Foundations 2 Week 5

Metabolic Pathways in Cancer Cell Glucose Metabolism

"Cancer metabolic pathways involved in glucose metabolism. The PPP is shaded in blue, and glycolysis is shaded in yellow. Red text is used to denote potential therapeutic targets. The green arrow indicates positive regulation of PFK1." Recent studies indicate that the activation of protooncogenes (e.g., Myc), signaling pathways (e.g., PI3K), and transcription factors (e.g., HIF-1), as well as the inactivation of tumor suppressors (e.g., p53), induce the Warburg effect in cancer cells". "Glycolysis generates ATP with lower efficiency, but at a faster rate, than oxidative phosphorylation. This enhanced rate of ATP generation has been postulated to be beneficial for rapidly proliferating cells". However, "mitochondria are the major source of cellular ATP in most cancer". "Thus, high glycolytic rates likely benefit proliferating cells through the production of glycolytic intermediates, which are shunted into subsidiary pathways to fuel metabolic pathways that generate de novo nucleotides, lipids, amino acids, and NADPH". "Glycolytic intermediates fuel several biosynthetic pathways that are essential for duplication of biomass during cellular proliferation. After cellular uptake through glucose transporters (GLUTs - especially GLUT-1 are upregulated leading to increased glucose import), glucose is phosphorylated by hexokinases (HKs), which produces glucose-6-phosphate. Glucose-6-phosphate can either proceed into glycolysis through conversion into fructose-6-phosphate by glucose-6-phosphate isomerase, or it can be shunted into the oxidative branch of the pentose phosphate pathway (PPP) by glucose-6-phosphate dehydrogenase. The oxidative branch of the PPP generates NADPH, which is used for the reduction of cellular glutathione pools to promote redox homeostasis and acts as a reducing agent for lipid, nucleotide, and amino acid biosynthesis. The nonoxidative branch of the PPP generates ribose-5-phosphate, which is used in the biosynthesis of nucleic acids. Back in glycolysis, phosphofructokinase-1 (PFK-1) irreversibly converts fructose-6-phosphate to fructose-1,6-bisphosphate. Fructose-1,6-bisphosphate is converted into glyceraldehyde-3-phosphate or dihydroxyacetone phosphate. The latter is a precursor to glycerol-3-phosphate, which is crucial for the biosynthesis of the phospholipids and triacylglycerols required for generation of cell membranes. Fructose-6-phosphate and glyceraldehyde-3-phosphate can also combine to generate ribose-5-phosphate through transketolases and transaldolases. Further down the glycolytic pathway, 3-phosphoglycerate can undergo oxidation to generate serine and NADH. Serine can be used to generate two critical amino acids, cysteine and glycine, and to generate important signaling molecules such as ceramide". "Proliferating cells up-regulate the expression of PFK-2, which generates fructose-2,6-bisphospate, a potent allosteric activator of PFK-1, thus maintaining high glycolytic flux in the presence of high ATP. High glycolytic flux is also maintained by the overexpression of lactate dehydrogenase (LDH), a transcriptional target of Myc. LDH generates NAD+ from NADH while reducing pyruvate to lactate. NAD+ regeneration is necessary for continued flux through glycolysis, as NAD+ is required for conversion of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate. The overexpression of LDH is sufficient to increase glycolytic flux. Although NAD+ can also be regenerated by mitochondria, this requires the transport of NADH into the mitochondria through various shuttles that are likely too slow to meet the high demands of the cytosolic NAD+ required to maintain glycolysis. "Pyruvate kinase (PK) plays an essential role in regulating the balance between glycolytic ATP generation and biosynthetic needs for proliferating cells. PK catalyzes the rate-limiting and ATP-producing step of glycolysis in which phosphoenolpyruvate (PEP) is converted to pyruvate. Cancer cells express higher levels of M2 isoform (PKM2) over the more catalytically active M1 isoform (PKM1), and cancer cells engineered to express PKM1 instead of PKM2 exhibit reduced tumor-forming ability".

Gleason grading for prostate cancer

1 = small uniform glands 2 = more stroma between glands 3 = distinctly infiltrative patterns 4 = Irregular masses of neoplastic glands 5 = rare glands Score and grade Well differentiated (I) = patterns 1 & 2, score 2 - 4 Moderately differentiated (II) = pattern 3, score 5 & 6 Poorly differentiated (III) = pattern 4 &5, score 7-10 The scoring system looks at the most common tumor pattern and then the second most common tumor pattern. The only variable is the histologic pattern. Pic- 0, 1, 2-3, 4-5

Questions about toxicology

1.When quinidine is given to a patient already receiving digoxin, the serum levels of digoxin may increase with a corresponding toxic reaction to digoxin. Which of the following caused the increase in digoxin levels? Displacement of digoxin from tissue binding sites. - Quinidine displaces digoxin from tissue binding sites 2. Which of the following compounds inhibits drug metabolism? ketoconazole. Can inhibit drug metabolism (e.g., verapamil) 3. A child arrives at the Emergency Department after an overdose of iron supplements, which one of the following treatments is appropriate for the patient? -Chelation therapy with deferoxamine, monitoring urine color and serum levels of the heavy metal 4. Which of the following is a toxin that is a toxic lectin extracted from castor beans gastrointestinal bleeding, dehydration, and tissue necrosis leading to death? Ricin- Toxic lectin extracted from castor beans 5. Which of the following is a definition of Biomagnification? A persistent environmental chemical pollutant is concentrated with each progressive link of the food chain. 6. The following is a list of experimental antidepressant drugs with their corresponding Margin of Safety (MOS). Which one is likely to have the lowest incidence of toxicity? CA-1a MOS = 10.4 ( highest number is safest)

Methotrexate

Anti-metabolite A Dihydrofolate Reductase Inhibitor Folic acid is reduced by DHFR to 5,6,7,8-tetrahydrofolate FH4 FH4 acts as carrier of one-carbon (methyl) groups for synthesis of purine nucleotides and thymidylate MTX inhibits DHFR and shuts off DNA/RNA synthesis resulting in cell death T is made by taking off H and replacing with a methyl. Left has active C1 unit. MTX is a competitive inhibitor. runs out of dTTP so it cant make DNA

Evasion via MIC and NK cells

Best chance to kill tumor is immediately. Otherwise, the tumor will develop mutations and evade the immune response, like it has in panel 3

Grading

Best used in: Prostate carcinoma, Breast carcinoma, Renal carcinoma, Astrocytomas, B Cell lymphomas Tumor grading to classifies cancer cells in terms of how abnormal they look under a microscope and how quickly the tumor is likely to grow and spread. There is no uniform grading system like there is for staging.

Cyanides

Binds tightly to heme iron in the Cytochrome a - a3 complex makes patient look pink. not absorbing oxygen properly Treatment Stabilize patient Amyl nitrite and/or I.V. sodium nitrite followed by sodium thiosulfate

Management of breast and ovarian cancer

Breast cancer: Monthly BSE beginning at age 18 CBE every 6-12 months starting at age 25 (or 5-10y before the earliest dx in family) Annual mammography and MRI starting at age 25 (or 5-10y before the earliest dx in family) Consider chemoprevetion (Tamoxifen) Consideration of prophylactic mastectomies Mastectomies reduces risk by 90%. Does not get rid of every breast cell Ovarian cancer: Recommend prophylactic BSO (removal of ovary and fallopian tubes) between ages 35-40 (consider reproductive desires) If delaying BSO: pelvic examination and transvaginal ultrasound with color Doppler imaging every 6 months beginning at age 30-35 (or 5-10 years prior to the earliest dx in the family) with concurrent serum CA-125 Consider oral contraceptive - discussion of risk/benefit Oral contraceptive decrease ovarian cancer, increase breast cancer. More dramatic for ovarian and screening is worse for ovarian so it's a good tradeoff

Evasion via MHC expression and CD8 T cells

CD8 T cells are the best way to eliminate a tumor. The tumor can lower its MHC expression to evade the CD8, but then it is subject to NK cells who cannot recognize the tumor cells as "self"

KRAS and EGFR in Colon Carcinoma

Cetuximab- blocks EGF in colon With muatted KRAS_ drug doesn't work Wild type- block EGFR signaling, kind of works

Collective cell migration

Collective cell migration is associated with well differentiated tumor. Cell clusters, strand with or without a lumen - poorly to moderately differentiated carcinomas

