ASTRO 2023 (4-15) Flashcards

Question

VI-5) The components typically required for the analysis of a standard, adherent cell clonogenic survival assay require all of the following, EXCEPT: A. Calculation of a plating efficiency B. Colony formation rates at a range of cell densities, for several radiation doses C. A cell line capable of multiple cell divisions D. Intact apoptosis pathways E. Nonirradiated control

Answer 1

VI-5) D The clonogenic survival assay measures the ability of single cells to divide continuously after a given exposure, and typically measures colony formation 7-14 days after exposure to the agent. It requires a normalization in which the number of colonies formed is divided by the number of cells seeded (in the absence of any DNA damaging agent), which yields the plating efficiency. Surviving fraction is then calculated for each dose of a given agent by dividing the number of colonies formed by the number of cells seeded and normalizing to the “0 Gy” plating efficiency. Multiple doses and cell densities typically are needed for the adequate analysis of cell survival. This is not a short-term growth delay assay, and thus a cell capable of multiple cell divisions is needed. DNA damaging-agents induce cell death via a number of pathways, including apoptosis. However, apoptosis is not the sole cell death pathway

Answer 2

VII-1) C Assuming that all cells in the cell population are identical and that cell killing is a random, probabilistic process that follows a Poisson distribution. One model that can calculate the radiation dose that produces an average of one lethal hit is the single-target single-hit model. From the equation that describes this model, S = e , the dose, D, at which there would be an average of one hit per cell would be equal to D0, the constant of proportionality. Therefore, S = e-1 ~ 0.37.

Answer 3

VII-2) E The / ratio represents the dose at which the D component of cell killing, assumed to result from single hit killing, is equal to the lethality produced by the D2 component of cell killing that results from multi hit killing.

Answer 4

VII-3) D Cells derived from an individual diagnosed with xeroderma pigmentosum are defective in nucleotide excision repair (NER). These cells are sensitive to UV radiation because this form of radiation produces damage such as pyrimidine dimers that are removed through the nucleotide excision repair pathway. Because DNA double-strand breaks are important lesions responsible for lethality in cells exposed to X-rays and because DNA double-strand break repair is generally normal in cells derived from a person diagnosed with xeroderma pigmentosum, their cells are not expected to be especially sensitive to X rays. People with Nijmegen breakage syndrome, LIG4 syndrome, ATR-Seckel syndrome and ataxia telangiectasia, who possess mutations in either NBS1, LIG4, ATR or ATM, respectively, are all characterized by defects in strand break repair or repair-related signaling. Therefore, their cells would be expected to be sensitive to X rays.

Answer 5

VII-4) E Parameters to define a radiation cell survival curve include: the initial slope (D1), a final slope (D0), and some quantity that is a measure of the width of the shoulder. This quantity can be the extrapolation number (n) or the quasi-threshold dose (Dq). If n=1, the survival curve has no shoulder and D37 (dose resulting in a survival fraction of 0.37) equals the D0. D0 is a measurement of radiosensitivity based on the exponential part of the survival curve. In a target theory, it can be defined as a dose that gives an average of one lethal hit per cell in a population. Thus, an increase of an exposed dose of D0 would result in a 37% further reduction of survival. A smaller D0 value indicates a higher sensitivity to radiation. For the same radiation dose, radiation delivered at a lower dose rate may produce less cell killing than radiation delivered at a higher dose rate because sublethal damage repair occurs during the protracted exposure. As the dose rate is reduced, the slope of the survival curve becomes shallower and the shoulder tends to disappear because does not change significantly but trends to zero in the linear quadratic model. The inverse of the D0, not the Dq, is equal to the final slope of the survival curve. For densely ionizing radiation (increasing LET), the shoulder of the survival curve tends to disappear. N, therefore, decreases, until it reaches a value of 1.0 for very high LET radiations. The D0 would not necessarily be a good predictor for the effect of fractionation on survival; Dq or n would be better.

Answer 6

VII-5) A Reoxygenation generally occurs over a period of hours to days. Little to no reoxygenation of hypoxic cells is therefore likely during irradiation performed at dose rates in the 1-10 Gy/min range since, for a total dose of 6 Gy, the irradiation times would only vary from 0.6-6 minutes. [](http://) If the total treatment time is long enough that significant repair of sublethal damage (half-time on the order of 0.5-1.0 hour) can occur during irradiation, repair does influence cell survival. The irradiation time would vary from 6-60 minutes for dose-rates in the range of 1-0.1 Gy/min and significant repair would occur. Movement of the surviving cells through the cell cycle (causing redistribution of viable cells from resistant phases into sensitive phases) can influence the radiosensitivity of the cell population when irradiation times are increased to several hours (for the dose-rate range of 0.1-0.01 Gy/min, times of 1-10 hours would be needed to produce 6 Gy. Repopulation can lead to an increase in the number of cells during irradiation and, hence, to an increase in the total number of surviving cells when a radiation dose is delivered over days (10-100 hours are required to produce a total dose of 6 Gy over a range of 0.01-0.001Gy/min.

Answer 7

VII-6) C For high LET radiation it can be assumed that the survival curve is exponential, or near exponential, and cell survival can be modeled using the single-target, single-hit equation (S = e), or the simplified form of the linear quadratic equation in which is zero (S = e-D). Using either of these equations, 3 Gy reduces the surviving fraction to 10-1, and a dose of 18 Gy therefore would reduce survival to 10-6. Therefore, irradiating 108 cells with 18 Gy would result in the survival of: (108 cells) x (10-6 surviving fraction) = 102 cells.

Answer 8

VII-7) B An exponential survival curve can be modeled using the single-target, single-hit equation (S = e), or the simplified form of the linear quadratic equation in which is zero (S = e-D). Since four 3 Gy fractions reduce the surviving fraction to 10-4, and assuming an equal effect per fraction, each 3 Gy fraction reduces the surviving fraction by 10-1. Accordingly, two additional 3 Gy fractions (producing a total dose of 18 Gy) would yield a surviving fraction of 10-6.

Answer 9

VII-8) B The survival curve for this cell line is exponential because each incremental dose of 3 Gy decreased the surviving fraction by an additional factor of 0.1. Thus, this survival curve can be modeled using an exponential equation which can be expressed as either S= e-D (linear quadratic model) or S = e-D/D0 (target theory model). The D0 is equal to the D10/2.3, or 1.3 Gy, not 3 Gy. The n and Dq values for this survival curve are equal to 1 and 0 Gy, respectively, and are therefore small, not large. The surviving fraction after a single dose of 3 Gy can be calculated from the colony forming efficiency of the irradiated cells (40/1000), divided by the plating efficiency (PE) of the unirradiated cells (40/100), which is equal to 0.04/0.4 or 0.1. Since this survival curve can be represented by S = e-D, the term of the linear-quadratic equation must approach zero, so the / would be very high, and in fact will be undefined if the term is actually zero.

Answer 10

VII-9) D eD10 is the dose required to kill 90% of population eD10 = 2.3 x D0 The eD10 is equal to the eD0 multiplied by 2.3, or 4 Gy x 2.3 = 9.2 Gy

Answer 11

VII-10) C When the irradiated cell population receives an average of 1 lethal hit, it results in 37% cell survival based on Poisson statistics.

Answer 12

VII-11) E In order to achieve a 90% tumor control rate, the total dose delivered must reduce the number of surviving clonogenic cells to an average of 0.1. This is based on the equation P = e-(N)(SF), where P is the probability of tumour cure (90% or 0.9 in this case), N is the initial number of tumor clonogens (10^7) and SF is the surviving fraction resulting from the irradiation protocol. Thus, for 10^7 clonogenic cells, a total dose that reduces the surviving fraction to 10^-8 (i.e., an average of 0.1 clonogen surviving in each tumor or 1 cell surviving out of every 10 tumors irradiated) must be used to achieve a 90% control rate. Since the effective survival curve is exponential with a D10 of 8 Gy, it would be necessary to use a dose of 64 Gy.

