritance giving around 80% risk of breast cancer and 45%/30% chance of ovarian cancer for BRCA1 and 2 respectively
- Aetiology: germline mutation of either BRCA 1 or BRCA 2 gene (TS genes) produce truncated proteins involved in DNA repair
- Clinical features: early onset of breast ovarian cancer and can affect multiple family members. Both cancers at breast and ovary can occur.
- Management: consider genetic counselling and mutation analysis afterwards. Testing of individuals at risk within a family with mutation, e.g. screening mammography, pelvic ultrasound and tumour markers etc.
• Males with BRCA gene: increased risk of prostate cancer by 6% with BRCA 1 and 2 and risk of breast cancer with BRCA 2 only.
• BRCA gene: mutation of BRCA greatly increases the risk of cancer, even more than family history. It does not present with 100% penetrance
• Other breast cancer: inherited syndromes include Li Fraumeni Syndrome (P53 mutation) and Cowden syndrome.
• Issues with cancer predisposition testing: ethical, legal and social aspects
- Potential for discrimination, e.g. employment, insurance
- Alienation, discrimination and loss of spiritual integrity
- Guilt feeling
• Advantages of genetic testing for familial breast cancer:
- Empowers patients to make choices
- Allow follow-up screening such as mammography and tumour marker testing
- Prophylatic surgery to remove the breast and ovary

Environmental Basis of Cancer

• Cancer induction experiments:
- Application of only inducer (no tumour)
- Application of repeated promoter (no tumour)
- Application of repeated promoter after inducer (tumour)
- Application of repeated promoter before inducer (no tumour)
- Application of repeated promoter a long time after inducer (tumour)
- Application of promoter with long interval separation after inducer (no tumour)
• Deductions: above experiments show that
- Initiator and promoter synergise induction of tumour
- Initiator has to be applied prior to promoter for tumour development
- Effect of initiator is irreversible
- Effects of promoters are reversible
• Initiators: factors that alter DNA sequence and permanently mutate the cells conferring potential carcinogenic damage
• Promoters: factors that damage proteins and affect cellular signal pathways (gene expression) temporarily and reversibly thus stimulating outgrowth of “initiated” cells.
• Complete carcinogens: cancer-inducing factors that is comprised of both the initiator and promoter
• Mouse squamous cell carcinoma model: application of PAH (polycyclic aromatic hydrocarbons) as the initiator followed by phorbol ester which is the promoter induced papilloma to grow on the back of mice. These eventually transformed into squamous cell carcinoma
• UV light radiation carcinogenesis: associated with basal cell carcinoma, squamous cell carcinoma and melanoma. Two components are:
- UVB: a complete carcinogen with smaller wavelength and weak tissue penetrance but causes sunburn and damages DNA
- UVA: a promoter with higher wavelength and strong tissue penetrance. Though it does not cause sunburn but generates reactive oxygen species
• UV Initiator: UV light causes mutation of the p53 tumour suppressor gene with C to T transitions (especially the dipyrimidine sequences) and CC to TT tandem changes.
- Adjacent pyrimidine bases absorb UV radiation forming cyclobutane dimers C<>C