Amanita mushrooms

Cyclopeptides inhibit RNA polymerase II which decreases protein synthesis that leads to cellular necrosis May need liver transplant

Types of Toxicology

Environmental toxicology: Bioaccumalution - (e.g., DDT in fat) increasing concentration of a persistent environmental chemical in the tissues of an organism as a result of the chemical physicochemical properties. Biomagnification is the increasing concentration of the chemical with each progressive link of the food chain. Ecotoxicology - the effects of agents in the environment on whole populations. a. Comprehensive Emergency Response, Compensation, and Liability Act (CERLA) - Superfund enacted shortly after the Love Canal incident. Forensic Toxicology - deals with the medicolegal aspects of toxicology. 1. Cause of death or injury due to exposure to an agent. 2. Under the influence? of drugs and alcohol. Hair grows ~1/2 inch per month Nails grow ~1/8 inch per month. arsenic- binds to nail and hair protein. can measure arsenic in hair and nails. Ethanol is eliminated at ~7 - 11 grams/hour. zero order elimination Clinical Toxicology - deals with emergencies such as overdoses and poisonings. 1. Compound identification and quantification. 2. Sign and symptom management

Extravasation

Facilitated by factors released from the primary tumor (TGF-β and TNF-alpha increase MMP expression in the tumor cells. Involves selectins cell adhesion molecules on the ECs and the tumor cells Differential expression of selectins may account for "homing" Homing of tumor cells: Mechanistic theory: determined by the pattern of blood flow (66% of the cases). "Seed and soil" theory: the provision of a fertile environment in which compatible tumor cells could grow (20% of the cases). Some tumors commonly metastasize to certain sites (organ tropism) Chemokines and trophic factors Unfavorable environment of other sites

Biomarker phase

Few biomarkers for cancer are thoroughly validated Drug companies are bundling the biomarker assay and the drug for it.

Ethylene glycol

Increased osmolar gap leading to anion gap acidosis Treat with ethanol or fomepizole produces crystals ethanol is competivie inhibiotr fomepizole inhibits AD

Pharmacokinetic properties

Intake: Direct interaction- Tetracyclines precipitate sulfonamides. Carbenicillin reacts with aminogylcosides GI absorption-laxatives or antibiotics Dermatomucosal absorption- DMSO or patches Distribution: Plasma protein binding- Salicylates displace warfarin and phenytoin. Displacement from tissue binding sites- Quinidine displaces digoxin Metabolsim: Induction - slow. Phenobarbital Inhibition - fast - covalent binding irreversible a.Chloramphenicol b.Ketoconazole c.Cimetidine Excretion: Weak acid transport in proximal tubules pH of urine- Na bicarbonate ↑ pH ↑ elim. weak acids Ammonia chloride & ascorbic acid ↓ pH ↑ elim. weak bases Diuretics ↓ tubular concentration ↓ renal toxicity (Cisplatin) Thiazides ↓ potassium (toxicity with digoxin)

Biomarker validation

Level I evidence is the highest. Level V is the lowest Well designed prospective, well-controlled clinical study. Every step of the process is standardized. tissue collection and purification high-throughput molecular ("omic") techniques data capture, normalization, statistical analysis and scoring.

Management of poisoned patient

Maintain the vital functions ("ABCD") Airway Breathing Circulation Dextrose - thiamine if alteration of CNS- give dextrose. if hyopglycemic, brain cells die quickly. thiamine- vitamin that is neuroprotective -syrup of ipecac to make vomit -enhancement of elimination - antidotes

Factors affecting response

Mercury is very toxic. except for mercury metals Parathion converted to paraoxon via moisture and sunlight. binds cholinesterase

Generation of tumor antigens

Mutations are one way to generate a tumor antigen. Another is through slicing ie the picture

Drug- Food and drug-drug interactions

NSAIDs - ginkgo biloba. increase risk of bleeding Calcium channel blockers (CCB) - grapefruit juice. Lowers CYP3A4, so higher CCB (verapimil) leads to hypotension & myocardial depression Also, drug-drug is CCB with ketoconazole. same as grapefruit juice Phenobarbital and warfarin- phenobarbital lowers warfarin effectiveness so must increase warfarin dose to get same result Ethanol-acetaminophen. pic. normally, goes left. in high concentratiosn, goes right. depletes glutathione and body cant compsenate. with ethanol- induces CYP2E1. so higher conc. of metabolite even at therapeutic amounts. use n-acetylcysteine to treat

Breast cancer biomarkers

Nuclear receptors in breast cancer Diagnostic Predicts response to chemotherapy Predicts response to hormonal therapy Look for 3 things for breast cancer. IHC for ER and PR. Estr and progest. And HER2/NEU Not prognostic, predictive of response to chemotherapy. Hormonal ie tamoxifen blocks the receptors. Aromatase inhibitor blocks androgen from tissue HER2/NEU: Membrane receptor - IHC staining Nuclear oncogene amplification - FISH Predicts response to receptor blockade Indiciator of poor prognosis. EGF on surface of tumor cells. Herceptin blocks this binding. Pink- upregulation of oncogene HER2/NEU antibody herceptin is patented. Drug manufactureres control testing and treatment Triple negative breast cancer- ER/PR, HER2 all negative. Patients will not respond to hormonal therapy or herceptin. Must use normal chemotherapy

Pharmacodynamic properties

Potentiation - (0 + 1 = 10); synergy - (1 + 1 = 3) antagonist and agonsist- naloxone antagonizes opiates Partial agonist = tamoxifen physiological alteration- Ethanol ↑ fluidity of membrane ↑ Cl- influx. GABA

Castor bean plant

Ricin disrupts protein synthesis by binding the 60S ribosomal subunit Administer activated charcoal

Intrinsic and extrinsic pathways

Some cross-talk between the 2 pathways

Iron

Symptoms Vomiting and diarrhea (often bloody) - massive blood and fluid loss Remission Shock, seizures, liver failure and death may be a remission after 12 hours, but can get abrupt relpase that leads to death treatment: Deferoxamine (chelating agent) - if serum Fe > TIBC Monitor urine color Monitor serum Fe levels TIBC-total iron binding capacity dark orange red color urine. when goes away, iron conc. goes down

Tumor-Associated macrophage

TAMs promote tumour development, growth and survival through innate immune mechanisms via the synthesis and secretion of a wide variety of products. Extensive cross-talk with tumor cells M1: involved with pro-inflammatory mediators. early stage tumor development M2: allows for tumor growth

Occupational (Industrial) Toxicology

TLV's - threshold limit values TLV - TWA - time weighted average values. safety of a coumpound throughout a work-week. problem arises when average is abive a certain limit TLV - C - ceiling values. can never go above it or else its toxic guidelines, not standards. PEL's - permissable exposure limits. posted in federal regulations. not changed like the others. regulated by OSHA.

Tumor stage

TMN Criteria T = tumor extent N = regional lymph node metastasis M = distant metastasis Pathologic stage (pStage) is based on tissue examined Clinical stage (cStage) is based on physical exam and imaging Primary Tumor (T) TX Primary tumor cannot be evaluated T0 No evidence of primary tumor Tis Carcinoma in situ (has not spread) T1, T2, T3, T4 Size and/or extent of the primary tumor Regional Lymph Nodes (N) NX Regional lymph nodes cannot be evaluated N0 No regional lymph node involvement N1, N2, N3 Involvement of regional lymph nodes (number and/or extent of spread) Distant Metastasis (M) MX Distant metastasis cannot be evaluated M0 No distant metastasis M1 Distant metastasis (cancer has spread to distant parts of the body) In most cases, we get just tumor and lymph node. Nodal status is critical. If nodes are negative, better prognosis For most solid tumors, staging is a better predictor of patient survival than grade.