Answer 13

VII-12) D A surgical excision of 99% of the tumor would reduce the initial number of clonogens to 10^5. Thus to achieve a 90% control rate, a dose of 48 Gy would be required, corresponding to a final clonogen surviving fraction of 10^-6.

Answer 14

VII-13) A A doubling of a tumor’s diameter reflects about an 8-fold increase in cell number, which would require 3 cell divisions to accomplish. Thus, if 18 days were required to complete 3 cell cycles, the cell cycle time must be 6 days.

Answer 15

VIII-1) D RBE decreases with increasing LET above approximately 100 keV/µm. This is thought to be due to the “overkill” effect in which many more ionizations (and damage) are produced in a cell traversed by a very high LET particle than are minimally necessary to kill it, thereby “wasting” some of the energy. Maximum cell killing occurs at an LET of approximately 100 keV/µm, not 1000 keV/µm. RBE shows the greatest changes for LET values between roughly 20 and 100 keV/µm. OER decreases slowly with increasing LET for low LET values, but falls rapidly after LET exceeds about 60 keV/µm and, therefore, does not follow a bell-shaped curve.

Answer 16

VIII-2) D There is little or no split-dose recovery following high LET radiation exposure because the single dose survival curves for high LET radiations have little or no shoulder. There is also little or no potentially lethal damage recovery, oxygen effect or radioprotection afforded by the presence of sulphydryl compounds. Delivery of a radiation dose at a low dose rate leads to less sparing for a high LET radiation compared with a low LET radiation.

Answer 17

VIII-3) B Relative Biological Effectiveness (RBE) is defined as: Dose of Reference Radiation (250 keV X-Rays)/ Dose of Test Radiation to give the same biological effect The reference radiation for calculation of RBE is low LET radiation, suchas 250 keV X-rays or Co-60. The dose of the reference radiation that will achieve the same level of cell killing as high LET particles in hypoxic cells will be greater because there is little to no oxygen effect for high LET radiation. The RBE is greater for neutrons than it is for protons in the therapeutic energy range because the high energy protons used in radiotherapy are of a relatively low LET and therefore possess an RBE of approximately 1.1. The RBE for carbon ions, or any other type of high LET radiation, is greater for fractionated irradiation compared with an acute exposure due to the substantial sparing exhibited with reference X-rays with fractionation. The RBE for charged particles is low at the beginning of the particle track and greatest near the end of the track, in the Bragg peak region. RBE does show a fractionation dependence; it decreases with increasing fraction size. The RBE for 4 MeV alpha particles will decrease with increasing dose because there is more sublethal damage repair with low LET X-rays at lower doses, and therefore more survival compared with high-LET radiation.

Answer 18

VIII-4) A 150 MeV protons have an LET of approximately 0.5 keV/m. 1 GeV Fe ions, 60Co -rays, 2.5 MeV -particles and 250 kV X-rays have LET values of approximately 143, 0.2, 166, and 2 keV/m, respectively.

Answer 19

VIII-5) D LET is a measure of local energy deposition along a track of medium. It is inversely proportional to the energy of a given charged particle. The local transfer of energy to medium is more probable at lower energies.

Answer 20

VIII -6) D LET is the quotient of the average energy that a particle lost in causing ionization to the average distance it travels between two consecutive ionizations. Photons, such as 250 KV X-rays, in passing through tissue produce no ionizations directly but only by setting in motion atomic electrons of tissue molecules. Electrons set in motion by incident photons have a broad energy distribution which is dissipated in tracts with LET ranging from about 0.4 to 40 keV/μm. Radiation therapy high energy photons can generate neutrons with energy between 0.1 to 2 MeV through photon interactions with nuclei of high atomic number materials that constitute the linac head and collimator systems. These neutrons in passing through tissue also produce no ionization directly but by setting protons in motion by knock on collisions with hydrogen nuclei of the cellular water molecules. Protons set in motion by photoneutrons dissipate energy over a range of LET up to about 70 keV/μm. Answer choice E mainly pertains to neutrons, not protons, where the average method and the energy method for calculating LET give significantly different numbers.

Answer 21

VIII-7) C On average, the formation of a three-ion cluster requires dissipation of 110 eV. Therefore, or 1 cluster every 20 Ǻ (1 μm = 10,000 Ǻ). This spacing of ion clusters along the silicon ion track corresponds to a 20 Ǻ diameter of the DNA helix.

Answer 22

IX-1) C In a typical respiring tissue, the approximate distance that oxygen can diffuse from a normally oxygenated capillary before cellular hypoxia is detectable is approximately 70-200 µm. The oxygen diffusion distance will depend on the partial pressure of oxygen in the capillary and on the rate of oxygen consumption by the tissue, and therefore shows some variability. Thomlinson and Gray measured 150 µm in their landmark experiments in 1955. Olive et al. (IJROBP 1992) determined that the maximum oxygen diffusion distance using solid tumor cubes incubated with fluorescent probes and found it to range from 107 um to 192 µm, depending on the cell line. Torres Filho et al. (Proc. Natl. AcadUSA 1994) measured in vivo oxygen concentration in a SCID mouse model and found hypoxia to occur at distances >200 µm.

Answer 23

IX-2) B The fraction of cells in a tumor that are hypoxic can be estimated using the paired survival curve method. This corresponds to the surviving fraction of cells irradiated in normally oxygenated tumors divided by the surviving fraction of cells from a tumor made fully hypoxic by asphyxiating the host with nitrogen immediately prior to irradiation, which is assumed to render all of the tumor cells radiobiologically hypoxic. Thus, the estimate for the fraction of hypoxic cells would be 0.001/0.1 = 0.01.

Answer 24

IX-3) B The Km value occurs at an oxygen concentration of roughly 0.5-1% or 3- 8 mm Hg.

Answer 25

IX-4) A The most dramatic change in radiation sensitivity occurs over an oxygen tension range of 0-30 mm Hg (Torr). Cells irradiated under an oxygen partial pressure at the low end of this range are maximally radioresistant, whereas irradiation at 30 mm Hg oxygen results in near maximum radiosensitization.

Answer 26

IX-5) A Since the high energy protons used in radiotherapy have an LET similar to that of X-rays, their OER values are also similar. However it is important to know that as LET increases, OER values decrease. An oxygen partial pressure greater than about 2-3% during irradiation will result in essentially full radiosensitization. The OER is defined as the ratio of the radiation dose needed to cause a certain biological effect in hypoxic cells divided by the dose needed to produce the same effect in aerated cells. Both acutely and chronically hypoxia cells can reoxygenate The increased cell killing resulting from irradiation in the presence of oxygen is thought to be the result of increased radical damage and damage fixation by oxygen. The initial number of ionizations produced by radiation in the aerated and hypoxic cells would be the same.

Answer 27

IX-6) C Since the X-ray OER is typically about 3 and the OER for 15 MeV neutrons is about 1.6, the ratio of the OERs is about 2.

Answer 28

IX-7) E Exposure of cells to hypoxia, as in other stress situations, leads to changes in expression of a number of stress genes, many of which are responsive to the transcription factor, hypoxia-inducible factor-1 (HIF-1) (HIF1A). Under normoxic conditions, HIF-1 is hydroxylated on proline residues by oxygen-dependent prolyl hydroxylases. The hydroxylated prolines bind to the von Hippel-Lindau (VHL) protein, which is a component of the E3 ubiquitin-protein ligase complex that ubiquitinates HIF-1 and targets it for degradation. Oxygen acts as a radiosensitizer principally through its ability to “fix” radiation-induced DNA damage but does not inhibit DNA repair The OER decreases with increasing LET, whereas the RBE increases with LET until reaching a maximum at approximately 100 keV/µm, and then decreases . Measurements with pO2 microelectrodes and bioreductive probes have demonstrated that hypoxic cells are often present in human tumors The Km of radiosensitivity for cells (i.e., the concentration at which there is 50% radiosensitivity compared to oxic conditions) is about 3mm Hg (about 0.5%), not 30mm Hg.