- Oxidative deamination of C generates U <>U
- Repair of U<>U forms U-U (behaves like T-T)
- With DNA replication, the U-U pairs with A-A hence permanently changing the DNA sequence
• UV promoter: altered DNA sequences will hinder or inhibit function of p53 to activate apoptosis of damage skin cells due to sunburn, i.e. peeling of skin and thus creates conditions favorable for mutants types
- Cells that suffered initiator damage of one allele of the p53 gene will be “tolerant” to apoptosis (due to defective protein) and hence survive while normal cells will die
- Successive exposure to UV light will expand the pool of mutant p53 cells (heterozygotes). Actinic keratoses are patches of p53 heterozygous cells on the skin.
- Eventually after repeated episodes of sunburn, the other p53 allele of the cell could be mutated creating homozygous malignant cells with loss of apoptosis
• Tobacco chemical carcinogenesis: associated with lung cancer especially adenocarinoma of mucus surfactant cells, metaplastic squamous cell carcinoma, anaplastic large cell adenocarcinoma and bronchial endocrine cells
- Benzapyrene: complete carcinogen
- Nicotine derivative: NNK (nitrosaminoketone) is a complete carcinogen
• Tobacco initiator: NNK causes G to A mutations in lung cells
- NNK is hydroxylated by cytochrome P450
- Intermediate breaks down to form methyldiazohydroxide H3C-N=H-OH with an electrophilic methyl group
- The methyl groups attaches to the O6 atom of G and the product O6-methyl G behaves like A.
- DNA replication will pair up the mutated G with T (which later on pairs with A) thus a G to A transition
• Evidence of initiator effect:
- Non-smokers with lung cancer due to cooking fires (high PAH) have frequent G to T transversions
- Smokers lacking the repair enzyme to repolarize O6-methyl G have frequent G to A transitions
• Tobacco promoter: nicotine and derivatives act as signaling molecules to promote cancer development.
- NNK is beta-adrenergic receptor agonist which induce cell division, suppress apoptosis via adenylyl cyclase and activate Ras oncogene
- Nicotine binds to nACh receptors in brain to cause addiction, adrenal medulla to stimulate release of catecholamine and beta-adrenergic receptor activation, induce lung epithelial cell proliferation, suppress apoptosis of cancer cells and stimulate blood vessel development.
• Viral carcinogenesis: associated with hepatocellular carcinoma
- Aflatoxin B1: fungal metabolite in poorly stored food is an initiator
- Hepatitis B virus: act as promoter
• Viral initiator: mutation of the p53 gene at codon 249 AGG to AGT (G to T transition)
- Metabolite of aflatoxin alkylates the guanine to thymine and mutations gives the host cell a growth advantage in the presence of a promoter, leading to selective proliferation of mutant clones
• Viral promoter: chronic hepatitis virus creates conditions to promote rapid cellular replication and potentiate carcinogenicity with the HBX protein
- Viral infection causes necrosis and inflammatory response that stimulate rapid regeneration and replication of cells, thereby expanding mutant cell types and minimize repairing.
- HBX protein inhibits DNA repair
- HBX binds to p53 and remove it from the nucleus (impairing its function)
- HBX indirectly activates Ras oncoprotein.