Colon cancer staging

This figure summarizes the various stages of colon carcinomas. Here T is not a function of size but of depth of invasion in and through the wall of the colon. 0 adenmoatous polyp 1 invading the wall 3 nodes becomes positive 4 metastases

Tumor mediated stimulation of angiogenesis

Through Hypoxia Oxygen demands exceed supply from surrounding vessels Hypoxia-Induced Transcription Factor (HIF) is activated Expression of proangiogenic factors like VEGF and bFGF Important target in cancer drugs Target: VEGFR-2 Bevacizumab (Avastin) - Antibody against VEGF Sunitinib (Sutent) - VEGFR kinase inhibitor (small molecule) Sorafinib (Nexavar) - VEGFR/ Raf kinase inhibitor (small molecule All small molecule kinase inhibitors end in -nib

Targets of apoptosis

Thymocytes that recognize self antigens Virus infected cells DNA damaged cells Defective lymphocytes Unnecessary cells during development Excess cells - neurons Obsolete cells Chemotherapeutic killing Syndactyly: webbed fingers/toes. Not proper apoptosis. Failure of cells between digits

Pericytes

Tumor vessels are dilated, tortuous, and highly permeable. Pericytes stabilize blood vessels, inhibit endothelial proliferation, maintain capillary diameter, regulate blood flow, and provide endothelial survival signals via heterotypic contacts and soluble factors. Abnormalities in pericytes most likely contribute to the relatively poor vascular perfusion and high interstitial fluid pressure in tumors.

Protein biomarkers

Used to monitor disease progression Serum: PSA CEA CA125 CA19-9 β-HCG. - pregnant. Biomarker for testicular cancer. Germ cell tumors 95% of Test. Cancers. 2/3 of those express HCG and Alpha-fetal protein (AFP) AFP Monoclonal Ig Tissue: ER - estrogen receptor PR - progesterone receptor HER2/NEU CD117 CD20 Monoclonal. Much better. MUGUS- Monoclonal gammopathy of undetermined significance (MGUS) is a condition in which an abnormal protein (monoclonal protein, or M protein) is in the blood. M protein is produced by plasma cells, a type of white blood cell. Not good for screeening

Grading breast carcinoma

Variables Nuclear grade Mitotic Count Tubule formation percentage Each variable score1-3 Grades - combined scores Low grade 3-5 Intermediate grade 6-7 High grade 8-9 Acini: Score 1 = >75% Score 2 = 10-75% Score 3 = <10% Mitoses: Score 1 = <7-10%/10 hpf Score 2 = 8-14%/10hpf Score 3 = >15%/10hpf Nuclear Atypia/Pleomorphism: Score 1 - small nuclei with min. pleomorphism, very inconspicuous nucleoli Score 2 - Larger nuclei with mild to moderate pleomorphism, small visible nucleoli Score 3 - Vesicular nuclei with prominent nucleoli, marked pleomorphism

Cancers associated with viruses

Viruses cause 15% of cancers. Many more people get these viruses without getting cancer. caused by many steps Mutagen - chemical or physical agent that damage DNA in such a way as to cause an increased rate of mutations. Carcinogen - mutagens that are known to increase the risk of cancer

Platinum Co-ordination Complexes

cisplatin, carboplatin, oxaliplatin Used in broad spectrum of tumor types In the cell hydrolysis of platin compounds occurs Reaction with DNA causing intra- or interstrand crosslinking e.g. N7 of guanine, guanine-guanine or guanine-adenine DNA adducts formed by cisplatin inhibit DNA replication and transcription, lead to strand break and miscoding

Chronic lead poisoning

decreases Hb synthesis. leads to anemia and retardation. Basophilic stippling and microcytic, hypochromic anemia. Peripheral neuropathy Segmental demyelination of peripheral nerves Treat by: Chelate with dimercaprol and/or calcium EDTA

Graded vs quantal response curve

graded- response in individuals. quantal- populations. look for absence or presence of phenomenon.

Drug trials

nonclinical can sometimes overlap with clinical Ames test- tests for mutagens. if there is one, bacteria grows Dominant lethal test- Expose males to mutagenic treatment.Observe the number of viable offspring Phase I: small group of normal healthy volunteers Phase II: small group with target disease Phase III: multicenter trials Phase IV: postmarketing surveillance factors influencing placebos- Color, Size, Route of administration, Physician's attitude, Cost Code of federal regulations (CFR)- informed consent, IRB, children may provide assent, selecting right investigators and sponsors, clinical blinding can be crossover or parallel. cross switches between placebo and treatments Types of controls- historical (death), positive (other treatments), negative (placebo) First-time in humans study- Randomized, double-blind, placebo controlled. Parallel design, single dose. Small cohorts (e.g., 2 placebo and 4 or 6 drug). escalate doses. Measure Safety, tolerability, and pharmacokinetics. not efficacy Typically males only, ages 18 - 40 or 45 Risk for pregnancy- Category A is best, Category X is contraindicated

Colon cancer case study

40 year old diagnosed with right-sided colon cancer No prior history of colon polyps Father reportedly had colon cancer at 52 Robert is not overly concerned about this history -At high risk 1 tumor more likely to be lynch syndrome IHC shows absence of MSH2 and MSH6 protein MLH1 and PMS2 present Very high likelihood he has LS Direct DNA testing on blood sample from Robert shows MSH2 mutation Robert's unaffected brother has 50% chance of having Lynch. After proved negative, has same risks as general population Sister's children have 50% risk of Lynch

Kinases

518 Kinases, Only the GPCR family is larger, 30-35% of Drug Discovery Programs Only the last 15 years. Used to not be regarded as good options because would be too general and block many kinases... best way to block enzyme is to block catalytic site. Important component in this site is ATP. If can prevent ATP binding to kinase, can inhibit kinase. All ATP binding sites are pretty similar.

Cost-benefit analysis (CBA)

A PE analysis where both cost and benefits are measured in monetary units 1. Net Benefit = Total Benefits - Total Costs Cost beneficial if Net Benefit > 0 2. Benefit-to-Cost Ratio = Total Benefits / Total Costs Cost beneficial if Benefit-to-Cost > 1 3. Internal Rate of Return (IRR) = The rate of return that equates the present value of benefits to the present value of costs 4. Break-Even Point = The time required to recoup the investment CBA is most useful when: Analyzing a single intervention to determine whether its total benefits exceed the costs, or Comparing alternative interventions to see which one achieves the greatest benefit. Major advantages: Can determine if benefits exceed costs of program - less subjective than CEA or CUA Can compare multiple programs with either similar or unrelated outcomes (anticoagulation and diabetes clinics) Disadvantage: Difficult to place a monetary value on health outcomes Different methods of doing so may elicit different estimates

Cost-utility analysis (CUA)

A PE analysis which measures outcomes based on years of life that are adjusted by "utility" weights (patient preferences); range [0, 1] Most common utility is the Quality-Adjusted Life Year (QALY) 1.0 QALY = 1 year of life in perfect health 0.0 QALY = death 0.0 < QALY < 1.0: a year when health is diminished by disease or treatment Quality Adjusted Life Years (QALYs) weight the life years remaining by the utility weight (QALY) Ex: 4 years of life post cancer treatment at 0.6 utility wt = 2.4 QALYs Average cost per QALY or average cost utility ratio (CER) Cost of intervention/QALY of intervention Incremental Cost per QALY (ICER) Cost of B - Cost of A/ QALY of B - QALY of A Must be less than $50,000 (or 75,000) per QALY gained to be cost effective. SO this example is not cost effective. but if only given to high risk stroke patients, proved to be under $50,000 and thus cost effective Common Applications CUA is useful when utility adjustments are needed, such as when: Length of life (quantity) and quality of life are different Length of life (quantity) is unaffected and quality of life is different Outcomes are very different CUA is not warranted when: Number of life years saved (quantity) is different but quality of each year of life is very similar Advantages: Can incorporate both morbidity and mortality Can compare multiple programs with either similar or unrelated outcomes (anticoagulation and diabetes clinics) Can use a threshold or cutoff cost per QALY (such as $50,000) and decide somewhat objectively if an intervention is cost effective Main disadvantages: No consensus on calculating utility weights Utility weights are "rough estimates" Many clinicians are not familiar with QALYs Whether or not negative QALYs make sense is debatable.

The Extrinsic, or Death Receptor-mediated Apoptosis Pathway

A second apoptotic pathway can be triggered by signals outside of the cell. This apoptotic pathway is known as the extrinsic apoptotic pathway and is plasma membrane receptor activated involving the activation of pro-apoptotic cell surface death receptors. There are 30 death receptors in the human genome, and they are members of the tumor necrosis factor (TNF) family of receptors, receptor ligands include TNF-a, TRAIL, and FasL. Ligand binding activates the cytoplasmic tail of the death receptor to bind a cytoplasmic protein known as FADD (FAS-associated death domain). Association of FADD with the receptor forms a complex known as the DISC (Death Inducing Signaling Complex). The DISC binds an initiator procaspase 8 (or initiator procaspase 10), and in a fashion similar to the apoptosome complex, allows for cleavage and formation of active caspase 8. Caspase 8 will activate the downstream executioner procaspases 3, 6, and 7. Activation of the death receptor pathway bypasses or circumvents the Bcl-2 mitochondrial pathway of apoptosis. The motifs in death receptors that allow dimerization with FADD are called death domains (DDs). FADD also has a second domain, the death effector domain (DED) that recruits procaspase-8 and allows for its activation.

