Genetics — Gene Therapy; Genetic Testing; and Cancer – Flashcards
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What are the three approaches to treating genetic disorders?
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(1) Addressing a genetic deficiency (augmentation); (2) eliminating positive harmful effects; and (3) altering disease susceptibility (prevention).
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What are some examples of how we can address a genetic deficiency through augmentation therapy?
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Cochlear implants; organ or cell transplants; or supplying a functional gene product.
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What is a positive-harmful effect?
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Describes the presence of any molecule which is harmful. Examples include excess protein buildup, or the presence of autoantibodies.
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What is the result of a mutation causing 21-hydroxylase deficiency?
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(1) a lack of mineralocorticoid and glucocorticoid hormone; and (2) buildup of precursor molecules and a resultant increase in sex hormones (a positive harmful effect).
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What is the treatment for 21-hydroxylase deficiency?
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Supply the hormones that are lacking (augmentation therapy) and eliminate excess sex hormones (eliminate the positive-harmful effect).
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What is type 1 tyrosinemia?
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A genetic disease characterized by a lack of FAH enzyme, and a resultant buildup of precursor molecules which leads to liver damage and neurologic crisis.
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What is the treatment for type 1 tyrosinemia?
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Inhibition of an upstream enzyme involved in the formation of harmful precursors (eliminate the positive-harmful effect). This is an example of inhibitory treatment.
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What are some ways to alter disease susceptibility?
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Remove the factor which causes the disease (for example: avoiding lactose in lactose-intolerance); or avoiding certain environmental exposures.
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What is genetic treatment of disease?
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The application of genetic manipulations to treat both genetic AND non-genetic disorders. Such techniques may be used in combination with conventional drugs or therapy.
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How is genotyping used in the treatment of disease?
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It can be used to determine a person's response to a drug or therapy, and determine their rate of metabolism for a particular drug (slow vs normal vs fast).
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How are genetic techniques used in the production of drugs and vaccines?
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Genes are cloned and expressed in suitable cell cultures in order to purify recombinant protein products (such as hormones, enzymes, or antibodies).
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What is gene therapy?
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Genetic modification of a person's cells for a therapeutic purpose -- this is the ultimate application of genetics in treating disease.
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What are the two categories of genetic treatment of disease?
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(1) The production of small molecule drugs; and (2) genetically engineered molecules.
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What are the two types of genetically engineered molecules?
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(1) Recombinant proteins; and (2) monoclonal antibodies.
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What is drug repurposing?
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Using an existing drug for a new purpose. This is useful because the drug's safety profile is already known; thus, lengthy and expensive clinical trials can be avoided.
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What is pharmacokinetics?
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What the body does to the drug; includes absorption, distribution, metabolism, and excretion.
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What is pharmacodynamics?
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What the drug does to the body; the therapeutic effect -- includes Phase 1 and Phase 2 metabolism.
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What is phase 1 metabolism.
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Reactions carried out by monooxygenases, which are deigned to make the drug more polar; can also include the activation of a prodrug into an active metabolite.
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What is phase 2 metabolism?
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Conjugation reactions catalyzed by transferases.
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What is pharmacogenomics?
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Studying the effects that specific genes have on drug metabolism and drug effects.
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What are the two types of gene therapy?
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(1) Germ-line gene therapy; and (2) somatic cell gene therapy.
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What is germ-line gene therapy?
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Modification of the DNA within a gamete, zygote, or embryo. This produces a permanent modification which can be transmitted to descendants. There are enormous ethical considerations with this type of therapy.
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What is somatic cell gene therapy?
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Modification of cells or tissues which are specific to the patient; thus the effects are confined to the patient.
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What are the two techniques in somatic cell gene therapy?
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(1) Selective modification of the disease cells; or (2) selective killing of the disease cells.
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How does access to the target cell prove to be a problem in gene therapy?
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Some cells, such as blood cells, are easily accessible. However, accessing the cells of internal organs is difficult and invasive.
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How does the replicative potential of a cell prove to be a problem in gene therapy?
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Cells which don't divide, such as muscle cells, require repeated injection and administration of gene therapy.
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What is viral gene therapy?
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Using a viral vector to introduced a particular gene. Viruses are used because they are self-replicating, and can integrate into the host genome.
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What are some problems with viral gene therapy?
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There is a package limit -- large genes cannot be used; we do not have control where the genes will insert -- harmful oncogenes may be activated.
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What is the difference between In vivo and Ex vivo gene therapy?
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In vivo gene therapy occurs entirely within the body; Ex vivo gene therapy involves taking cells out of the body, manipulating them, and returning them to the body.
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What are some of the different reasons that we employ genetic testing?