Cellular Basis of Malignancy

• Epithelial to mesenchymal transition: progression of normal epithelial cells that adhere to one another and their basement membrane to loose and motile carcinoma cells freed into the ECM
• Adheren junction: intercellular adhesion units made up by E-cadherin, beta-catenin and alpha-catenin.
- E-cadherin is a calcium dependent junction and the molecule penetrate membrane to link to cytoskeleton
- Alpha and beta catenin are the linking molecules to the cytoskeleton
- Carcinoma demonstrates loss of adheren junctions and function
• Aetiology of diffuse cancer: irreversible EMT producing dissociated tumour cells
- Mutational inactivation of the E-cadherin gene, e.g. diffuse gastric carcinomas
- Methylation of the E-cadherin promoter
- Mutation of the alpha-catenin gene
• Focal dissociation: concentration of dissociated malignant cells in only one area (usually the invading margin) of a solid tumour. The cause is due to stimulation of growth factors leading to reversible EMT and loss of adheren junctions.
• Role of growth factors: cancerous cells can express their own growth factor and promoter as a self-driven mechanism to growth and invade. A common influential GF is the HGF (hepatocyte growth factors) whose receptor is a transmembrane tyrosine kinase. Its functions are:
- Activates Ras oncoprotein and induces proteins that repress E-cadherin gene
- Phosphorylate E-cadherin and beta-catenin causing them to degrade and dissociate from junction
• Mechanism for excessive HGFR:
- Over-expression due to altered regulation in late cancers
- Gene amplification
- Point mutation which increase receptor activity and activation
- Co-expression with HGF producing autocrine loops
• Mechanism of invasion: hydrolytic enzymes allow cancer to break out of tissue compartment. Proteolytic and cell adhesion molecules are assembled on the tumour cell surface protrusions (podosomes). Intracellular signaling and positive feedback maintain invasiveness. Two systems are:
- Urokinase-type plasminogen activator
- Matrix metalloproteinases
• Urokinase-type plasminogen activator: a serine protease that is upregulated by cancer cells and binds to a high affinity receptor uPAR to cleave plasminogen to plasmin. Active plasmin can cleave:
- Pro-uPA to uPA (positive feedback)
- Pro-MMP to MMP’s
- Pro-HGF to HGF which stimulate Ras and transcription factors to upregulate uPA and uPAR (and some MMP)
- Plasmin can also degrade fibres and ECM tissue.
- Ras increase expression of snail which suppress E-cadherin
• Matrix metalloproteinases: proteases that degrade components of the ECM with Zn2+ at their active site. These are secreted in a latent form by stromal fibroblasts or inflammatory cells (leukocytes) and are concentrated at the edge in the direction of migration. Functions include:
- ECM degradation: carves out a pathway for cell migration and free bound GF to cause inflammation
- Laminin 5 degradation: release chemotactic fragments which promote migration
- E-cadherin degradation: suppresses epithelial cell adhesion
- Latent growth factors degradation: degrades binding proteins to release growth factors
• Cancer environment: cancers often outgrow their blood supply, creating outer regions of hypoxia and necrosis.
• Features of hypoxia cancer cells:
- Resistance to radiotherapy: oxygen is required to convert radiation damage to lesion in DNA
- Resistance to chemotherapy: cessation of proliferation
- Stimulate angiogenesis: activation of hypoxia-inducible factor (HIF1-α) leads to production of VEGF
- Stimulate tumour aggressiveness: p53 gene is induced under hypoxia and induced apoptosis so mutated cells without p53 function will pose survival advantage and be selected for
- Risk of metastasis: HIF1-α induces HFGR which stimulate invasion
• Tumour angiogenesis: oxygen can only diffuse 200 µm, hence cancer only grow by invading, recruiting or forming new blood vessels. Triggers of angiogenesis include:
- Metabolic stress, e.g. hypoxia means HIF1- α does not bind to pVHL and self-degrade, hence promoting angiogenesis
- Inflammation
- Activation of oncogenes or loss of TS genes which control angiogenesis regulators
• Mechanism of angiogenesis:
- Angiogenic sprouting: appropriate stimulus (e.g. VEGF) cause endothelial cells to degrade a patch of the basement membrane and invade the blood vessel. Proliferating cells migrate behind the zone of degradation, following differentiation to form a lumen. Tubes coalesce into capillary loops.
- Co-optation of existing blood vessels: tumours such as astrocytomas initially grow around existing blood vessels but when it develops to highly malignant glioblastomas, blood supply is overwhelmed leading to massive cell loss. Remaining tumor initiate angiogenesis
- Intussusception: columns of tumour grow into the lumens of pre-existing vessels and physically forcing them to remodel and expand, i.e. reform around the tumour
- Incorporation of EC precursors: haemangioblasts home into tumours from the bone marrow via blood and differentiate into ECs, smooth muscles (PDGF induced) and pericytes.
- Capillary formation: trans-differentiation of cancer cells allows them to express characteristics of ECs and form capillaries. Cancer capillaries lined by mosaic of EC and cancer cells and can be shed into circulation.
• VEGF: expressed generally by hypoxic cells and cancer following the loss of p53. It stimulates tyrosine kinase receptor, promotes EC proliferation and increases blood vessel density and induces vascular permeability.
• Inhibitors of angiogenesis: usually fragments of larger proteins, such as angiostatin (fragments of plasminogen), endostatin (fragment of collagen XVIII) are released by primary tumours to suppresses angiogenesis of metastases.