Caspase-Independent Pathway to Apoptosis

AIF: Apotosis-inducing factor AIF is located in the intramembranous space of mitochondria. When mitochondria are permeabilized by Bax/Bak activity, AIF is released. AIF then travels to the nucleus where it specifically induces nuclear chromatin condensation and DNA fragmentation. The absence of certain growth factors may trigger the onset of apoptosis. The PI3K-Akt pathway (that is stimulated by growth factors like insulin) aids in cell survival by Akt-mediated phosphorylation and inactivation of Bad (via its sequestration by 14-3-3). Withdrawal of these growth factors can trigger the intrinsic apoptosis pathway.

Immunosurveillence or Cancer Immunosurveillence

Ability of the immune system to detect and eliminate tumors at an early stage. Only a small amount of malignancies actually cause damage. most are killed off early enough by immunosurveillance. similar to an innate and adaptive immune response, except it just takes longer to notice

Nitrogen mustards

Alkylating agent mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil Covalanetly link drug to DNA. Can do it twice to cross-link either intra or interstrand. Effect integrity of DNA Reaction with guanine on N-7 occurs to the greatest extent Serious systemic toxicity also follows exposure: leukopenia dissolution of lymphoid tissue ulceration of the GI tract First anti-cancer drugs. Creates vesicles in the lungs filled with fluid.

Nitrosoureas

Alkylating agent carmustine, lomustine, semustine Alkylation of DNA and carbamoylation of proteins Carbamoylation of proteins on lysine residues can lead to their functional inactivation Consequences of DNA alkylation: Serious DNA damage because of: Mispairing of guanine with thymine residues during DNA synthesis Depurination by excision of guanine residues Cross linking of DNA, no replication (bifunctional alkylating agents- only, e.g. nitrogen mustard)

MHC is critical in immune recognition of tumors

Alloantigens are recognized as foreign and elicit adaptive immune responses to eradicate the antigen CD8 T cells kill tumor if it doesn't recognize the MHC

Anthracyclins

Antibiotics Mechanism of Action: Intercalate with DNA which results in effects on DNA and RNA synthesis Induction of single and double strand breaks Maximal effect during S phase but generally effective throughout the cell cycle Cardiomyopathy is a critical toxicity which is related to total dose Damage both mammalian and bacterial cells because have DNA/RNA. Another antibiotic is bleomycin Glycopeptides Induce fragmentation of DNA...accumulation of cells in G2 Minimal myelo- and immunosupression....used therefore in combination with other cytotoxic drugs

Cell cycle non-specific agents (CCNS)

Antibiotics (Anthracyclins and others) Alkylating Agents: Nitrogen mustards (e.g. mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil) Nitrosoureas (e.g. carmustine, lomustine, semustine) Platinum co-ordination complexes (e.g. cisplatin)

Monoclonal antibodies

Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) Antibodies attach to antigen on cancer cell. This recruits cytotoxic T-Cells that kill the cancer cell. Fuse B cell and myeloma cell, form hybridoma. Will produce antibody and also confer immortality because of myeloma cell. Pro: Highly selective But: Need to be injected Expensive to manufacture Useful only against extracellular targets- Cannot cross plasma membrane -mab = monoclonal antibody

5' Flourouracil

Antimetabolite Pyrimidine analog Uracil has H instead of F. needs to be activated. Must be ribosylated (top). Then phosphorylated. Flourine is incorporated into RNA, affects integrity of RNA. FdUMP- flourine harder to pull off than H. dTTP lowers. Tri-phosphate- effects DNA integrity

Cell-cycle specific agents (CCS)

Antimetabolites - Inhibit specific enzymes involved in DNA/RNA metabolism (S phase) Methotrexate, 5-fluorouracil, cytosine arabinoside, 6-mercaptopurine, 6-thioguanine, fludarabine, cladribine, pentostatin, gemcitabine Vinca Alkaloids and Taxanes - inhibit microtubules (M) vincristine, vinblastine, docetaxel, paclitaxel Epipodophyllotoxins - inhibit topoisomerase II (G2) etoposide (VP-16), teniposide Camptothecins - inhibit topoisomerase I topotecan, irinotecan

Advantages and liabilities of tumor metabolism

As the early tumor expands, it outgrows the diffusion limits of its local blood supply, leading to hypoxia and stabilization of the hypoxia-inducible transcription factor, HIF. HIF initiates a transcriptional program that that shifts cell metabolism toward glycolysis by the increased expression of glycolytic enzymes, glucose transporters, and inhibitors of mitochondrial metabolism. In addition, HIF stimulates angiogenesis. Gene muatations alter the cells metabolism. Shunting Is for macromolecule synthesis. Still have ATP in mitcohindria Hypoxic tumor cells shunt glucose to lactate. Glutamine can be used to drive the TCA cycle or used for lipid synthesis While aerobic proliferating cells use glucose and glutamine for biomass production through the TCA cycle, hypoxic cells shunt glucose to lactate and rewire glutamine metabolism. Glutamine can be used to drive the TCA cycle independently of glucose or contribute to lipid synthesis via IDH-mediated reductive carboxylation of ketoglutarate generated from glutamine

Hereditary Breast and Ovarian Cancer Syndrome (HBOC)

At risk: Multiple cases of early onset breast cancer Ovarian cancer Breast and ovarian cancer in the same woman Bilateral breast cancer Male breast cancer Ashkenazi Jewish heritage Increased risk of multiple tumor types Breast cancer (females):50-85% Breast cancer (males): 6-16% Ovarian cancer: 20-40% Rare tumors: pancreatic, melanoma, prostate Caused by mutations in BRCA1/BRCA2 Tumor suppressor genes Repairs double stranded DNA breaks Signals the presence of these lesions to the cell Autosomal dominant 3 mutations. 1 in 40 for jews.

Mutations Predicting Increased Cancer RISK

BRCA1 and BRCA2 Increased risk for breast and ovarian cancer MLH1, MLH2, MSH6, and PMS2 (Lynch syndrome) Increased risk for carcinomas in the colon, stomach, small bowel, biliary tract, ovary,and endometrial, and brain cancer Start with IHC. Lack of staining. Can then refer patient to further testing TP53 (Li-Fraumeni syndrome) Increased risk for breast carcinoma, acute leukemia, sarcomas, brain cancers, and adrenal carcinomas Multiple Mutational Analyses: Genetic analysis of similar tumors to see if they are the same. Ie the same chromosome. metastatic vs separate primary tumor

Bcl-2

Bcl-2 promotes survival, not growth and proliferation Balance of anti- and pro-apoptotic proteins maintains proper cell survival Higher ratio of anti-apoptotic proteins - cell survives Higher ratio of pro-apoptotic protein - cell dies Bcl-2 exerts apoptotic effects by: 1. BH3-only family proteins are activated by many types of cellular stresses. ie hypoxia, DNA damage, P53 mutation, Ca influx 2. This results in release of BH3-only proteins from sites where they are sequestered. (Some BH3-only proteins can be sequestered by dynein light chains (DLCs). Others, like Bad, can be sequestered dependent on their phosphorylation state. When Bad is phosphorylated, it binds its sequestering protein, 14-3-3. Another protein, Bid, is inactive until it is cleaved into a truncated form by granzyme B or caspase-8). 3. BH3-only proteins now are free to bind pro-survival proteins like Bcl-2. This keeps the pro-survival proteins from binding and inactivating Bax family proteins (Bax, Bak). 4. Free Bax and Bak create channels in the mitochondrial outer membrane that allow cytochrome C to leak out. Bax/Bak knockout yielded large lymph nodes and spleen

Apoptosis

Biochemical pathway inducing cell death Programmed cell death during development Triggered in response to cell damage DNA damage Hypoxia Nutrient and/or ATP deprivation Transcriptional blockage Loss of apoptosis results in growth of defective cells Lots of apoptosis in brain during development. Apoptotic cells die without damaging their neighbors or inducing inflammation. Apoptosis results from the activation of a cascade of proteases within the cytoplasm. Morphology of apoptosis: Chromatin condensation Progressive cell shrinkage Plasma membrane blebbing Apoptotic bodies Phagocytosis, but no inflammation In the final stages of apoptosis, cellular fragments and debris are phagocytized by macrophages

Biomarkers

Biomarkers are substances produced by tumor cells or by other cells of the body in response to cancer, or to a benign or noncancerous condition. Found in blood, urine, or tissue 3 ways to improve health: better patient understanding of disease/risk, healthier patient behaviors, better clinical decisions Uses: Risk assessment, Screening, Diagnostic, Prognostic, Predictive , Monitor Benefit >>>> toxicity risk. Must be cost-effective Biomarkers must be able to subclassify patients for individualized treatment, reproducible, high sensitivity and specificity, and the use is validated.

