Definitions

Question 1

Plating Efficiency (PE)

Answer 1

% of cells seeded that grow into colonies

= # of colonies counted / # of unirradiated or untreated cells plated initially

Question 2

Survival Fraction (SF)

Answer 2

SF = # of colonies counted / (# of cells plated * PE)

(PE = plating efficiency = # counted/#plated)

(When plotted on Y-axis with X-axis for dose, curve has exponential shape…. don’t be fooled, when plotted as the log SF vs. Dose it is a straight line!)

Ln (S.F.) = # shots/ mean lethal shots = Dose given/D₀

(D₀ = mean lethal dose; N → 0.37N)

Question 3

Quasi-threshold dose (D_q)

Answer 3

dose at which there is minimal B effect (measure of radioresistance)

D_q= D₀ Ln (n)

Can use this to calculate the extrapolation # (n)

D_q is the straight part of the Survival curve

Ln (n) = D_q/D₀

or

log_en = D_q/D₀

(as D₀ ↑, n ↓ and as D_q ↑, n ↑)

Question 4

extrapolation number (n

Answer 4

of targets in a cell required to be damaged before the cell loses clonogenicity

large for survival curves with broad shoulders (i.e. x-rays)

small for curves with narrow shoulders (neutrons, α-particles)

Ln (n) = D_q/D₀

or

log_en = D_q/D₀

(as D₀ ↑, n ↓ and as D_q ↑, n ↑)

Question 5

Linear Quadratic Model

Answer 5

S (fraction of surviving cells) = e^{-αD - βD^2}

Example: α=0.3Gy^-1 β=0.03Gy^-1 , what percentage of cells survive a single fraction of 2Gy?

S = e ^{-((0.32) - (0.032^2))} = 49%

Example: With the above survival, what % of cells would survive 5 fractions of 2Gy each assuming complete repair but no proliferation ?

(.49)⁵ = 3%

Also know that S = e^-D/D₀ (assuming β = 0)

Example: For TBI of 6Gy, assuming D₀ = 1Gy for hematopoetic stem cells, what is the surviving fraction of stem cells?

S = e^-D/D₀ = e^-6/1 = e^-6 = 0.0025

Question 6

Relative Biological Effectivness (RBE)

Answer 6

Relative Biological Effectiveness (RBE): allows comparison of a test radiation with a standard radiation. It is the ratio of dose between the radiations to give a certain biological effect, for instance a grade of skin reaction or death of mice. Standard radiation is either 250 keV x-rays or 1.17/1.33 MeV 60Co gamma rays

RBE = D_250kv/ D_{test <strong>radiation</strong>}

Things that affect RBE:

1) LET (As LET increases towards 100 keV/μm, RBE also increases, up to 4 - 5 when compared with low LET radiations. This increase is also dependent on the other factors which influence RBE (such as dose rate, fractionation and the chosen biological effect). Over 100 keV/μm, the RBE falls as LET increases. This is due to cell overkill - too much dose is deposited in individual cells, wasting radiation which could be used elsewhere.)
2) Dose
3) Dose Rate: (lower dose rate decreases RBE for low LET radiation, but lower dose rate increases RBE for high LET radiation (less overkill!!)
4) Dose fractionation: (decreases with increasing fraction SIZE; but remember that HIGH LET radiation has increased RBE when fractionated because it is being compared to X-rays (250keV photons) which have a lower RBE with fractionation due to shoulder)
5) Biologic system or endpoint: RBE is high for tissues that accumulate and repair a lot of sublethal damage (and low for those that do not)

Other Facts to know:

*RBE for high LET particles is greater for hypoxic cells than for well-oxygenated cells of the same type because there is little or no oxygen effect for high LET radiation (but hypoxia will dramatically reduce cell kill for photon based low LET radiation)

*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

Question 7

D₁₀

Answer 7

N (initial) → 0.1N

Question 8

D₀

Answer 8

D₀ = mean lethal dose

N (initial) → 0.37N

Question 9

S.F. Equations

Answer 9

Single Hit - Multi Target model:

SF = 1 - (1- e^-D/D0)ⁿ

Linear Quadratic Model:

SF = e ^{-αD - βD^2}

(α = linear ; β = quadratic)

SF = e^-D/D₀

Question 10

Key to a SF graph

Answer 10

1) n (extrapolation #) = # of hits required to kill
2) Dq (the hump) = quasi-threshold dose
3) D₀ = mean lethal dose = N → 0.37 N
4) D₁₀ = N → 0.1 N
5) α
6) β

Question 11

D_T

Answer 11

D_T = D_q + 2.3 D₀ (# logs)

also

D_T = D_q + D₁₀ (#logs)

Remember, to get a 90% chance of kill, you have to get to one cell, then you have to kill THAT last cell (so add one more log)… to get 99% chance add two logs once you get to one cell left!!

Question 12

Damage per Gy of radiation

Answer 12

1000 base alterations

1000 ssDNA breaks

40 dsDNA breaks

Question 13

Cyclin / CDK parings in cell cycle

Answer 13

G1: cyclinD/cdk4 + CyclinE/cdk2

*Rb binds to the transcription factor E2F to keeps cells in G1/G0… only when Rb gets phosporylated twice does it let go of E2F which then results in expression of S phase associated genes, allowing cells to proceed to S (each of the above cyclin/cdk complexes phosphorylate Rb once)

S: CyclinA/cdk2

G2: CyclinA/cdk1 +CyclinB/cdk1

M: Dephosphorylated CyclinB/cdk1 (de-Phos by cdc25)

To remember: DEAABB and 4–>2–>1–>1 (G–>M)

Question 14

Oxygen Enhancement Ratio (OER)

Answer 14

OER = dose_{hypoxic cells} / dose_{aerated cells}

OER is by definition never less than 1.