Answer 29

IX-8) B Hypoxia in tumors has been detected using both imaging and direct electrode measurements.

Answer 30

IX-9) C Amifostine is a drug whose active metabolite contains a sulfhydrl moiety and acts as a free radical scavenger. It has been studied as a radioprotectant in several clinical and preclinical settings. As a radioprotectant, it does not sensitize hypoxic cells. Carbogen is a mixture of 95% oxygen and 5% carbon dioxide and has been used to mitigate chronic hypoxia. Nicotinamide, used concurrently with carbogen, is intended to mitigate acute intermittent hypoxia seen in tumor vessels by preventing intermittent vessel closure Perfluorocarbons such as perflubron have been shown to improve tumour oxygenation in preclinical cancer models but have not yet shown clinical utility Both misonidazole and nimorazole are nitroimidazoles that have radiosensitizing properties in hypoxia cells.

Answer 31

IX-10) D OER for energized ions should be 1.0. By definition, OER cannot be smaller than 1.0.

Answer 32

IX-11) D There is no difference in the uptake of the chemical by aerobic and hypoxic cells, but there is an obvious difference in the action of cell kill due to the amount of oxygen available in the cells. Tirapazamine (TPZ) is a benzotriazine di-N-oxide which is reduced (i.e. gaining an electron) preferentially under hypoxia with the help of an intracellular reductase forming the reactive, TPZ radical as well as the very potent hydroxyl radical downstream. In contrast, under aerobic conditions, the initial TPZ radical is rapidly back-oxidized (i.e. losing electrons) to the parent compound losing its reactive potency, ultimately driving the selectivity for hypoxic cells. A byproduct of this reversal is superoxide which may be responsible for the muscle cramps seen in patients.

Answer 33

IX-12) C In the SWOG 0222 trial, tirapazamine was associated with higher rates of esophagitis compared to historical estimates. Tirapazamine was developed as a hypoxic cytotoxin to potentially enhance tumor responses and showed promise in single-arm phase II trials in several disease sites. However, the phase III trials failed to show efficacy above the control arms. These include GOG 219, which examined the efficacy of the drug when added to standard chemoradiation for locally advanced cervical cancer. The trial failed to accrue due to lack of drug availability. Of a planned accrual of 750 patients, only 379 were eligible and evaluable; no difference in overall or progression-free survival was seen, but there was an increase in grade 3+ leukopenia, GI toxicity, and hepatic/renal dysfunction. The TROG examined tirapazamine’s utility and safety in head and neck cancers in the HeadSTART TROG 02.02 Trial. There was no overall survival or failure-free survival benefit of tirapazamine when added to cisplatin-based chemoradiation in this trial. The authors reported more frequent muscle cramps, diarrhea, and skin rash in the experimental arm.

Answer 34

IX-13) A The DAHANCA 5-85 Trial examined the addition of nimorazole to conventionally fractionated radiotherapy for pharyngeal and supraglottic larynx cancers. The trial demonstrated that nimorazole improved locoregional control and disease-specific survival compared to placebo but did not significantly improve overall survival. Nimorazole was reasonably well-tolerated in the DAHANCA 5-85 Trial, although only 60% of patients completed the treatment, and it was associated with a higher rate of nausea/vomiting, flushing, dizziness, and skin rash; patients also had trouble swallowing the large capsules of the drug. There was no increase in mucositis or late complications with the addition of nimorazole. Additional subset analyses of the trial showed that high osteopontin concentration was associated with worse disease-specific mortality, but also improved response to nimorazole in terms of locoregional control and disease-specific mortality. The benefit of nimorazole also appears to be isolated to p16-negative tumors, whereas p16-positive tumors did not appear to benefit.

Answer 35

X-1) D Bromodeoxyuridine, a synthetic nucleoside analogue, when incorporated into cellular DNA in place of thymidine acts as a radiation sensitizer, so cell killing would be enhanced. S-phase is the most radioresistant phase of the cell cycle, so cell killing would be decreased relative to that for an asynchronous population. Oxygen is a radiation sensitizer, so cell killing would decrease in cells made hypoxic before irradiation. Splitting the dose into two fractions separated by 24 hours would allow sublethal damage recovery and possibly enable cellular proliferation to take place between fractions. Cell killing would therefore be less than if the total dose had been delivered acutely. Cysteine is a sulfhydryl-containing compound that scavenges radiation induced free radicals; it therefore acts as a radioprotector and reduces cell killing.

Answer 36

X-2) A Therapeutic radiation at a low dose-rate of ~1 Gy/hr is associated with increased cell survival compared to higher dose-rates primarily due to DNA repair - especially the repair of DNA double-strand breaks produced as a result of radiation.

Answer 37

X-3) E Potentially lethal damage recovery is operationally defined as an increase in cell survival under environmental conditions not conducive to progression of cells through the cell cycle for several hours after delivery of a large, single radiation dose. If non-cycling cells are forced to re-enter the cell cycle immediately after irradiation, rather than remaining quiescent, potentially lethal damage will be “expressed” and therefore the surviving fraction will be lower. Sublethal damage recovery is operationally defined as an increase in cell survival noted when a total radiation dose is delivered as two fractions with a time interval between the irradiations, i.e. splitting the dose, as opposed to a single exposure. Repair of DNA damage and rejoining of chromosome breaks presumably underlie both the sublethal and potentially lethal damage recovery. Cell cycle reassortment has a sensitizing effect on a population of cells receiving multi-fraction or protracted irradiation regimens. This is because surviving cells that were in a resistant phase of the cell cycle during the initial irradiation may progress through the cell cycle between fractions and reassert into a more sensitive phase of the cell cycle by the time of delivery of the next fraction. This process is irrelevant under the conditions described here, in which only a single radiation dose was administered. Translesion DNA synthesis is an error-prone process during which certain DNA polymerases synthesize DNA using a damaged DNA strand as a template, resulting in error-prone DNA synthesis.

Answer 38

X-4) C When a dose of 5 Gy is delivered at a dose rate of 1 Gy/min, irradiation requires 5 minutes. When 5 Gy is delivered at 1 Gy/hr, irradiation requires 5 hours. Extensive repair of sublethal damage will occur during the low dose rate, but will not be able to occur during high dose rate irradiation. Essentially, the chance for separate lesions to interact with one another forming more complex, lethal lesions increases at the higher dose rate. As a result, the component of cell killing will increase. More cell killing would therefore occur when a dose of 5 Gy is delivered at a high dose rate rather than a low dose rate. The surviving fraction would change the least for a cell line with a radiation survival curve characterized by a high, not low, / ratio. Treatment with an agent that inhibits DNA repair would have little impact during the 5-minute period of irradiation that would occur at the high dose rate. In contrast, such a treatment would markedly reduce cell survival for the 5-hour irradiation required at the low dose rate since, in the absence of the agent, substantial repair would take place during the course of the irradiation. The increase in the surviving fraction for this low dose rate irradiation is primarily a consequence of sublethal damage recovery and not repopulation, as the repopulation would only occur for overall treatment times on the order of days. The total number of ionizations produced is a reflection of the total dose delivered and does not vary with the dose rate.