B cell Lymphoma

CD20 Diagnostic for B cell non-Hodgkin lymphomas Monoclonal antibody to CD20 (rituximab) is used in various chemotherapy regimens for B cell lymphomas Lymphoma in stomach. Can also use antibody for autoimmune disease against B cells. Also use to look at lymphocytes in peripheral blood. Flourescent antibody staining

ECM proteins

Cadherins - increased invasiveness of cells and metastasis and are related to reduced survival and poor prognosis. Hepatocyte growth factor - Regulates cell proliferation, migration, survival, tumor angiogenesis, and invasiveness. TGF-β - Promotes tumor cell survival, invasiveness and metastasis by targeting fibroblasts, myofibroblast and immune cells in tumor microenvironment. Tgfb3 in fetuses, can do surgery and there wont be a scar. Osteopontin - Increased cellular and invasion behaviors, protection from apoptosis, increased metastasis and induction of tumor-associated inflammatory cells. Galectins - Promote neoplastic transformation and progression toward metastasis in breast, colon, stomach and thyroid Hyaluronic acid - binds to CD44 on tumor cells promoting invasion MMPs- functions are critical in tissue remodeling, organ development, regulation of inflammatory processes, wound healing, and in cancer. The various types of MMPs in the tumor-microenvironment degrade and remodel the ECM.. Proteolysis of matrix substrates generates degradation fragments that regulate specific pathways involved in invasion, angiogenesis, immune regulation lymphangiogenesis, and metastasis. TIMPs control MMPs and thus tissue remodeling under normal conditions. The normal MMPs/TIMP shifts toward MMPs in cancer

Immuno-modulation

Cancer Vaccines Prophylactic: Cervical Cancer Gardasil, Cervarix anti-Human Papilloma Virus (HPV) Therapeutic: Prostate Cancer Provenge Antigen Presenting Cells (APCs) harvested from the patient are "loaded" with Prostate Specific Antigen (PSA). Subsequently, the APCs are re-infused into the patient to elicit an immune response. Activation of the immune system: Ipilimumab: Melanoma (Yervoy) monoclonal antibody directed against CTLA-4 on T-Cells. CTLA-4 may downregulate the activity of T-Cells which is overcome by ipilimumab.

Individual cell migration

Cell migration is a dynamic process mediated by cytoskeleton coupling with cell surface receptors that engage with surrounding tissue structures; thus, "the cytoskeleton serves as the cell's engine, and the cell surface receptors act as its transmission". "The physicochemical steps in single-cell migration are coordinated within the same cell body and executed in a synchronous, often pulsatile manner, which allows the cell body to protrude and generate traction in an oscillatory manner". Must have contact with ECM. Lay down, pick up Single cells or multicellular, mesenchymal tumors, carcinomas after EMT hematopoietic or neuroectodermal

Caspases

Cysteine-dependent aspartyl-directed proteases When active, cause rapid cell death, therefore must be tightly controlled Synthesized as zymogens (inactive enzyme precursors) that must be cleaved to be activated Regulatory signal pathway controls activation Endogenous cellular inhibitors modulate activity 14 human caspases (3 categories) -Initiators: caspases -2, -8, -9, -10 -Effector/executioner: caspase -3, -6, -7 Inflammation control, processing of cytokines and not involved in apoptosis: remaining 7 caspases Initiator caspases activate other caspases in a cascade. Effector or executioner caspases do the damage to cellular structures that result in apoptosis.

Dendritic cells

Dendritic cells are normal components of all types of tissues. Cancer cell derived chemokines attract immature dendritic cells. VEGF can influence their maturation in such a way as induce CD4+ T cells that promote expansion of cancer cells (procancer) at the expense of CD8+ T cells that can cause tumour regression (anticancer).

Epithelial to Mesenchymal Transition (EMT)

EMT- Before a neoplastic epithelial cell is able to invade through the basement membrane it must first detach itself from its neighboring cells and the basal lamina Tumor cells at the invading interface of the tumor are more likely to have undergone EMT as compared to those "inside" the tumor. Cellular changes during EMT: Lost or altered: E- cadherin alpha-catenin, beta-catenin frequently translocates to nucleus (Wnt) Apico-basal polarity Circumferential F-actin fibers Epithelial cytokeratins Gained: N-cadherin alpha-v-beta-6 integrin Intermediate filament protein - Vimentin Matrix metalloproteinases secreted, produced Fibronectin secretion alpha-smooth muscle actin (myofibroblasts) Motility, Invasiveness due to MMPs The tumour microenvironment promotes the EMT of carcinoma cells. This involve integrin signalling, growth factor binding, binding of TGF superfamily molecules to TGF receptors (TGFR), and cleavage of E-cadherin by MMP3.

Lynch syndrome

Early age at CRC diagnosis (mean=45) Tumors in proximal colon predominate Extracolonic cancers: endometrium, ovary, stomach, urinary tract, small bowel, bile ducts, sebaceous skin tumors Autosomal dominant 50% risk to offspring and siblings Surveillance (colonoscopy) Reduces Risk of CRC in HNPCC Families Testing: Genes - DNA mismatch repair (MMR) First, screen tumor for MSI; IHC Microsatellite instability (MSI) results 95% of HNPCC associated tumors are MSI+ vs. 15% of sporadic tumors Immunohistochemistry (IHC) results Loss of protein indicates possibility of mutation in specific MMR genes IHC results can direct gene testing e.g. MSH2/MSH6 absent - just do testing for these genes in step-wise fashion -MSI alone isnt enough to make diagnosis. IHC looks for gene products Gene testing is gold standard. MSH2 first, if negative, go to MSH6 Many hospitals do routine IHC on all or a sub-set of colon cancer specimens Gene testing averages about $1000/gene for full mutation analysis; $2000-$4000 for colorectal cancer gene panel (multiple genes); $200-$450 for mutation specific analysis Colonoscopy beginning age 20-25* q 1-2 years *Age 30 yrs for MSH6/PMS2 carriers Annual TVU and endometrial aspirate starting at age 30 -35. Consider TAH/BSO after childbearing

Endothelial Cells

Form tumor-associated (angiogenic) vessels Control leukocyte recruitment Endothelial cells express various MMPs and TIMPs that influence tumor progression. Distinctive gene expression profiles and various signaling pathways are involved in the angiogenic switch of tumor endothelial cells ECs of cancer and tumor cells are very different than normal ECs

Gastroinstential Stromal Tumor

GIST - gastrointestinal stromal tumor - spindle shaped tumor cells CD117 (c -kit) protooncogene (tyrosine kinase) staining Diagnostic Predicts response to targeted therapy with imatinib (TKI)

Response to gefitinib in the primary tumor and thoracic involved lymph nodes

Gefitinib response of erlotinib-refractory lung cancer involving meninges—role of EGFR Mutation Response not a cure Also helps BAC-bronchioalveolar carcinoma

The Granzyme B-mediated Apoptosis Pathway

Granzyme B is a serine protease that is released by cytotoxic T cells and natural killer cells, causing apoptosis of virally infected cells. Granzyme B is released with another protein called perforin, which aids the granzyme B in entering infected cells. Granzyme B then can activate the BH3-only protein Bid by cleaving it, along with directly activating caspases -3 and -8. A third apoptotic pathway is triggered by the action of cytotoxic T lymphocytes and natural killer cells of the immune system. These immune cells can activate death receptors or release granzyme B and perforin that are taken up by the target cell. Once in the cytoplasm, granzyme B activates initiator procaspase 8 along with the BH3-only protein Bid. Granzyme B also directly activates executioner caspase 3.