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To check identity or biological relationship; for forensic or legal purposes;to trace ancestry; and to better understand normal genetic variation within different populations.
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How do we evaluate a particular genetic test?
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We use the ACCE Framework; it stands for: Analytical validity, Clinical validity, Clinical utility, and Ethical validity.
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What is analytical validity?
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Assesses how well the test measures what it claims to measure; it includes sensitivity and specificity.
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What is sensitivity?
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The proportion of people who are correctly identified as having a disease; if a genetic test lacks sensitivity, it will produce false-positive results.
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What is specificity?
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The proportion of people who are correctly identified as NOT having a disease; if a genetic test lacks specificity, it will produce false-negative results.
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What is clinical validity?
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Assesses how well the test predicts the projected health outcome.
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What is clinical utility?
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Assesses the usefulness of a genetic test.
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What is ethical validity?
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Assesses whether or not the test meets expected ethical standards.
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What are the two broad categories of genetic testing?
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(1) Those which detect large-scale DNA changes; and (2) those which detect small-scale DNA changes.
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What are the two tests used for detecting large-scale DNA changes?
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(1) Microarray-based genomic copy number analysis; and (2) chromosome FISH.
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Why can't we use standard karyotyping to detect large-scale changes in DNA?
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It will not detect deletions and duplications involving less than 5-10 Mb of DNA.
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What is the principle behind microarray-based genomic copy number analysis?
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It involves the application of microarray-based hybridization to scan the DNA of each chromosome for changes in copy number.
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Describe the process of microarray-based genomic copy number analysis:
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DNA fragments from a test sample and DNA fragments from a control sample are combine on one chip; the chip is analyzed for a 1:1 binding ratio. If there is a deletion the ratio is reduced and if there is an insertion the ratio is increased.
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How are changes in copy number analyzed with microarray-based genomic copy number analysis?
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A change in copy number is denoted by a color fluorescence; a computer analyzes the results and tells you where the abnormalities are located.
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Why can't this technique be used for all large-scale changes in DNA?
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It cannot detected balanced chromosomal rearrangements in which there is no net gain or loss of DNA -- for this we must use chromosome FISH.
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Describe the process of chromosome FISH:
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Chromosomes are fixed to a slide and then denatured; fluorescent probes of interest are hybridized; the location of fluorescent signals are recorded against a background stain that binds to all the sequences.
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What is an example of a chromosomal aberration that can be detected using chromosome FISH?
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Presence of the BCR-ABL gene that is characteristic of CML.
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What three genetic tests are employed to detect small scale changes in DNA?
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(1) Amplification Refractory Mutation System (ARMS); (2) oligonucleotide ligation assay; and (3) pyrosequencing.
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What is the underlying principle of ARMS?
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PCR cannot proceed if the oligonucleotide probe does not bind to the 3' end; this technique involves monitoring for a PCR product in order to determine if a mutation is present.
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Describe the process of ARMS:
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PCR is conducted using both a common primer and an allele specific primer; if a mutation is present you will get no PCR product; you can then repeat the experiment to identify which nucleotide substitution is present.
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What is a limitation of the ARMS technique?
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It requires you to have prior knowledge of the mutation location.
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What is oligonucleotide ligation assay?
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A genetic test which employs the same concept as ARMS; this technique includes an extra molecule that will produce a fluorescent signal when a PCR product is produced.
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What is the underlying principle behind pyrosequencing?
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When a nucleotide is incorporated into a growing strand, pyrophosphate is released and can be detected.
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Describe the process of pyrosequencing:
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dNTPs in separate reactions are supplied in a defined order; normal and mutant sequences are discriminated by the presence of a fluorescent signal.
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What are the six practical applications of genetic testing?
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(1) Testing of affected individuals and their close relatives; (2) prenatal diagnosis; (3) pre-implantation genetic testing; (4) pre-symptomatic testing; (5) genetic carrier screening; (6) cancer diagnostics.
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What are the three important ethical considerations in regards to genetic testing?
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(1) Consent for genetic testing; (2) sharing of genetic information; and (3) confidentiality of test results.
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What are some special consent consideration in regards to children?
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Children should NOT be tested for adult-onset disorders; children should NOT be tested for carrier status.
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What does the concept of beneficence assert in regards to genetic testing in children?
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We should only test for relevant and actionable variants; disclosure of other genetic information could infringe on the child's future autonomy.
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What are designer babies?
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The use of in vitro fertilization and pre-implantation to preferentially select embryos with desired qualities and reject the rest; this process is considered unethical.
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What are the two types of tumors, in regards to clonal origin?
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Polyclonal and monoclonal.
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What is a polyclonal tumor?