Answer 39

X-5) B Compared to the cell surviving fraction after the single 8 Gy dose, the increase in cell survival noted for the two 4 Gy doses delivered with a 2- hour interfraction interval was due to sublethal damage repair (SLDR). Although SLDR also occurred when the interfraction interval was 8 hours, cells surviving the first dose start progressing through the cell cycle and reassort from the radioresistant phases they were in at the time of the initial irradiation (e.g. late S) into more radiosensitive phases (e.g. G2 and M), thereby resulting in an overall surviving fraction for the 8-hour interval that was lower than that for the split dose protocol with a 2-hour interval between fractions. It is unlikely that much repopulation would take place during the total time of 8 hours needed to complete the irradiations. Reoxygenation would not be an issue for cells maintained in a well-aerated 95% air environment.

Answer 40

X-6) D Sublethal damage recovery is operationally defined and demonstrated using a split dose protocol. Potentially lethal damage repair is detected by changing the post irradiation environment and observing the effect on survival. Incorporation of tritiated thymidine into DNA would not specifically measure PLDR, but would reflect DNA synthesis and other forms of DNA repair. Reoxygenation would best be assayed by performing repeat measurements during the course of radiotherapy by using an oxygen electrode or by treating with a hypoxia maker, such as pimonidazole, that is metabolized and incorporated exclusively into hypoxic cells. Cell cycle age response is best demonstrated by performing cell synchronization followed by irradiation of cohorts of cells in particular cell cycle phases and then performing the clonogenic survival assay as a readout. Repopulation can be assayed in vitro by counting the number of cells present as a function of time after irradiation. The mitotic shake off technique is used to collect synchronous populations of cells for use in experiments examining age response functions

Answer 41

X-7) B Ataxia Telangiectasis Mutated (ATM) serves as the central orchestrator of the signal transduction response to DSBs. Cells deficient in ATM activity display cell cycle checkpoint defects and sensitivity to ionizing radiation.

Answer 42

X-8) C PLD is believed to be complex double strand breaks (DSBs) that are repaired slowly as compared to simple DSBs. Therefore, cells that are left in stationary phase after irradiation display enhanced survival as they have time to repair complex DSBs before resuming progression through the cell cycle.

Answer 43

X-9) B Sublethal damage (SLD) is the term used to describe the increase in cell survival that is observed if a given radiation dose is split into two fractions separated by a time interval, i.e. allowing time for repair. However, this benefit in survival can be offset when the time between doses extends so much that reassortment, reoxygenation, and repopulation kick in. Ultimately, the outcome of split-fraction experiments depends on the interplay between Repair of DNA damage, Reassortment through the cell cycle and Repopulation (requires a split time period greater than the cell cycle length). S-phase, especially the latter part of S-phase is the most radioresistant cell cycle phase therefore most survivors of the first fraction are generally in S phase. SLD is seen to a far higher-degree in low-LET irradiated cells than in high-LET irradiation. The fraction of cells surviving a split dose increases with increasing time between the two doses because of the repair of SLD, hence B is false.

Answer 44

X-10) C The effect of a given dose of radiation is less if it is split into two fractions, delivered a few hours apart which is due to the repair of sublethal damage. Most drastically this is observed within the first hour when most damage is repaired, starting to plateau after 2h for most proliferating mammalian cells. The majority of surviving cells will come from S-phase as this is the most radioresistant cell cycle phase. Potentially lethal damage repair is not assessed in split dose experiments but is more about post-exposure conditions and the time available for repair in arrested populations before cell cycle resumes. (single-dose experiment)

Answer 45

XI. Solid Tumor Assay Systems Answers & Explanations XI-1) B An increase in the number of tumors per animal would be a reflection of metastatic spread of the tumor, and would not necessarily reflect the radiation response of the primary tumor per se. All of the other assays can be used to quantify the response of tumors to irradiation. The lung colony assay is a technique to measure the radiosensitivity of tumor cells that are irradiated in situ. Following irradiation, the tumour is removed, made into a preparation of single cells, and then injected into the mouse tail vein. Cells that retain clonogenic capacity form colonies in the lung that are countable. This allows comparison of clonogenic survival in various in situ experimental scenarios Similarly, colony forming ability of cells explanted from a tumour are radiated in situ; however, radiation response is tested by counting the surviving colonies grown in vitro, also known as the in vivo/in vitro assay. Lastly, tumor growth assays, quantifies radiation response through the measurement of tumor diameter at various time points following radiation compared to untreated tumors.

Answer 46

XI-2) E The TCD50 assay quantifies the dose required to cure 50% of a group of matched tumors and is therefore a highly relevant endpoint for extrapolation to the clinic. The assay can be conducted using mouse tumors or human tumor xenografts, although suppression of the host immune system when using xenografts is crucial in order to minimize misleading results due to rejection of implanted cells. The TD50 assay can be used to measure the number of cells required to cause a tumor in mice and has historically been used to determine tumor cell survival curves, to assess the number of clonogens in a tumor, and to study host factors that influence tumor development

Answer 47

XI-3 B CD133 has been described as a putative marker for cancer stem cells in glioblastoma. Injection of 100 CD133+ cells is sufficient to initiate tumour formation in >30% of nude mice, supporting CD133 as a potential cancer stem cell marker. The unsorted bulk cells contain cancer stem cells; given, however, that 100-1,000 more cells are required in order to form the same number of tumors as that seen when purified CD133+ cells are injected, it appears that <1% of the cells in the tumor are stem cells. CD133- cells were derived from a glioblastoma and are therefore not normal, although they possess a very limited ability to form tumors de novo. In this experiment, no data are provided regarding the sensitivity of the cell lines (or sublines) to radiation.

Answer 48

XI-4) D Results from tumor transplantation experiments indicate that only a small proportion of all cancer cells have an unlimited proliferative capacity and demonstrate the capacity of self-renewal. In analogy to in vitro assays, tumor cells that demonstrate the ability to achieve a local recurrence following radiotherapy have been termed “clonogenic cells” and correspond to putative “cancer stem cells,” or “tumor initiating cells.” The existence of cancer stem cells, defined by the ability for self-renewal and generation of the heterogeneous lineage of cells within a tumor, has been hypothesized.

Answer 49

XI-5) D It has been suggested that a small proportion (< 1%) of all cells in a tumour are cancer stem cells. If correct, this hypothesis suggests that all cancer stem cells must be killed or lose replicative potential in order to achieve permanent local tumor control. Thus, the rate of permanent local tumor control is a direct measure of radiation response of cancer stem cells. In contrast, tumor shrinkage and growth delay are dominated by the response of the bulk of cancer cells and not specific for the radiation response of cancer stem cells. Cancer cells with a limited proliferative capacity, as well as doomed cancer stem cells, might undergo a number of cell divisions before they permanently stop proliferating and ultimately die. Determination of proliferating cells will therefore not provide information regarding the radiation response of cancer stem cells. Cancer cells can die following exposure to radiation in different ways, including interphase death (i.e. apoptosis) and mitotic catastrophe (apoptosis, autophagy, or necrosis). None of these modes of cell death is likely to be specific for cancer stem cells. Given that many solid tumors exhibit resistance to undergoing apoptosis and the controversial data from studies comparing the rate of apoptosis with radiation response of tumors, it is unlikely that the rate of apoptosis after irradiation will be a proper parameter to determine the response of irradiated cancer stem cells.

Answer 50

XI-6) A There are indeed some examples in the literature showing a discrepancy between growth delay and tumor control probability. In these experiments, various molecular targeting approaches in combination with radiation were investigated. Though difficult to prove, the assumption of a differential effect on cancer stem cells and non-cancer stem cells is the most likely explanation for these results. It is likely that the drug reached the tumor since there was an effect on tumor growth. Cancer cells generally express EGFR and cell survival following irradiation is affected by vascular supply. The observed discrepancy between growth delay and local tumor control in some experimental settings suggests that the latter assay is the preferable endpoint to evaluate new therapeutic approaches with curative intent.