Lecture 50: Tumour and the Immune System
• Immune response to cancer: premalignant and malignant cells are kept under control by the immune system. This can be show as compromise of the immune system leads to an increased risk of cancer, e.g. transplant patient taking immunosuppressive drugs are at increased risk of malignant lymphoma.
• Human Tumour-associated antigens: antigens that are expressed only in tumours intrinsic to human body. These are present either on surface or intracellularly. Antigen such as melanoma can cause strong immunogenic T lymphocyte response.
• Innate immune system: non-adaptive components of the body immunity (i.e. non-specific). It is capable of regulating immune response and communicates with adaptive immune system by secretion of cytokine, chemokine and interleukin. Natural killer cells are the most important against tumour.
• Natural killer cells: cytotoxic cells that do not require a specific antigen to kill. They are specific to tumour cells and infected cell and also contribute to immunoregulation by secreting influential lymphokines.
• Mechanism of NK action: antibody-dependent cell mediated cytotoxicity
- Release of granules containing perforin and granzyme that inserts holes in the target cell’s membrane causing apoptosis (primarily in spleen and lung). IL-12 and BCG activates NK cells to destroy tumours.
- Death receptors ligands such as Fas ligand, TNF and TRAIL which bind to death receptors expressed on cancer cells (e.g. Fas) and mediate apoptosis.
- Secretion of effector cytokines such as IFN (interferon), TNF-gamma which recruit granulocytes and macrophages. IFN upregulates MHC I and II molecules and activate T-cells to which enhances adaptive immune system and block angiogenesis (necrosis of tumour).
- NK cell have inhibitory receptors called killer inhibitor receptors that when bound to the same MHC class I molecules, have their cytotoxicity and cytokine function blocked – termed HLA restriction. Tumours that down regulate MHC I as an evasive measure are then susceptible to NK killing.
• Adaptive immune system: antibody centralized immune system in which T-lymphocytes aid clonal expansion of cells that are specific for the tumour cell.
• Dendritic cells: antigen presenting cells or macrophages that take up apoptotic bodies derived from tumor cells and are processes and peptides are presented on MHC class II molecules.
- Licensing: activation of dendritic cells from the antigen and the presence of danger signals by CD4+
• Toll like receptors: single germline encoded transmembrane receptors that recognize and bind pathogen-associated molecular patterns which are small molecules that are consistently found on all pathogens.
- Action: these are expressed on dendritic cells and once bound, interpret the molecule in context of a danger signal, e.g. LPS binds to TLR4, and activate the APC (license) to present the tumour peptide on MHC class II and express co-stimulatory molecules CD40, CD80, and CD86.
• T-lymphocyte function:
- Crossing priming: dendritic cells act as a messenger between CD4 and CD8, i.e. T-helper cells provide costimulator molecules that activate the dendritic cells to present other epitopes of the antigen to cytotoxic T cells.
- Cytotoxic T cells: cells that mediate killing of any cells bearing the specific antigens it recognizes (selected through MHC class I)
• T-regulatory cells: T- cells that block the activity of helper T cells after clearance of the disease to prevent autoimmunity.
• B-lymphocyte activation: antigen is presented to CD4 cells which activate corresponding B-cells with specificity for the same antigen to replicate.
• Tumour cell evasion: tumours grow as they are able to evade the immune response. Mechanism include
- No induction of pro-inflammatory cytokines required to licence APC
- “Self” cells and express MHC class I, so resistant against NK.
- Tremendous genomic instability to change antigen and characteristics to evade immunity
- Lack Fas receptor, so Fas proteins release by T cells binds to T-cells own receptors and cause suicide
• Tolerance: T cells become anergic as a result of lack of co-stimulatory molecules on APC.
- Tumour cells secrete substances to cause APC to produce IL-10. IL-10 suppresses T-cell activity and hence no CD8 and inflammation
- Increased level of T-regulatory cells in cancer which block autoreactive T-cells to replicate
• Immunotherapy: vaccine used to reduce cancer however tumour can become tolerant through natural selection.
- Interleukin 2 and BCG: IL-2 activates T cells and cause proliferation. BCG upregulates macrophages.
- Passive transfer of antibodies: humanized monoclonal antibody engineered against antigens on B lymphocytes which are clonal cells in non-hodgkins lymphoma. Death of target cells due to activation of complement pathway and apoptosis.
- Dendritic cell vaccine: dendritic cells which have been given cancer antigens in context with danger signals will be activated and can be applied directly to a patient or incubated with cytotoxic T cells.
- Cytotoxic T cell: generated after exposure to activated APC and grown in labs and be given back to the patient.
• Effective procedure of vaccination:
- Optimally present relevant tumour antigen
- Manipulate immune regulation to facilitate CTL response
- Prevent CTL neutralization at site of tumour