Fibroblasts and cancer associated fibroblasts

Growth Factors, ECM Proteases, and Chemokines activate normal fibroblasts. Fibroblast activation in response to TGF-β, platelet-derived growth factor (PDGF) and fibroblast growth factor 2 (FDF2) secreted by tumor cells. CAFs function in: Initiation of cancer Tumor progression EMT and metastasis Myofibroblasts - contraction induces "stiffening of the ECM - defined by their expression of alpha-smooth muscle actin. Stiffening allows for better adhesion CAFs induce an altered extracellular-matrix microenvironment, which potentially provides additional oncogenic signals, probably leading to accelerated cancer progression Cross talk is critical. The growth factors play an important role

Pharmaco-economics

Identifies, measures, and compares the costs and consequences of pharmaceutical interventions (products, services, and programs) to health care systems and society. Helps answer the question: Is the added benefit of one pharmaceutical intervention worth the added cost of that intervention? PE is a tool in decision making; not the final answer. PE is sometimes referred to as cost-effectiveness analysis (CEA), although CEA is actually just one type of PE. Must compare 2 different things. . A pharmacoeconomic analysis requires measuring and comparing the costs of a pharmaceutical intervention (i.e., a drug, a product, or a service) and the outcomes that result from the pharmaceutical intervention Although growth rate has been lowering, Prescription drug costs are projected to continue to exceed the growth rates for hospital care and other professional services. Spending on medications is 10% of the national health expenditures Many other countries provide health care coverage to all its citizens via a single, centralized, nationalized system funded primarily by the government (taxes). This single payer system has a finite tax revenue and, therefore, an incentive to determine the cost-effectiveness of pharmaceuticals and medical technology for the citizens of its country. US does not do this PE overlaps with both health care economics and pharmacy-related outcomes research. The outcomes may either be clinical measures, such as percent of patients cured, or humanistic measures, like patients' quality of life or satisfaction with their healthcare.

Evasion via anergic T cells

If a tumor antigen is presented by an activated macrophage or mature dendritic cell, B7 (CD80/86) will be present on the antigen presenting cells (APCs) and the tumor-specific CD8 T cell will be able to become activated and kill tumor cells. However, APCs are often not activated by tumors and therefore, the APCs remain in an immature or inactivated state. These APCs will not express the costimulator molecule B7 (CD80/86) and therefore, T cells specific for the tumor antigens will not become activated. They will become anergic or unresponsive, allowing the tumor to go undetected by CD8 T cells

Gleevec

Imatinib mesylate The bcr-abl fusion gene is the result of a reciprocal chromosomal translocation between chromosome 9 and chromosome 22 (Philadelphia chromosome) Bcr-abl is the critical underlying mutation for Chronic Myeloid Leukemia (CML) The inhibition of the abl kinase activity triggers apoptosis in CML cells. ABL kinase is always on now. Divide, divde divide, survive, survive etc.. Immune system mostly holds up at first. But then turns very lethal. Can live normal life with pill. active in cancers with mutated tyrosine kinases: Bcr-abl CML C-kit Gastrointestinal Stromal Tumor PDGFRB CMML PDGFRA Eosinophilic Leukemia Paradigm: Mutationally activated tyrosine kinases confer oncogene addiction (tumor cells dependent on kinase. Take away signal, cell dies). That makes mutated kinases great cancer drug targets. Relatively selective kinase inhibitor, but not completely selective. T315I. Single point mutation. From threonine to isoleucine. Lines gleevec binding pocket. Pre-existing mutation in patient. Small pocket of cells gains resistance and becomes dominant. Second generations bcr-abl inhibitors have been developed to overcome Gleevec resistance (e.g. Dasatanib, Nilotinib)

P53 in apoptosis

In addition to stress-mediated responses, the intrinsic apoptotic pathway can be triggered by activation of p53. In response to cell damage, DNA damage, checkpoint activation, or anoxia, p53 levels are increased. As a transcription factor, p53 can induce the synthesis of Bax or BH3-only pro-apoptotic proteins Noxa and Puma that will trigger the intrinsic apoptotic pathway. In addition p53 can also upregulate transcription of factors that are secreted by the cell and bind to survival cytokines. This effectively blocks the survival cytokine signal pathway, and can trigger apoptosis. Lastly, p53 can also increase the transcription of death receptors that mediate the extrinsic pathway. p53 is a tumor suppressor. Mutations in p53 are present in about half of human cancers. Its own expression is upregulated by multiple types of cell stress. In the nucleus, it can upregulate the transcription of certain BH3-only proteins, promoting apoptosis. In the cytosol, it can interact with Bax and Bak to promote their oligomerization and cause mitochondrial membrane permeability. p53 can also promote transcription of various death receptors.

Metabolism in resting vs proliferative cells

In resting cells, catabolic metabolism is met through fatty acid oxidation and the oxidative metabolism of glucose. Proliferating cells increase glucose and glutamine uptake for energy needs, biosynthesis of nucleotides, lipids, and proteins, as well as the maintenance of redox homeostasis. Need more energy and need more building blocks. Up-regulation of glucose transport into cells ~ GLUT1 Glucose and glutamine support cell growth and proliferation. The carbon skeletons from glycolysis and the TCA cycle are used for the synthesis of macromolecules - amino acids, lipids, and nucleic acids.

Necrosis vs apoptosis

In some pathological conditions (e.g. ischemic injury), both necrosis and apoptosis can occur. The "decision" to undergo necrosis or apoptosis is probably determined by the extent of cell damage: greater insult = necrosis; lesser insult = apoptosis

Intrinsic mitochondria dependent apoptotic pathway

Includes steps 1-4 from previous card. -After step 4, Seven molecules of an adaptor protein, Apaf-1, combine with seven cytochrome C protein molecules (released from mitochondria) to form the apoptosome, or the "wheel of death". The apoptosome then recruits procaspase-9 (an inactive form), which then self-processes and becomes activated. Regulatory proteins: IAP - Inhibitor of Apoptosis, cytoplasmic protein Function to inhibit caspases Smac, a.k.a. DIABLO: promotes apoptosis It is a mitochondrial protein that is released with cytochrome C. Binds to and inactivates IAPs

Epi-genome targeting

Inhibit DNA methylation, up regulate tumor suppressors Both increase suppressors

EGFR Mutation analysis

Inhibitor blocks EFGR. In colon. Drug inhibits cancer proliferation in colon. Doesn't work in the lung Use tyrosine kinase in lung

All kinds of migration

Integrins functions as the main adhesion and mechanotransduction system for tumor cell invasion through the ECM.

Breast cancer case study

Jane should have genetic testing Negative for 5- site (most common mutations) BRCA1 Negative for BRCA2 Hard to determine if she needs a mammogram. Test other positive family members Target other family members. When possible - there are always exceptions. Identifying a familial mutation allows for targeted testing of family members. Improved result interpretation Limited family structure. Only men on left side. Doesn't actually skip generations- just lower penetrance for men Sister's breast cancer was ER/PR/Her-2neu negative (triple negative) Jane's sister has BRCA1/2 testing. Deletion of exons 21-24 on BRCA1 Jane must do additional testing to see if she has this mutation. After testing, true negative Should not order mammogram. basically has general population risk. Start at age 40 If jane's sister had a negative result, Jane has higher risk than general population based on family history Maternal and paternal equally important

Cancer of the immune system

Leukemia - cancer of circulating immune cells Lymphoma - solid lymphoid tumors Myeloma - tumor of plasma cells in bone marrow

Immune cells in tumor development

MDSC- Myeloid derived suppressor cell

Vinca alkaloids

Microtubule Destabilizers Vinblastine and Vincristine Bind to tubulin and induce microtubule depolymerization (Disassembly) Cells are arrested at metaphase as no chromosome segregation is possible Have activity in a multitude of cancer, e.g. Hodgkin's disease, breast, germ cell Vincristine has revolutionized the treatment of childhood leukemia 1. Vinca alcaloids bind to tubulin dimers at a specific recognition site on the protein 2. Tubulin form paracrystaline aggregates 3. Concentration of the free dimers is reduced 4. Equilibrium of growth/shrinkage of tubules shifted to the shrinkage 5. Mitotic spindles can not be formed from rosy periwinkle plant Vinca rosea (native to Madagascar);