Answer 51

XII. Tumor Microenvironment Answers & Explanations XII-1) D In the absence of reoxygenation it is unlikely that all hypoxic cells would be eliminated following a typical course of radiotherapy from a tumour possessing even a small percentage of hypoxic cells because hypoxic cells demonstrate approximately 3-fold greater radioresistance compared with aerated cells. Hypoxic regions in tumors can be detected using a labeled nitroimidazole compound. Bortezomib (Velcade) is a proteasome inhibitor and would not label hypoxic regions within tumors. Although not without exceptions, as tumors increase in size the hypoxic fraction generally also increases. This is because the typically abnormal tumor vasculature is insufficient to maintain oxygen demand. Regions of acute or transient hypoxia may develop in tumors due to intermittent blood flow via the intermittent closing down of blood vessels. Chronic hypoxia, on the other hand, is defined as diffusion-limited hypoxia due to the inability of oxygen to diffuse farther than 100 m from a blood vessel. Acutely hypoxic cells tend to exhibit rapid reoxygenation, whereas chronically hypoxic cells generally reoxygenate more slowly.

Answer 52

XII-2) E Bevacizumab (Avastin) binds to and neutralizes vascular endothelial growth factor (VEGF)-A ligand, thereby preventing its interaction with cell surface receptors, including the VEGF receptor (VEGFR). The fibroblast growth factors (FGFs) are a family of pluripotent growth factors that stimulate proliferation of mesodermal or neuroectodermal cells and can play a role in angiogenesis. FGFs have yet to be successfully targeted pharmaceutically. Hypoxia-inducible factor (HIF)-1 is a transcription factor that detects hypoxia and enhances angiogenesis. The Von Hippel-Lindau (VHL) protein belongs to a complex that is involved in the ubiquitination and degradation of HIF The Ras proteins are a family of small GTPases involved in the activation of signaling cascades following activation of receptors. Ras is frequently mutated in human cancers but is difficult to target pharmacologically

Answer 53

XII-3) D An increased apoptotic index is often observed in hypoxic regions of tumors. The gene for vascular endothelial growth factor (VEGF/VEGFA) is one of the major genes under the control of the hypoxia responsive promoter, HRE, which binds the transcription factor, hypoxia-inducible-factor (HIF)-1 Studies in animal models have indicated that treatment with anti angiogenics can cause “normalization” of tumor blood vessels and result in a transient improvement in tumor oxygenation before vessels start to deteriorate. Pre-clinical studies with animal models as well as clinical studies have linked increased hypoxia in tumors to increased tumor aggressiveness and metastatic potential Exposure to severe hypoxia halts progression of cells through the cell cycle and therefore inhibits proliferation.

Answer 54

XII-4) A Chronically hypoxic regions in a tumor can be targeted for elimination by administering certain bioreductive drugs that preferentially kill hypoxic, but not aerobic, cells. Chronically hypoxic cells tend to be sensitive to hyperthermia. This is because they exist in an acidic (low pH) microenvironment Acutely hypoxic cells are a consequence of intermittent blood flow in tumors It has been shown via model calculations of oxygen consumption rates in respiring tissues and through the use of hypoxia markers that chronically hypoxic cells rarely appear closer than about 70 m from capillaries

Answer 55

XII-5) C Expression of vascular endothelial growth factor (VEGF) and downstream angiogenesis is induced under hypoxic conditions via hypoxia-inducible transcription factors that bind to the VEGF promoter to stimulate its transcription. In the absence of angiogenesis, tumors would only be expected to reach a diameter of about 2 mm, not 2 cm Microvessel density, a measure of angiogenesis, has been correlated positively with metastatic spread for most tumor types Angiostatin and endostatin are inhibitors of angiogenesis while basic fibroblast growth factor (bFGF) is a positive regulator of angiogenesis

Answer 56

XII-6) E Hypoxia-inducible factor-1 (HIF-1) is a heterodimer that acts as a key regulator of several oxygen-responsive proteins, including erythropoietin and vascular endothelial growth factor (VEGF). HIF-1 was first identified as a DNA-binding protein that mediated the up-regulation of the erythropoietin gene under hypoxic stress. Subsequent studies have implicated HIF-1 in the regulation of a broad range of oxygen responsive genes including VEGF, VEGF receptors, angiopoietins, nitric oxide synthase, fibroblast growth factors and platelet-derived growth factor (PDGF). Under aerobic conditions, HIF-1 is hydroxylated by HIF prolyl hydroxylases. Hydroxylation at two prolyl residues targets HIF-1 to the von Hippel-Lindau (VHL) E3 ubiquitin ligase and results in HIF-1 ubiquitination and subsequent proteosomal degradation, thereby limiting upregulation of target genes. Because the hydroxylation catalyzed by prolyl hydroxylases requires molecular oxygen, HIF-1 escapes inactivation under hypoxic conditions.

Answer 57

XII-7) E When administered as a single agent, several anti-angiogenic drugs have not yielded a long-term survival benefit. In contrast, delivery of anti angiogenic agents with chemotherapy has produced a significant survival benefit in colon cancer and previously untreated lung and breast cancers. If the anti-angiogenic agent were destroying tumor vasculature in combination regimens, one would expect decreased tumor blood flow and compromised delivery of chemotherapy to the tumor. The survival benefits produced by the addition of an anti-angiogenic drug to traditional chemotherapeutic regimens therefore appears paradoxical. One possible explanation for this has been termed the “normalization hypothesis.” Under the pressure of pro-angiogenic factors, tumor vasculature is structurally and functionally abnormal. Anti-angiogenic therapy (transiently) restores the balance of pro- and anti-angiogenic factors. Consequently, immature and leaky blood vessels are pruned, pericyte coverage increases, and the basement membrane becomes more homogenous and normalized. As a result, the resultant vascular bed achieves greater organization by being less leaky, dilated, and tortuous. These morphological changes also result in functional changes, including decreased interstitial fluid pressure, increased tumor oxygenation, and improved penetration of drugs into the tumor parenchyma. Due to improved drug delivery, chemotherapy is more efficacious. Sustained or high-dose anti-angiogenic therapy, however, may drive an imbalance favoring anti-angiogenic factors leading to inadequate tumor blood supply and compromise of the efficacy of systemic therapies.

Answer 58

XII-8) C Solid tumors develop regions of increased hypoxia (decreased pO2), decreased pH, decreased glucose, and increased (not decreased) interstitial fluid pressure. Low oxygen levels shift glucose metabolism from oxidative phosphorylation to glycolysis, which increases the production of metabolic acids. Oxygen can diffuse about 70 µm from the arterial end of a capillary.

Answer 59

XII-9) B Paclitaxel stabilizes microtubule polymers and protects them from disassembly. As a result, mitosis is consequently blocked and apoptosis is activated. Paclitaxel has been shown to increase the radiation sensitivity of tumors by inducing apoptosis, increasing oxygenation of hypoxic cells in tumors, arresting cells in the radiosensitive G2/M phase of the cell cycle, and decreasing interstitial fluid pressure. Some of these studies have been conducted in animal models while others have been performed in clinical trials of human breast cancer patients. No studies have demonstrated upregulation of HIF-1 following treatment with paclitaxel; indeed, one might expect HIF-1 to be degraded more rapidly following reoxygenation.

Answer 60

XII-10) E Tumor masses exhibit abnormal blood vessel networks that fail to provide adequate and homogeneous nutritional support.

Answer 61

XII-11) B Since oxygen is unable to diffuse more than approximately 70 µm from the arterial end of a capillary, tumors require the ability to develop new blood vessels in order to grow. This process is normally regulated by a balance of both pro-(including VEGF, FGF, PDGF) and anti-(thrombospondin, angiostatin, endostatin) angiogenic molecules.