Neoplasia – Grading, Staging and Classification

• Diagnosis of cancer:
- Pathology
- Neoplasticity
- Malignancy
• Cytological features of cancer: observation with a microscope after cells are smeared on to a slide
- Increased nuclear staining (hyperchromasia)
- Increased nuclear size (up to 2:1 ratio of nucleus to cytoplasm)
- Variation in nuclear shape (pleomorphism - normal nucleus all appear the same)
- Mitoses (visible spindle fibers)
- Decrease cell cohesion (falling apart)
- Example: leukemia with neutrophils been darker and larger, chromatin is coarse.
• Architecture feature of cancer:
- Disorganized
- Invasion of normal tissue (infiltration)
• Microscopic features of cancer: monoclonality are strong indications for neoplasm
• Banding test:
- Kappa light chain: increased mono-clonal band from inflammation to neoplastic
- Gamma light chain: decreased mono-clonal band from inflammation to neoplastic
• Tumour type diagnosis: exact classification of the tumour in terms of all its characteristics such as origin, malignancy, histology etc. This process is important as each cancer has different behaviours, and identification is necessary for treatment.
• Tumour grade: a measure of the rate of tumour growth based on tumour histology, such as nuclear changes and mitotic activity.
- Grade 1 to 3: nuclear becomes bigger and uglier (enlargement), more varied in size and shape (pleomorphism), loss of tubular differentiation and cancer progress faster with mitosis
- High grade cancer are undifferentiated, anaplastic (loss of cell features), and pleomorphic
• Tumour staging: measure of the extent of tumour based on clinical, radiological and pathological features. The common relationship is rate × duration = extent so stage is determined by both grade and duration.
- Indicators of spread: mitotic count (number of cells in cell cycle), serum markers (cancer cell in blood)
- Duke staging: three stage of A, B C – A (small tumour in mucosa to submucosa); B (tumour invading muscular layer); C (tumour pass into muscle coat and into lymph node)
- TMN staging: T represent duke stage in form of T1, T2 etc, N represent lymph node affected in form of N1, N2 etc, Mx represent metastasis
• Spread of cancer:
- Lymphatic spread
- Venous spread
- Serosal cavity
- Nerve spread
• Histogenetic classification: tumour classification based on cellular origin
- Epithelium: surface – papilloma/carcinoma; glandular – adenoma/adenocarcinoma
- Melanocyte: melanocytic naevus/malignant melanoma
- Connective tissue: fibrous tissue – fibroma/fibrosarcoma; bone – osteoma/osteosarcoma; fat – lipoma/ liposarcoma; blood vessel – haemangioma/angiosarcoma
- Lymph node: lymphoma (malignant)
- Bone marrow: leukaemia and multiple myeloma
- Centra nervous system: glioma
• Carcinoma features:
- Preceded by an in situ carcinoma growth phase with either flat or benign tumour appearance.
- Invasion through natural barriers and demarcations such as basement membrane and tend to spread via lymphatics
- Later stage enter blood stream and spread to liver, bone etc
- Treatment is mainly surgical resection and varied response to chemotherapy
• Microscopic features of carcinoma:
- Resembles normal epithelial cells
- Sometimes keratin production, e.g. squamous cell carcinoma and gland differentiation with mucin secretion e.g. adencarcinoma
- Stains for cytokeratin and epithelial membrane antigens
• Melanocytic tumour features:
- In situ phase of malignant melanoma applicable as melanocytes are located in the dermo-epidermal junction
- Melanocytic naevi are common
- Spreads via lymphatics to regional lymph nodes and travel later in blood stream to metastatic sites such as skin, brain and small bowel etc.
- Treatment also mainly surgical
• Microscopic melanocytic tumour features:
- Fine brown melanin pigment in the cytoplasm
- Pleomorphic, round cells
- Negative for cytokeratin but positive for S100 proteins
• Connective tissue tumour features:
- Very common cancer with unidentifiable in situ phase
- Cancer occurs mainly in the deep tissue of the limbs
- Sarcomas spreads primarily by blood stream so lymph node are unaffected
- Treatment involves radiation, chemotherapy and other surgery
• Microscopic connective tissue tumours:
- Sarcomas resemble connective tissue but more cellular
- Cells may have spindle, round and bizarre
- Cytokeratin and S100 are negative
• Lymphoma features:
- Involves mainly lymph nodes and some exranodal site such as skin, stomach and small intestine.
- In situ phase is not recognisable and no benign counterpart
- Enlarged lymph node though this is not a good indicator
- Treatment with chemotherapy and radiotherapy
• Microscopy of lymphomas:
- Tumours consist of masses of non-cohesive around cells
- negative for cytokeratin and S100 but positive for leukocyte common antigens
• Leukemia features:
- Neoplasms of haematopoetic cells which infiltrate and replace the normal bone marrow
- In situ phase is absent along with benign counterpart
- Treatment with chemotherapy and prognosis depends on the time
• Glial tumours:
- Arise from astrocytes, oligodendrocytes and ependymal cells restrictedly within the CNS
- Diffusely infiltrative hence unresectable
- Respond well to radiation and chemotherapy as palliative measures
• Diagnosis of undifferentiated tumour: two types of cancers
- Tumours that is similar to its tissue of origin, e.g. stratified squamous epithelium of the epidermis still show keratin production and other similar features
- Primitive tumours that lack resemblance to the tissues of origin, e.g. poorly differentiated squamous cell carcinoma of the skin.
• Technique of identification: tumour may appear undifferentiated when observed under a light microscope but will reveal features of differentiation when applied with special methods:
- Electron microscope: melanosomes present melanoma
- Immunohistochemical: identification of antigenic molecules located on the surface, cytoplasm or nucleus of tumour cell. This is achieved through using antibodies, i.e. antibodies to antibodies of the cellular antigen is fluorescently tagged (immunofluorescence technique – capable of demonstration of subclass lymphomas antigen)
- Monoclonal antibody technology: large expansion of antibodies