Taxanes

Microtubule stabilizers Paclitaxel, docetaxel Mechanism of action = mitosis block: promote microtubule assembly and super stability action is opposite to vinca alcaloids, but effect is the same Came from pacific yew tree. hard to synthesize in lab. taxol Discovery of close analog, Baccatin III, in the leaves of European species of ornamental shrub (Taxus baccata) made clinical trials possible

Informed consent for genetic testing

Minimum discussion points to attain informed consent: Syndrome, genes to be tested, associated risks Possible results and implications (including VUS) Options to assess risk without testing Implications for family members - importance of sharing information Accuracy of testing Cost Psychological implications Genetic discrimination risks Options and limitations of result-based management Follow up plan Until June 2013, Myriad genetics held BRCA patent Now: multiple labs and multiple options New methods: next generation sequencing Difficult to detect deletions Panels include 6-51 genes w/ some combo of: Other high penetrance genes associated with breast cancer (TP53, PTEN) Low penetrance genes associated with breast cancer (CHEK2, ATM) Cancer genes that are associated with other syndromes - maybe not breast cancer (Lynch genes, thyroid genes) Other genes that are less studied and may/may not contribute to risk Complicates informed consent process

Cells of the tumor microenvironment

Myofibroblasts / Fibroblasts: Tumor initiation, growth and tumor invasion by incorporation of α-smooth muscle actin fibers Adipocytes : Adipokines lead to increased tumor cell migration and tumor invasion by regulating expression and activation of MMPs Tumor endothelial cells: Leucocytes recruitment and tumor cell behavior and metastasis Pericytes: Stabilize blood vessels, inhibit endothelial cell proliferation, maintain capillary diameter, regulate blood flow, provide endothelial survival signals. Tumor associated macrophages : Immune regulation, promote cell growth and promote tumor development Dendritic cells: Induce vascularity and involved in tumor immune-pathogenesis Immune cells: Promote growth and progression of cancer Mast cells: Promote tumor development by disturbing the normal stroma-epithelial communication, facilitating tumor angiogenesis, releasing growth factors, and inducing state of immunosuppression.

MMR proteins

Normally present (brown stain) If protein is absent, gene is not being expressed (mutation or methylation)

Nuclear hormone receptor signaling

Nuclear hormone receptors are transcription factors. Binding of their respective ligands (e.g. estrogen, testosterone) leads to translocation from the cytoplasm into the nucleus. In the nucleus, hormone receptors promote the transcription of specific target genes. Biological insight: Breast tumors are often dependent on estrogen receptor signaling. Selective Estrogen Receptor Modulators (SERMs) are used to antagonize the estrogen receptor in breast cancer (e.g. tamoxifen) Prostate tumors are often dependent on androgen (testosterone) receptor signaling. Selective Androgen Receptor Modulators (SARMs) are used to antagonize the androgen receptor in prostate cancer (e.g. bicalutamide) These are one reason why death rates are so low for these cancers.

Intravasation

Occurs at the primary site. Invasion of tumor cells through the basement membrane and across or between the endothelial cells into either the blood or lymph. Active via TAMS factors (e.g.) TNF-alpha and chemokines and nutrients gradients Passive due to increased vascular permeability and pushing? Intravascular survival- pic. Once in the bloodstream circulating tumor cells must overcome many threats. Cancer cells are coated with platelets which provides protection from stress and immune cells. Platelets guide tumor cells to site of inflammation or tissue injury (easier to invade the damaged tissue)

Cost-effectiveness analysis (CEA)

PE analysis where outcome is measured in natural or clinical units like life years gained, mm Hg blood pressure, mmol/L blood glucose. CEA is most common type of PE analysis Two methods of reporting cost-effectiveness: Average Cost-Effectiveness Ratio (CER) = Cost of Intervention/ Effectiveness of Intervention Incremental Cost-Effectiveness Ratio (ICER) = Cost of Intervention B - Cost of Intervention A/ Effectiveness of Intervention B - Effectiveness of Intervention A CER looks at one intervention while ICER looks at two Study: Average CER: With the vaccine and subsequent screening, the cost would be $42,683 per case of cancer prevented vs. $39,964 for the current screening program, about a 7% increase in cost per case of cancer prevented. Incremental CER: The incremental cost-effectiveness is $57,800 per additional case of cancer prevented by employing the vaccine + screening alternative instead of the screening only alternative. You can think of it like this: assume that with either alternative, the cost of each case of cancer prevented for the first 2.78% of women avoiding cancer is the same, $39,964. For each case of cancer prevented beyond 2.78% and until 3.28% of women avoiding cancer, the cost is $57,800. Is preventing one additional case of cervical cancer worth $57,800? This is a judgment call but our policy makers have said yes. Avoiding cervical cancer can save lives; also, there are other benefits to the HPV vaccine to consider, such as the avoidance of genital warts. Common CEA application: medications with the same type of primary outcomes, and most often for treatment of the same types of health condition CEA is only performed when the outcome of one intervention is both better than another AND the cost is greater. Advantages: Health units are common outcomes routinely measured in clinical trials - familiar to clinicians Outcomes are easier to quantify than CUA or CBA Disadvantages: Interventions with different types of outcomes cannot be compared Can't combine more than one important outcome Difficult to collapse both the effectiveness and the side effects into one unit of measurement CEA estimates extra cost associated with each additional unit of outcome, but who is to say that added cost is worth added outcomes? Requires judgment call.

Cost-minimization analysis (CMA)

PE analysis where outcomes of two or more interventions are assumed to be equivalent Thus, only costs of intervention are compared Objective: choose the least costly alternative Example: Substantial cost savings were found with prostaglandin E2 therapy in an outpatient rather than an inpatient setting for patients who required an induction of labor and were candidates for outpatient cervical ripening Common CMA application: Cost comparison of two generic medications rated as equivalent by FDA Cost comparison of same drug therapy in different settings Not appropriate for comparing different classes of medications Advantage: simplest analysis to conduct Disadvantage: cannot be used when outcomes of each intervention are different

Diseases linked with apoptosis

Suppression of apoptosis: Cancer Atherosclerosis- suppression of macrophages Autoimmune disorders Increased apoptosis: Viral infections (AIDS) Neurodegenerative disease (Alzheimer's) Multiple sclerosis Ischemic injury (Myocardial infarction) Toxin-induced diseases (alcohol-induced hepatitis)

Cancer treatment options

Surgery- most straight forward Radiation Therapy Cytotoxic Chemotherapy (Target: DNA Metabolism) Molecularly Targeted Therapy (Target: Proliferation and Survival Pathways) Biologics (especially Antibodies) Vaccines

Telomeres

Telomeres are a tandem repeats of TTAGGG units from 5-15 kb on the ends chromosomes. Telomeres protect the open ends of chromosomes from enzymatic digestion and associate with each other. These associations are involved in transcription regulation, facilitate sister chromosome pairing at mitosis, anchor chromosomes to nuclear membrane. They facilitate complete replication of the DNA and serve as a "mitotic clock" by shortening by 50-100 bp per replication. When you don't have anymore telomere, the cell dies Overexpression of telomerase is key for the telomeres to stay intact long after they are supposed to be degraded because it keeps adding bases to the telomeres. When telomere falls off, telomerase puts on another. Allows cancer cells to become immortal Stem cells have long telomeres that are not shortened. Have telomerase. The clinical implications on this process include impaired healing and repair, and age-related disease. Telomerase is a reverse transcriptase (TERT) that contains an RNA template (hTR) that adds TTAGGG repeats to chromosome ends. Normal cells don't have mRNA or protein from the hTERT gene, it is present in embryonic and adult stem cells, and in cancer stem cells.

Testing children

Testing minors for adult-onset cancer predisposition is not generally recommended unless it will alter management Eg. If youngest colon cancer in the family was at age 20 for instance Some cancer syndromes affect children and testing is recommended Insurance discrimination concern GINA protects health insurance and employment GINA does NOT protect life, long term care, or disability insurance For this case - sister's children should not be tested yet

Proteasome targeting

The inhibition of the proteasome is thought to disrupt the balance of signaling and survival pathways which can result in an anti-tumor effect. Tumor suppressor genes instead of oncogenes like kinase inhibitors. By inhibitng proteasomes, maybe increase tumor suppressor proteins.