Answer 62

XII-12) D Colchicine is an anti-inflammatory agent that binds tubulin. Colchicine itself induces vascular damage but only at doses that are limited by toxicity and therefore not used in the clinical setting for this purpose.

Answer 63

XII-13) D Whereas angiogenesis is the sprouting of endothelial cells from nearby blood vessels, vasculogenesis is the formation of blood vessels from circulating endothelial progenitor cells (i.e. the bone marrow). Vasculogenesis is of particular importance following treatment with anti angiogenic agents as well as following irradiation. During early tumour development, both vasculogenesis and angiogenesis are likely utilized. Because tumor irradiation abrogates local angiogenesis, the tumor must rely on the vasculogenesis pathway for re-growth following irradiation. Tumor irradiation produces a marked influx of CD11b+ myeloid cells into tumors and are critical in order for a tumor to achieve formation of blood vessels after irradiation as well as for tumor recurrence.

Answer 64

XIII. Cell and Tissue Kinetics Answers & Explanations XIII-1) A CDK1 (and cyclin A/B) is associated with the G2 to M cell cycle phase transition. The other CDKs and Cyclins are appropriately paired as follows: o G1 phase S phase: CDK4 and Cyclin D1; CDK2 and Cyclin E o S phase G2 phase: CDK2 and Cyclin A o G2 phase M phase: CDK1 and Cyclin B/A

Answer 65

XIII-2) C A dose that kills 90% of the cells in the population would leave a surviving cell population heavily enriched in the radioresistant cells in late S phase. Radiosensitivity across the cell cycle is ranked as follows from least to most sensitive: Late S, Early S, G1, G2≈M.

Answer 66

XIII-3) B The typical TC for tumor cells in vivo is generally in the range of 1-5 days.

Answer 67

XIII-4) A The cell loss factor (Φ) is the percent of newly produced cells that die or fail to continue dividing. Most human tumors have a ϕ of around 77 % and explains why tumors grow in-vivo far more slowly than would be expected based upon cell doubling time. CLF=1‐Tpot/Td Where: Tpot= potential doubling time = the time necessary to double the number of proliferating tumor cells in the absence of spontaneous cell loss. Td= volume doubling time = the actual doubling time observed. Cell loss factor (CLF, ϕ) often appears to decrease several weeks after the start of radiotherapy, which has the net effect of slowing tumor regression. The growth fraction is the ratio of the number of proliferating cells to the sum of proliferating and quiescent cells . If the observed tumor volume doubling time (TD) is 60 days and the potential doubling time (Tpot) calculated from the cell cycle time and the growth fraction is 3 days, then the cell loss factor is 95% (Answer Choice C). Although Tpot (as measured from a tumor biopsy derived from patients previously given bromodeoxyurdine) has not proven to be a robust predictor of long-term outcome after accelerated radiotherapy, it might still be useful for the pre-selection of patients most likely to benefit from accelerated treatment. For carcinomas, the cell loss factor is usually the major determinant of the discrepancy between a tumor’s potential doubling time and its overall volume doubling time.

Answer 68

XIII-5) E The question refers to the ‘percent-labeled mitoses technique’ using tritiated thymidine. The radiolabeled thymidine is added to the growth medium of cells for about 20 minutes (flash labeling). Only those cells in S-phase take up the label. The length of time required for the first radioactively-labeled S-phase cells to first enter mitosis, as measured using the percent-labeled mitosis technique, would correspond to the duration of the G2 phase (TG2). The additional time required for the cells to completely fill the mitotic compartment (i.e., 100% labeled mitoses) would be equal to the length of M (TM). The time to reach 50% of the maximum point, therefore, corresponds to TG2 plus TM/2.

Answer 69

XIII-6) B Using the equation MI = TM/TC, where MI is the mitotic index, TM is the length of mitosis and TC is the total cell cycle time, then TM = (MI)(TC/) = (0.05)(14 hours)/0.7 = 1 hour. Of note, the TM for most mammalian cells is typically ~1 hour.

Answer 70

XIII-7) A A tumor’s volume doubling time rarely equals its potential doubling time because most tumors have a high cell loss factor. Formation of metastases represents only one of many reasons for cell loss, and usually is only a minor contributor. Human tumor cells typically have cell cycle times of a few days whereas tumor volume doubling times are generally on the order of months. The presence of a high hypoxic fraction would probably contribute to a low growth fraction, which would affect both Tpot and volume doubling time. If hypoxia were a significant cause of cell death, it would affect the cell loss factor and therefore affect the volume doubling time. The presence of non-proliferating cells affects both the tumor volume doubling time and the potential doubling time and does not cause a difference between them. In addition, non-viable cells (whether hypoxic or aerobic) have similar effects on the tumor volume doubling time and the potential doubling time.

Answer 71

XIII-8) E The cell loss factor (Φ) is equal to 1-(Tpot/TD). Therefore, if the cell loss factor were zero, then the Tpot would equal the TD. The mean TC is shorter than the Tpot becauseTpot also considers the presence of quiescent cells, and the growth fraction in tumors is generally less than 100% For solid tumors the Tpot is generally much shorter than the TD because the cell loss factor is typically quite high. The GF is taken into account in the determination of Tpot, so it does not affect the relationship between the Tpot and the TD. Tpot can be calculated from the labeling index (LI) and the duration of S phase (TS) using the equation Tpot = TS/LI. (where λ is a constant ranging from about 0.6 to 1.0). It has been suggested that tumors with short pretreatment values for Tpot, (suggesting the presence of rapidly proliferating cells and a high growth faction), would be most likely to benefit from accelerated radiotherapy, but this has not been confirmed in clinical trials performed to date

Answer 72

XIII-9) C Regulation of the G2 checkpoint by ATM is thought to occur via the activation of CHK2, which phosphorylates CDC25C phosphatase thereby preventing it from dephosphorylating CDK1 (CDC2), a step necessary for the progression from G2 into M phase. The remaining proteins listed are all targets for phosphorylation by the ATM kinase, and, consequently, are implicated in various cell cycle control pathways although not the G2 checkpoint. CHK2 and MDM2 are involved in control of the G1–S phase transition. ATM also phosphorylates MDM2, which reduces the ability of MDM2 to negatively regulate p53. NBS1 and CHK2 are implicated in S phase progression. Upon phosphorylation by CHK2, p53 is stabilized and causes cell cycle arrest in G1. PUMA (“p53-upregulated modulator of apoptosis”) is a pro-apoptotic gene that can induce cell death via a p53- dependent pathway.

Answer 73

XIII-10) A Bromodeoxyuridine (BrdU) is incorporated into DNA in place of thymidine, so it can be used to label cells in S-phase. The incorporated bromodeoxyuridine is assayed using a fluorescently-labeled anti-BrdUrd antibody. Propidium iodide fluoresces when incorporated into DNA. The amount of fluorescence is directly proportional to the DNA content, which, in turn, is a reflection of the cell cycle phase in which the cell is located.

Answer 74

XIII-11) C Tumor types with a high growth fraction and short cell cycle time would be expected to grow more rapidly. Such a tumor would also be expected to regress rapidly after irradiation since irradiated cells generally die as they attempt to progress through mitosis.

Answer 75

XIII-12) B The volume doubling time (TD) of human tumors is characteristically 40 to 100 days, while the cell cycle time is relatively short, generally between 1 to 5 days. This has important implications, which often are overlooked, in the use of cell cycle-specific chemotherapeutic agents or radiosensitizing drugs for which it is the cell cycle time that is relevant.