Principle of cancer therapy

• Common cancers: makes up 65% of cancers
- Prostate
- Colon, rectum and anus
- Breast
- Melanoma of the skin
- Trachea, bronchus and lung
• Clinical presentation of cancer:
- Primary tumour: predominantly local effects with expansion of mass and breach of epithelial surfaces, narrowing of body tubes and invasion
- Metastasis: distant effect involving lymph nodes, lungs (breathlessness), brain, liver or bone (localized pain)
- Paraneoplastic syndromes: generalized effects due to hormonal, autoimmune or undefined mechanism, e.g. myastheniva graves is an immune response to nicotinic receptors of the muscular endplate, hypercalcemia
• Principle of cancer diagnosis and investigation:
- Diagnosis: identifying the cancer through tumour biopsy and histopathology to exclude benign pathology, and recognize the origin, grade and prognostic markers
- Staging: determining the extent of cancer involvement according to derived systems, e.g. TNM system
- Functional assessment: assessment of how patients is likely to cope with disease treatment, e.g. lung resection to assess whether the patient can survive
• Principle of cancer treatment: assessment for the realistic probability of cure to justify disfiguring procedures such as amputation
- Curative or palliative
- Best treatment modality
- Possible options, e.g. mastectomy or lumpectomy
• Principles of cancer surgery:
- Cure: surgery most effective cancer treatment (40% success) with complete excision with margin of normal tissue
- Other benefits: biopsy (for diagnosis and staging), local control and palliation (remove obstruction)
• Principle of radiation therapy:
- Ionizing radiation: energy from radiation damages DNA and generates free radicals from water which can damage membrane protein and organelles of tumour cells.
- Therapeutic radiotherapy: external beam radiotherapy managed according to dose, treatment fractions
• Principle of cancer chemotherapy: using chemicals to kill only disease causing cells, e.g. bacteria, fungi, viruses, cancer
- Drug therapy: using chemical to modulate body processes, e.g. arterial blood pressure, mood
• Selective toxicity: toxicity produced directs only at spermatic cancer cell and not affecting host cells. It is achieved through exploiting differences between host cells and tumour cells:
- Unique target in pathogen, e.g. penicillin targeting bacterial cell walls
- Target is structurally different in the pathogen
- Target is functionally different in the host
• Therapeutic Index: a indicator of selective toxicity measured by the ratio of dose required to produce toxic effect divided by dose require to produce desired effect

TI = ED50 for toxicity / ED50 for activity where ED50 is the dose that cause 50% of max effect

- Higher the TI means for the same amount of killing effect, the dosage needed to kill cancer is much lower than that needed for normal – better therapeutic
• Classification of cancer chemotherapy drugs:
- Alkylating agents: binds DNA
- Platinum-based drugs: binds DNA
- Antimetabolites: inhibit DNA synthesis
- Topisomerase-interactive drugs: inhibit topoisomerases
- Antimicrotubule: bind microtubules
- Hormonal agents: block production or action of sex steroids
- Targeted therapies: block oncogenic proteins
- Vascular targeting therapies: inhibit angiogenesis
• Time constant of cancer division: time takes for one cancer cell to divide into two and is constant
- clinical evidence: 108 cells
- lethal: 1012 cells
• Chemotherapy killing: each does kills a constant proportion of tumour cells and repeated doses required to eliminate cells.
- Continued dose even after clinical disappearance of disease to ensure no survival adaptive tumours
• Combination chemotherapy: drugs used in combination for additive effect. However criteria include:
- Differing mechanism of action
- Different side effect profile so toxicity does not add up
- Activity as a single agent
- Example: combination therapy for testicular cancer uses Bleomycin (induce DNA breaks and affect lung), Etopside (topoisomerase II poison), CisPlatin (induces DNA crosslinks and affect peripheral nerve)
• Adverse effect of chemotherapy: damage mainly results from the pharmacological action of the drug
- Used to determine the dose and dosing interval of the chemotherapy to ensure safety
- Most are reversible and manageable, e.g. chemotherapy induce nausea and vomiting to expel toxin
• Types of adverse effects:
- Antiproliferative: myelosuppresion, hair loss and sterility
- Mutagenesis: teratogenecity (mutagen to foetus), induce further cancer
- Microtubule disturbance: peripheral neurotoxicity
- Sex steroid deficiency: decreased libido, impotence and flushing
• Indications for chemotherapy:
- Cure: high cure rates such as in acute lymphoblastic leukemia
- With surgery: adjuvant chemotherapy for node-positive breast and colorectal cancer
- With radiotherapy: for localized cancer
- Palliation: improve symptoms and survival time, e.g. Lung cancer