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A tumor which forms when multiple cells transform to malignancy to become ancestors of several distinct sub-populations of cells within a tumor mass.
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What is a monoclonal tumor?
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A tumor which forms when only a single cell is transformed to malignancy to become the ancestors of all cells within the tumor.
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What are the three pieces of evidence implicating adenoma to carcinoma progression?
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(1) Carcinoma can often be seen growing directly out of adenoma; (2) Removal of colonic polyps has been shown to reduce the risk of developing colon cancer by 80%; (3) Among three premalignant carcinomas in situ, only those of the LLL of the lung develop into squamous cell carcinoma.
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What is asymmetric division of stem cells?
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Describes division in which one daughter cell remains a stem cell, and the other spawns a transit-amplifying cell.
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What type of stem cell division typically occurs in adult tissue?
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During injury -- symmetric division repopulates the stem cell pool; if an organ is growing -- symmetric division increases the size of the pool; during normal tissue function -- asymmetric division maintains the organ.
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How are stem cells typically organized in tissue?
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They are anatomically shielded from potential damage; for example, GI stem cells are organized into gastrointestinal crypts.
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What are the two distinct phases of the inasion-metastasis cascade?
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Phase 1: physical dissemination of core cells from the primary tumor to the parenchyma of distant tissue; Phase 2: colonization -- adaptation of disseminated cancer cells to the microenvironment of the new tissue.
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What is the R-point of a cell?
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A discrete window of time in which cells respond to extracellular growth factors and inhibitory factors; begins at the onset of G1 and ends just before the onset of G1.
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What occurs during the R-point?
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Cells commit to either advance through the M-phase, remain in G1, or retreat to G0.
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What is the cell cycle clock?
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Denotes cellular circuitry operating in the nucleus which integrates a variety of signals and decides whether or not to enter active cell division.
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What types of signals determine whether or not the cell will divide?
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Growth factor receptors; monitors of genome integrity; and monitors of cell metabolism.
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What is P53?
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A tumor suppressor gene whose expression increases in response to a variety of physiologic stresses.
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Which factors can activate P53?
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Lack of nucleotides; UV; ionizing radiation; oncogene signaling; hypoxia; and blockage of transcription.
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How does P53 expression suppress tumor formation?
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Accumulation of P53 protein can induce cell-cycle arrest, mobilization of DNA repair proteins, mobilization of proteins that block angiogenesis; or initiation of apoptosis.
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What is the major determinant of the tendency for metastasis formation in distant tissue?
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The microenvironment of distant tissue; the layout of the circulation can also play a role (for example: presence of the portal vein increases the potential for GI cancer to spread to the liver).
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Describe the use of expression arrays in breast cancer?
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Expression arrays and bioinformatics analyses have been used to identify 70 prognosis genes; a threshold was developed to separate tumors with a good expression signature from those with a bad expression signature.
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How does the presence of self-renewing tumor stem cells influence the response to treatment?
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Failure to destroy cancer stem cells can result is relapse at both primary and secondary sites; both cancer stem cells and other cancer cells must be destroyed in order to provide a cure.
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What are the three ways to classify tumors?
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Based on (1) differentiation state; (2) embryonic origin; and (3) biological behavior.
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What are the classifications based on differentiation state?
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Epithelial; non-epithelial; or mixed.
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What are the classifications based on embryonic state?
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Ectoderm; endoderm; or mesoderm.
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What are the classifications based on biological behavior?
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Benign or malignant.
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What is another way to categorize tumors?
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Based on the tissue or cell of origin (fibroblasts = fibroma/fibrosarcoma; bone = osteoma/osteosarcoma; cartilage = chondroma/chondrosarcoma; endothelial = hemangioma/hemangiosarcoma).
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What are the two ways classify cancer genes?
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As either positive (classic oncogenes; telomerase) or negative (P53) regulators of differentiation.
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What are the ten biological capabilities which are hallmarks of cancer cells?
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Self sufficiency in growth signaling; insensitivity to signals suppressing growth; avoid apoptosis; replicative immortality; genome instability; induction of angiogenesis; tissue invasion; avoid immune destruction; induce inflammation; aerobic glycolysis.
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What are driver mutations?
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Mutations which are responsible for the multistage evolution of cancer; each driver mutation results in an expanded clone -- this causes carcinoma to develop over several decades.
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What is PTEN?
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An important tumor suppressor gene whose loss of expression can have variable effects on the development of cancer.
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How is therapy chosen for cancer?
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Therapy is aimed at addressing each of the 10 hallmark abnormalities (for example: telomerase inhibitors; VEGF inhibitors; aerobic glycolysis inhibitors, etc.)