Answer 76

XIII-13) C The growth of a tumor depends on the fraction of cycling or proliferating cells (growth fraction), the cell cycle time, and cell loss (the number of newly produced cells that die or fail to continue dividing). Physical stresses such as low intra-tumoral oxygen concentration, low pH and high temperature both lengthen the cell cycle and slow down tumor growth. The high rate of cell loss in human tumors largely accounts for the great disparity between Tc and the volume doubling time (TD). Values for the cell-loss factor vary from 0% to more than 90% for tumors in laboratory animals.

Answer 77

XIII-14) E Cell migration within a tumor has recently been described from the study of microbeam radiation therapy (MRT). Since cell migration occurs within 200 m (the interspace between microbeams), the cells still stay in the tumor mass without affecting cell loss from the tumor.

Answer 78

XIII-15) D The cell loss factor (Φ) is equal to 1-(Tpot/TD). Rearranging this, TD = Tpot/(1-Φ). TD = 3 days/(1-0.8) = 15 days.

Answer 79

XIII-16) A A low value for SF2 indicates that the surviving fraction of tumor cells following irradiation with 2 Gy is low. This should be advantageous for radiotherapy, as it suggests that the tumor cells are relatively radiosensitive. That being said, a consistent, positive correlation between low SF2 and high tumor control probability has yet to be established. The potential doubling time (Tpot) is the time required for a tumor to double in size, taking into account the number of cells in the cell cycle and the speed of progression through the cell cycle. A short Tpot would be deleterious to tumor control because it suggests a high potential for vigorous repopulation during the course of treatment. Slow reoxygenation may also limit the effectiveness of treatment as hypoxic cells would remain hypoxic and radioresistant for a longer portion of the overall treatment time than if they had reoxygenated rapidly and efficiently. A large number of clonogenic cells would require a higher total dose for eradication; this might increase the probability of adverse normal tissue effects. Early onset of repopulation would also be deleterious as the tumor cells would be proliferating for a longer time during the course of radiotherapy and the cell population that must be killed to cure the tumor would therefore be larger.

Answer 80

XIII-17) B An agent that arrested cells in the radiosensitive G2 phase of the cell cycle could increase tumor response to a fractionated treatment protocol if provided prior to each dose of radiation. Prevention of cell cycle redistribution would diminish response to fractionated radiotherapy because surviving cells would remain in a radioresistant portion of the cell cycle rather than being permitted to traverse into a more radiosensitive phase of the cell cycle. Inhibition of reoxygenation would reduce tumor response to fractionated radiotherapy due to the prevention of the conversion of surviving radioresistant hypoxic cells to more sensitive aerated cells. Radioprotection of normal tissues would have no bearing on tumour response per se, although it could improve the therapeutic ratio overall, assuming the tumor was not similarly protected Stimulation of DNA repair would reduce tumor response to fractionated radiotherapy because a greater proportion of tumor cells may survive if treated with an agent with enhanced capacity for DNA repair.

Answer 81

XIII-18) B Variations in the duration of G1 phase are responsible

Answer 82

XIII-19) D The purpose of the cell cycle checkpoints is to slow down the cell cycle following DNA damage, hence facilitating DNA repair to maintain genomic integrity

Answer 83

XIV. Molecular Signaling Answers & Explanations XIV-1) C ATM is a kinase that is activated in response to the presence of DNAdouble-strand breaks, such as following exposure to ionizing radiation. Activated ATM phosphorylates multiple distinct target proteins, including histone H2AX, p53, BRCA1, and Artemis. Phosphorylation of H2AX (to -H2AX) results in chromatin modification that facilitates the recruitment of factors needed for DNA repair. The tumor suppressors, p53 and BRCA1, activate cell cycle checkpoint and/or DNA repair processes in response to genotoxic stress. VEGF is a secreted factor that promotes angiogenesis and is not a direct target of ATM phosphorylation.

Answer 84

XIV-2) D Epistasis is a form of gene interaction in which an allele for one trait (at one locus) influences the expression of an allele, at a different locus, for a separate and independent trait; this process is unrelated to ATM phosphorylation. Inactivation of the VHL (von Hippel-Lindau tumor suppressor) gene results in overexpression of many environmental stress-inducible mRNAs, including those involved in energy metabolism, apoptosis, and angiogenesis via the activation of vascular endothelial growth factor (VEGF). Cyclin D1 repression is associated with anti-proliferation effects. Its overexpression has been observed in human cancers, including pancreatic, lung, and esophageal. Release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria into the cytoplasm is a primary mitochondrial apoptogenic activity. MicroRNAs (miRNAs) are small non-protein-coding RNAs that function as negative regulators of gene expression under normal physiological conditions. Mis-expression of, or mutations in, miRNAs are associated with the development of a variety of human cancers, including B-cell chronic lymphocytic leukemia, colorectal cancer, and breast cancer.

Answer 85

XIV-3) C FOS is a transcription factor that has been shown to modulate a variety of genes involved in stress responses but has not been shown to modulate BRCA2. HIF-1 is a hypoxia inducible factor known to regulate the expression of the VEGF gene and thus the regulation of angiogenesis. p53 is a transcription factor that induces expression of p21. E2F is known to regulate a large number of proteins involved in cell cycle progression, including CDC25A. PUMA is the major mediator of p53-dependent apoptosis following ionizing radiation in most cell types. PUMA, a pro-apoptotic BH3-only member of BCL2 family protein promotes BAX/BAK and mitochondria dependent apoptosis in various cell types.

Answer 86

XIV-4) D Cytokines are proteins released by cells, including irradiated cells, that stimulate tissues to mount a biological response. NF-B is a transcription factor (not a cytokine). TGF-1 (TGFB1) is an important example of one of the cytokines that has been associated with the development of lung fibrosis following irradiation. A paracrine response is the result of a cytokine that acts upon a cell, other than itself, within a tissue or organ. In contrast, an autocrine response is the result of a cytokine targeting the cell from which it was produced. Cytokines generally do not have tyrosine kinase activity.

Answer 87

XIV-5) C Nuclear factor (NF)-B generally exerts a pro-survival influence through interference with apoptotic signals. It accomplishes this via the TNF receptor signaling pathway which, upon activation by an apoptotic signal, is coupled via the FADD adaptor to a caspase cascade involving the initiator caspases-8 or -10. In some cell types, however, this may not occur, since it may be opposed through the parallel triggering by TNF of a signaling pathway that activates NF-B via the TRADD and TRAF adaptors. Active NF-B induces transcription of a set of genes that encode the anti-apoptotic IAPs (“inhibitors of apoptosis”). NF-B can also exert an anti-apoptotic effect by inducing transcription of anti-apoptotic proteins, such as Bcl-xL (BCL2L1), which act to prevent cytochrome c release and the subsequent caspase-9 activation. IB binds to NF-B to prevent its translocation to the nucleus. Following formation of DNA double-strand breaks and reactive oxygen species in irradiated cells, kinases (including ATM) phosphorylate IB, targeting it for ubiquitination and degradation, which allows NF-B to translocate to the nucleus from the cytoplasm where it can act as a transcription factor. NF- B can exist as hetero- or homodimers of five different subunits. Different heterodimers activate different sets of genes while p50 and p52 homodimers, lacking transactivation domains, can selectively repress expression of their target genes. Post-transcriptional modifications and cofactor binding also help shape the specificity of the NF-B response. Competition between p53 (TP53) and NF-B for CBP/p300 may play an important role in determining the balance between apoptosis and cell cycle arrest following irradiation.

Answer 88

XIV-6) E Many genes are both up- and down-regulated following irradiation in both a time and tissue-dependent manner. In addition, variation is also seen between cells derived from the same tissue and between tissue samples taken from different individuals. This inter-individual variation is seen both in the response to stressors such as ionizing radiation and in the normal basal gene expression patterns. One of the major driving factors in the science of microarray profiling is the hope that a better understanding of this variability in gene expression may lead to a more “personalized” diagnosis of disease, prognosis and prediction of the best therapeutic approach for cancer and other diseases.