Targeted Cancer Therapy

• Targeted cancer therapy: biological treatments that interfere with specific molecules needed for tumour development/progression. The process can be individualized while possibly more effective and safer.
• Cancer phenotypic hallmarks: 6 features that characterize nature of cancer
- Self-sufficiency in growth factors
- Evading apoptosis
- Insensitivity to anti-growth signals
- Tissue invasion and metastasis
- Limitless replicative potential
- Sustained angiogenesis
• Self-sufficiency: growth and division of normal cells requires growth factors to be bound to tyrosine kinases and transducer a growth signal. However mutations in the pathway means the enzyme is constantly turned on
• Sustained angiogenesis: cancer cells need blood supply to cope with the intake and excretion of waste. The tumour cells release angiogenetic factors such as VEGF, to bind the receptors and stimulate proliferation and sprout
• Examples of targeted therapies
- CML: Imatinib targeting Bcr-abl
- Non-small cell lung cancer: Gefitinib targeting mutant EGFR
- Breast cancer: trastuzumab targeting HER-2
- Renal cell carcinoma: sunitinib targeting VEGF
• Chronic myelogenous leukaemia: abl of chromosome 9 translocates to bcr of chromosome 22 and abnormal fusion gene encodes for an abnormal protein tyrosine kinase, e.g. constantly activated
- Mechanism of Imatinib: competitive binding of the drug to the location in which ATP binds to prevent phosphorylation of tyrosine kinases. However, it is not entirely selective and inhibits c-Kit and PDGF-R.
• Pharmacology of Imatinib:
- Clinical uses: treating CML and gastrointestinal stromal tumours
- Tyrosine kinase activation: CML is chromosomal translocation while GIST is point mutation activating c-kit
- Adversities: well tolerated as target is not present in normal body cell so less toxicity
• Non-small cell lung cancer: mutations for EGFR such as point mutations, deletions and gene amplification or Ras which both can activate signal pathways. EGFR mutation causes the receptor to be hyper-sensitive to EGF
- Mechanism of Gefitinib: binds to block ATP binding region
• Clinical pharmacology of Gefitinib:
- Features: small molecular weight selective inhibitor of EGFR to stop action of growth factor
- Adversities: mechanism-based skin and GI toxicities
• Breast cancer: genetic amplification of the human epidermal growth factors
- Mechanism of Gefitinib: binds to the extracellular domain of the HER-2 receptor causing no further interaction,
• Clinical pharmacology of trastuzumab:
- Method: Recombinant humanized monoclonal antibody
- Adversities: immune reaction is to waste the brain
• Renal cell carcinoma: renal cell induce angiogenesis, with mutations of von-Hippel-Lindera gene scattered around NZ
- Mechanism: loss of VHL increases HIF-alpha (hypoxia induced factor) to secrete angiogenetic substances such as VEGF and PDGF. Normal cells is hydroxylated by oxygen and recognized by VHL and HIF-alpha is degraded (ubiquitin pathway).
• Clinical pharmacology sunitinib:
- Method: recombinant humanized monoclonal antibody
- Support: selective targets inhibits the function of VEGF, suppressing formation of new blood vessels
- In conjuction: in combination with standard chemotherapy for advanced blood vessels
- Adversities: hypertension, thromboembolism, bowel perforation and proteinuria
• Features of Sunitinib: chemotherapy can damage normal cells as VEGF and PDGF are expressed on normal blood vessels.