Carcinoma and ECM

The stromal cells and the extracellular matrix combine to form the tumor microenvironment. A critical concept to understand is that there is a great deal of bilateral communication (i.e. crosstalk) between the malignant cells and the stromal cells and extracellular matrix. The tumor microenvironment is an essential part of a tumor, and its relative contribution to the tumor mass often exceeds that of neoplastic cells (here invasive squamous cell carcinoma). The microenvironment consist of various stromal cells (seen here as cancer-associated fibroblasts, inflammatory cells (macrophages, neutrophils, mast cells, and T and B lymphocytes), endothelial cells, pericytes), the components of the acellular extracellular matrix (ECM), blood vessels, and numerous growth factors, cytokines/chemokines and remodeling proteins that are dispersed within it. Together they tumor cell proliferation and invasion. There is a significant clinical relevance of the tumor microenvironment to tumor progression. Tumor progression involves tumor cells must be able to: move and degrade the ECM and initiate angiogenesis. There is extensive "cross-talk" between the tumor cells the stromal cells and ECM of in the microenvironment. This "cross'talk" is translated into transcription programs leading to epithelial-mesenchymal transition, movement through the ECM and establishment of metastatic foci. Leading edge of tumor looks different. Due to cross talk. Have a different function

Cross-Talk

This cross talk involves a wide variety of factors, many of which control wound healing, and signalling pathways. This cross talk is highly regulated at by those tumor cells actively invading the extracellular matrix. Cancer-associated fibroblasts play a central role in the cross talk. Their factors regulate angiogenesis, and enhance tumor cell proliferation and invasion while inhibiting apoptosis. These factors include growth factors, cytokines, proteases, and extracellular matrix proteins. These include FGF-2 (fibroblast growth factoir-2) VEGF (vascular endothelial growth factor), TGF-β (tumor growth factor-beta), HGF (hepatocyte growth factor), and the MMPs. You have seen these same factors in wound healing.

Tumor microenvironment progression

This figure demonstrates the changes in the stroma (cells and ECM) supporting development of a tumor of the breast. A) A basement membrane separates the normal glandular epithelium from the underlying cellular and acellular components of the stroma. B) In DCIS (ductal carcinoma in-situ), the lumen contains neoplastic cells. The wall of the duct is irregular, the myoepithelial cell layer is indistinct, but present, and the basement membrane is intact. During this phase, the stromal components are modified to support epithelial cell proliferation and mutation. There is activation and proliferation of fibroblasts, macrophages and other leukocytes, as well as angiogenesis. C) Ductal carcinoma is characterized by tumor cell invasion through the basement membrane. Stromal invasion by the tumor cells is characterized by collagen degradation, inflammatory cells increases and fibroblasts differentiate to myofibroblasts resulting in the expression of growth factors, matrix components and degrading proteases. Angiogenesis is maintained resulting in the development of new blood vessels.

Camptothecins

Topoisomerase I inhibitors topotecan, irinotecan Topoisomerase I produces reversible single-strand breaks in DNA These single-strand breaks relieve torsional strains, and allow DNA replication to proceed. Topotecan binds to the topoisomerase I-DNA complex and prevents ligation of the DNA strand, resulting in DNA breakage during the elongation and cell death TOPO I is not required for the viability of cells (TOPO II can substitute).Therefore, TOPO I - negative cancer cells are viable and resistant to topotecan

Epipodophyllotoxins

Topoisomerase II inhibitors Etoposide / Teneposide: Inhibition of cell division in late S-G2 phase of the cell cycle. Inhibition of topoisomerase II results in DNA damage through strand breakage induced by the formation of a ternary complex of drug, DNA and enzyme. Topoisomerase II binds to supercoil that allows one arm to rotate around the other, reduces torsional strain. Double strand

Chronic Myelogenous Leukemia

Translocation between 9 and 22 Fusion is yellow BCR/ABL has tyrosine kinase activity. Lots of proliferation and decreased apoptosis Gleevec is a tyrosine kinase inhibitor. Great drug

Invasion and metastasis

Tumor cell invasion begins with activation of signaling pathways that control cytoskeletal dynamics, and alteration of expression of cell adhesion molecules involved with cell-matrix and cell-cell adhesions. Then come invasion through the ECM. Metastasis occurs when the invading tumor cells penetrate basement membranes and endothelial walls of blood and lymph vessels, and disseminate through the circulation to colonize distant organs. The process is highly inefficient as indicated by the fact that less than 1 in 10,000 cells that enter the circulation form a metastatic focus. Detachment from the primary mass - epithelial - mesenchymal transition (EMT) Invasion through the basement membrane and migration through the extracellular matrix Penetration (intravasion) of blood and/or lymphatic vessels Survival within the circulation Arrest in the distant organ's microcirculation (capillaries, sinuses or venules) Penetration of vessels at the distant site Invasion through the distant site's parenchyma Tumor cell invasion is a heterogenous and adaptive process, i.e. plasticity characterizes the process. Tumor cells migrate in two major ways. Individual cell migration - no cell adhesion Collective cell migration - multicellular group when cell adhesion molecules are retained Involves cytoskeletal coupling with cell surface receptors (integrins)

Mast cells

Tumor mast cells have a marked impact on the various hallmarks of cancer via the synthesis and release of: various growth factors (e.g. EGF, PDGF, stem cell factor (SCF), TGF-β TNF-alpha, FGF), VEGF, MMPs, and chemokines (e.g. IL-8)

Warburg Effect

Under conditions where oxygen is plentiful, cancer cells metabolize glucose by glycolysis rather than by oxidative phosphorylation. It is a less efficient way to produce ATP. Leads to increase uptake of glucose. Useful for PET (positron emission tomography), using 2-deoxy-2(18F)-fluoro-D-glucose, scans that demonstrate a significant increase in glucose uptake in tumors and proliferating cells compared with adjacent normal tissue. 2D instead of 3D but it helps to identify tumor cells needing extra glucose. Do this because they need to produce the macromolecules as much as they need energy. Pic- 1 population secretes the lactate, the other takes it up. Recent studies indicate that the activation of protooncogenes (e.g., Myc), signaling pathways (e.g., PI3K), and transcription factors (e.g., HIF-1), as well as the inactivation of tumor suppressors (e.g., p53), induce the Warburg effect in cancer cells

Stem cells

Undifferentiated stem cells have 2 daughter cells: a clone that allows for self renewal of the stem cell, and a progenitor cell that can differentiate The stochastic model calls for cancer to be a proliferative disease due to serial gene mutations that promote proliferation and reduce apoptosis, and that all tumor cells can produce a tumor. The Cancer Stem Cell Model calls for tumors to arise from a distinct population of cancer stem cells that have the properties of normal stem cells. Mutations of normal stem cells leads to altered regulation of self renewal that results in unregulated proliferation. This further supports the concept that tumors are a clonal disease. Both normal stem cells and cancer stem cells have telomerase, and they share the same cell signalling pathways, but they are dysregulated in the cancer stem cells Mutations in a normal stem lead to a cancer stem that is needed for the initiation and long term maintenance of the tumor. Responsible for the expansion of the tumor - incapable of long term maintenance of the tumor. Cancer stem cells may arise due to mutations in specific stem cells or early stem cell progenitors. It is also possible that Cancer Stem Cells can be derived from late progenitors and more differentiated cells that acquire the ability to self-renew. Have stem cells in the bone marrow and in the basal lamina of epithelium. It is the adult stem cells that cause cancer

Monoclonal antibodies detect and treat cancer

monoclonal antibody - antibodies produced by a single B cell clone and thus are all identical in structure and antigen specificity. Can become flourescent and show where tumors/metastases exist For non-hodgkins lymphoma, anti-CDC-20 conjugated to radionuclide. The radioactive antibody binds to the malignant B cells and irradiates them. The radiation damages the cell's DNA and kills them; only kills the malignant cells, not the rest of the normal cells

Cancer genetics

most cancers are not inherited 15-20% familial 5-10% inherited- one strong acting gene 70-80% sporadic- lower penetrant genes. Not really strong acting genes. Lifestyle factors cause increased risk Clues: -Cancer in 2 or more close relatives (on same side of family) -Early age at diagnosis (under 50) -Bilateral/multiple cancers (not metastases. Separate cancer diagnoses) -Multiple rare cancers -Multiple primary tumors (breast and ovary; colon and uterus) -Evidence of autosomal dominant transmission. incomplete penetrance

Evasion via TGF-beta

neutralizes CD4 and CD8 T cells in microenvironment. T reg down regulates both CD8 and CD4, produces anti-inflammatory cytokines