Answer 89

XIV-7) A p21 is one of the most strongly p53-transactivated genes, and codes for the p21 protein. It responds robustly at both the mRNA and protein levels to ionizing and UV radiation as well as to most other stress-inducing agents. p21 is a CDK inhibitor and also binds to PCNA to prevent entry of cells into S phase. The predominant role of p21 appears to be in mediating G1 phase arrest, although it also plays roles in differentiation, senescence, and regulation of apoptosis.

Answer 90

XIV-8) B Prostate cancer is characterized by its dependence on androgen receptor (AR) signaling as well as frequent activation of PI(3)K signaling. AR transcriptional output is decreased in human and murine tumors with PTEN deletion. In addition, PI(3)K pathway inhibition activates AR signaling by relieving feedback inhibition of HER kinases. Similarly, AR inhibition activates Akt signaling by reducing levels of the Akt phosphatase, PHLPP. These two oncogenic pathways therefore cross regulate each other by reciprocal feedback. Inhibition of one pathway leads to activation of the other thereby maintaining tumor cell survival. Combined pharmacologic inhibition of PI(3)K and AR signaling causes near complete prostate cancer regression in a PTEN-deficient murine prostate cancer model and in human prostate cancer xenografts, indicating that both pathways coordinately support survival.

Answer 91

XIV-9) A “Oncogene addiction” was first coined by Bernard Weinstein and refers to the dependence of some tumors on a single dominant oncogene for continued growth and survival and that inhibition of this specific oncogene product is sufficient to halt the neoplastic phenotype. Answer Choice A is correct because imatinib is correctly paired with its target, BCR-ABL. The other answers are examples of oncogene-addicted cancers that are incorrectly paired with agents that do not target the dominant oncogene product. EGFR-mutant lung adenocarcinoma patient can be treated with tyrosine kinase inhibitors such as gefitinib or erlotinib. A BRAF-mutant melanoma patient can be treated with a BRAF inhibitor such as venvurafinib or dabrafinib. An EML4-ALK positive lung adenocarcinoma patient treated with an ALK inhibitor such as crizotinib or alectinib. Lastly, a FLT3-mutated AML patient can be treated with FLT inhibitor, midostaurin, along with induction chemotherapy.

Answer 92

XIV-10) C The promoter region is the regulatory portion of a gene that plays a critical role in directing whether a gene is transcribed or not. Tumor suppressor genes are generally inactivated in many cancers, typically resulting in a loss of control over cell proliferation. Exons are the expressed, or coding, regions of genes, whereas introns are the non-coding sequences. The protein encoded by the EGFR (epidermal growth factor receptor) gene is a cell surface tyrosine kinase receptor that is activated by epidermal growth factor (EGF) ligand, among others, and is important for cell proliferation. Loss of heterozygosity is a common mechanism by which tumor suppressor genes are inactivated. Oncogenes are generally activated by mechanisms including deletion/point mutation, chromosome rearrangement, retroviral integration, or gene amplification.

Answer 93

XV. Cancer Answers & Explanations XV-1) B RB1 is the product of the RB1 tumor suppressor gene (not an oncogene). Once phosphorylated by CDK4/6, RB1 releases E2F, which then activates genes associated with the G1 checkpoint. RB1 is functionally inactivated in virtually all human cancers, either directly or indirectly, via p53 (TP53). p53-dependent induction of p21 (CDKN1A) regulates cyclin E/CDK2 and cyclin A/CDK2 complexes, both of which phosphorylate RB1. The RB1 and p53 signaling pathways are dysregulated in the majority of human cancers.

Answer 94

XV-2) C Carcinogenesis is a multistep process with multiple genetic alterations occurring at particular stages of cancer progression. Alterations in PTCH are associated primarily with basal cell skin carcinoma and medulloblastoma. EGFR and VEGF are frequently overexpressed in colon cancer, but their lack of a relationship with progression and survival has led to their prognostic value being questioned. For example, over 90% of human pancreatic cancers harbor an activating point mutation in the K RAS gene at codon 12. Hereditary medullary thyroid carcinoma (MTC) is caused by autosomal dominant gain-of-function mutations in the RET proto-oncogene. BRAF mutation is present in 50% of cutaneous melanomas and provide the therapeutic target for vemurafenib. Alk mutation is present in ~5% of lung adenocarcinomas and provides a target for crizotinib.

Answer 95

XV-3) D WT1 is a transcription factor which, when mutated or absent, is associated with the development of Wilms tumor. Loss of APC plays a role in gastrointestinal carcinogenesis due to its normal involvement in cell signal transduction. RB1 and p53 are both tumor suppressors that regulate cell cycle progression; p53 also regulates apoptosis. BRCA1 protein is part of the DNA repair complex, but likely has several other functions as well, including regulation of the cell cycle and maintenance of genomic stability.

Answer 96

XV-4) E p53 is modified post-translationally by phosphorylation or by acetylation in response to DNA damage. p53 is encoded by a tumor suppressor gene (not an oncogene) that is inactivated in more than half of all human cancers. The DNA repair pathways that regulate p53 include not only NHEJ and HRR, but also MMR, BER, and NER so that p53 plays a universal role in DNA damage surveillance and repair. DNA damage causes p53 to become stabilized and active, not inactive. p53 increases expression of GADD45A, p21, and PCNA. Viruses that contain proteins that inactivate p53 include HPV, SV40 and adenovirus, but not EBV.

Answer 97

XV-5) A Retroviruses, viruses with genomes composed of RNA instead of DNA, can cause cancers in animals (example: Rous sarcoma virus [RSV] in chickens). Usually, this occurs because the retroviruses contain modified (often mutated) proto-oncogenes captured from the genomes of their vertebrate hosts.

Answer 98

XV-6) E ABL is an oncogene whereas PTEN, BRCA2, WT1, and NF1 are all tumour suppressor genes.

Answer 99

XV-7) D p53 stimulates the activity of BAX and BID in irradiated cells, resulting in apoptosis. MDM2 binding to p53 stimulates degradation of p53. Irradiation of cells activates ATM to add phosphate groups to p53. Following irradiation, p53 inhibits CDC25C which inhibits the G2 to M phase transition. Lymphocytes and thymocytes with a mutant p53 tend to be more radioresistant than their normal counterparts.

Answer 100

XV-8) E p14 ARF inhibits the MDM2-mediated degradation of p53. p16INK4A is a cell cycle inhibitor that prevents phosphorylation of RB by CDK4. p14ARF is an MDM2 inhibitor thereby causing p53 levels to increase, resulting in greater cell cycle inhibition. p16 INK4A is encoded by a tumor suppressor gene.

Answer 101

XV-9) A The oncogene addiction model postulates that some tumors rely on the continued activity of single dominant oncogene for growth and survival. Thus, according to the oncogene addition model, inactivation of this key single oncogene will halt malignant proliferation by inducing cell-cycle arrest, differentiation, senescence, or other forms of cell death, depending on tissue context. Each of the listed oncogene products in this question is an addictive oncoproteins in human cancer, however Choices B-E are incorrectly paired to the listed cancer. The receptor kinases KIT and/or PDGFR display activating mutations in more than 90% of gastrointestinal stromal tumors (GIST). This observation supported the use of the multi-target small-molecule tyrosine kinase inhibitor imatinib mesylate (Gleevac) in GISTs. The correct matches in other choices are: translocated ABL1 in chronic myeloid leukemia; mutated, not amplified, EGFR in non-small-cell lung carcinoma; translocated ALK in non-small cell lung carcinoma; mutated Notch1 in T-cell acute lymphoblastic leukemia

ASTRO 2023 (4-15) Flashcards

(125 cards)