Clinical impact of cancer

• Cancer cure: if cancer is removed for 5 years with no recurrent episodes, it is high likely to be cured for life.
- 55% of cancers are cured: 30% by surgery, 13% by chemotherapy and 12% by radiation therapy
• Clinical presentations of cancer:
- Laryngeal cancer: change in voice
- Esophageal cancer: difficulty in swallowing
- Stomach cancer: early satiety (stomach thickening and difficult to expansion, hence lowered appetite), dyspepsia and weight loss
- Lung cancer: SOB, cough, haemoptypsis (coughing up blood)
- Colorectal cancer: unexplained change in bowel habit, i.e. bloating and pain. Patient becomes anaemic with RH tumours and bowel obstruction with LH.
- Melanoma: changes in skin lesion
- Breast cancer: breast lumps and nipple discharge
- Testicular cancer: lumps on testicles and scrotum enlargement. Gynaecomastia (enlargement of mammary gland of the male breast) is also possible due to imbalance of hormone production from the testicular cells.
- Cervical cancer: vaginal bleeding
- Prostate cancer: prostatism
- Non-hodgkins lymphoma: dark patches of lymphoma visible on the surface of the skin
- Thyroid cancer: massive lump at the neck region due to invasion of thyroid cancer into lymph nodes
• Systemic effect of cancer: general unwell feeling overall with advance stages of cancer
- Unexplained weight loss
- Unexplained and progressive pain (especially at night)
• Abdominal cavity cancer: tumour deposited around the abdominal cavity stimulate fluid secretion which can cause ascites leading to abdominal distention
• Testicular cancer: usually due to secondary metastasis from lung or lymph nodes. Treatment involves resection of malignant tissues.
• Cancer syndromes:
- Compression
- Hollow viscus obstruction
- Metabolic
• Spinal cord compression: impaired nerve supply as cancer (usually metastatic bone disease) puts pressure on the fibers. As a result, patient may suffer a range of symptoms related to dysfunctional nerve activity, e.g. numbness.
- concurrent neck or back pain, radicular pain, Lhermitte’s sign (electrical sensation down the back to the limb when bending the neck forward). Mid thoracic pain is a classic sign.
- bladder, bowel and motor dysfunctions (lack of parasympathetic stimulation)
- reduced anal tone, urinary retention (also loss of parasympathetic)
- treatment of steroids, decompressive surgery RT or chemotherapy
• IVC compression: very common as seen with pancreatic cancer or due to other retroperitoneal or para-aortic mass. Drainage of blood is obstructed causing extended lower limbs. However condition is not life threatening but can lead to thrombosis.
• Mediastinum compression: may involve one or both trachea and SVC occlusion.
- Tracheal compression presents an oncology emergency as tumour blocks the airway and patient unable to breath.
- SVC compression is not immediately life threatening but early treatment is necessary to prevent thrombosis and worsen mediastinal compression
- Common compressive cancer such as lymphoma is high responsive to chemotherapy
• Obstruction of gastric outlet: cancer growth such as gastric, pancreatic, billiary disease can obstruct GI tract
- treatment: surgery removal of cancerous part, stent to open tract open, chemo/radiotherapy treatment for responsive cancers
• Obstruction of billary tract: caused by pancreatic, billary or nodal disease and compromise pancreatic secretion and hence results in malnutrition
- Treatment: stent, external drainage, surgery
• Obstruction of Bowel: large bowel is primarily obstructed by colon cancers while small bowel by adjacent peritoneal metastasis such as ovary or GI cancer.
- Treatment: bowel rest followed with surgical removal of tumour or stent. Chemotherapy can be used if proven effective against disease.
• Metabolic hypercalcemia: caused by an imbalance of synthesis and resorption of bones, i.e. secretion of factors from tumours that promote bone resorption (release of calcium) and stimulate renal tubular re-absorption of calcium filtered.
- symptoms: polydipsia (excessive consumption of fluid), polyuria (excessive urination), constipation, confusion and abdominal pain
- treatment: intravenous injection of fluid and bisphophonates
• Tumour lysis: massive release of intracellular phosphate, potassium, calcium and uric acid as a result of death of tumour cells after treatment.
- Symptoms: imbalance of body ion levels can cause major complications, e.g. hyperkalemia cause severe muscle weakness while hyperphosphatemia cause acute renal failure (crystallization)
- Treatment: intensive care support with intravenous fluids and alkalinisation to counter change in concentrations.
• Other clinical cancer syndrome:
- Bleeding: disseminated intravenous coagulation, venous arterial thrombosis (damage to vessels by cancer), migratory thrombophlebitis (variable locations of vein inflammation due to blood clot), acute tumour bleed
- Ascites: peritoneal metastasis from GI or ovarian malignancy
- Pleural effusion: metastasis from lung, breast
- Pericardial effusion: fluid build up in pericardial cavity (secreted from tumour)
- Pain: pressure of cancer on surrounding structures
- Cachexia: loss of weight and muscular atrophy
- Paraneoplastic: systemic syndrome on the body due to the presence of cancers, e.g. myasthenia gravis and acanthosis nigricans (hyperpigmentation of the skin around axilla as a result of insulinoma)

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