OER for various radiation types:

• X-ray: 3.0
• Neutron: 1.6
• Proton: 3.0
• Energized Ions: 1.0
• Alpha-particle: 1.0

Initial proportion of hypoxic cells = SF_aerated / SF_hypoxic

*Higher LET radiation has lower OER (oxygen has less of an impact): X-ray (low LET) OER is typically about 3 and the OER for 15 MeV neutrons (high LET) is about 1.6

*Higher dose has a higher OER (2.5 vs. 3.5) –> more damage for O₂ to “fix”

*Need very low O₂ partial pressure (as low as 3mm Hg or 0.5% O₂) KNOW THIS!!!!. irradiation at 30mm Hg (this is ~3% O₂) is essentially near maximum radiosensitization.

*Thickness of well oxygenated tumor is never larger than 180μm (this is the maximum distance that oxygen can diffuse from a capillary) but is typically around 70μm

Hypoxia causes increased HIF (hypoxia inducible factors) production (need 2 subunits HIF1β and either HIF1α or HIF2α) → increased trascription of VEGF

OER decreases slowly with increasing LET for low LET values, but falls rapidly after LET exceeds about 60 keV/μm

OER is slighly higher for S phase (resistant cells) with a value of ~2.9 vs. an OER at G₂ of ~2.3 and G₁ of ~2.6

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!!!

Question 15

Acute Radiation Syndromes (Exposure)

Answer 15

1) Hematopoietic syndrome: ≥2 Gy (usually 2.5.5-5 and <8 Gy total body)

• Death at 30-60 days after exposure due to loss of immune function
• potential role for bone marrow transplant if exposure is 8-10Gy
• role for antibiotics if exposure >5Gy

2) Gastrointestinal syndrome: ≥8 Gy (usually** ≥**10Gy) total body

• Death at 5-16 days after exposure (usually 7-10 days) in humans; 3-4 days in mice
• Death due to loss of intestinal epithelia leading to infection and sepsis
• Manage with antiemetics, antibiotics, antidiarrheals, oral nutritional support

3) Cerebrovascular Syndrome: >20 Gy (usually 40-100Gy) total body

• Death within 24-48 hours post exposure
• Death due to neurological and cardiovascular breakdown

Other Facts:

• LD_50/60 (50% lethal @ 60 days):
  
  • Mouse = 7 Gy
  • Rat = 6.75 Gy
  • Monkey = 5.25 Gy
  • Dog = 3.7 Gy
  • Human = 3.5-4 Gy (with antibiotics/best supportive care maybe up to ~7Gy)
• Larger LD₅₀ doses correlate with higher relative hematopoietic stem cell concentration, lower body weight, lower required rescue dose of stem cells in bone marrow transplant
• Prodrome of the radiation syndrome: a spectrum of early symptoms that occur shortly after whole body irradiation, lasts for a limited amount of time and varies in time of appearance, duration and severity depending on the dose. GI symptoms such as anorexia, nausea and vomiting occur when an individual is exposed to doses near the LD50; at higher doses symptoms such as fever and hypotension are also seen
• After exposure to 2 Gy, 50% or less will experience nausea and vomiting. Typically this occurs within 2-6 hours of exposure. Serotonin-receptor antagonists (Zofran) may help the nausea and vomiting
• Immediate diarrhea, fever, or hypotension indicates a supralethal exposure
• If you survive a total body radiation exposure, the increased risk of cancer is ~8%/1Gy

Question 16

Base Excision Repair (BER)

Answer 16

Base Excision Repair (BER): Process by which abnormal or damaged bases are removed

Example: removal of Uracil from DNA

• Can remove one base (major pathway) or several bases (minor pathway)
• Intermediates include abasic DNA and SSB (these may be more toxic than the original damaged base) but process is concerted to avoid cell exposure to reactive intermediates.
• Key actors: APE1 (AP endonuclease), DNA PolB (DNA glycosylase)

Question 17

Nucleotide Excision Repair (NER)

Answer 17

Nucleotide Excision Repair (NER): Process by which several damaged bases can be removed

-counters nucleotide instability (NIN)

Example: removal of Thymine dimers from genomic DNA

Key Actors: RNA Pol II, XPC, TFIIH, XPA/XPG (ERCC5), XPF (ERCC4 endonuclease)

-Defects are associated with Xeroderma Pigmentosum

Question 18

Non-homologous Enjoining (NHEJ)

Answer 18

Non-homologous Endjoining (NHEJ): repair in the G₁ phase of cell cycle

• Defects lead to chromosome aberrations, immune deficiency, and ionizing radiation sensitivity
• Fast component (10 minutes) and slow component (4-6 hours)
• May or may not result in deletion of DNA (cuts off the scraggly ends!)
• Overall lower fidelity (loss or gain of a few nucleotides can occur) than HR
• Cells with genetic defects in NHEJ (such as mutation of DNA-PK, XRCC4, or DNA ligase IV) display a more pronounced hypersensitivity to ionizing radiation than cells defective in HR
• Key Actors: Artemis (repair endonuclease that cleaves 3’ and 5’ nucleotide overhangs in slow component once it is activated/bound by DNA-PKcs), MRN (repair nuclease in slow component; includes NBS1, Mre11,Rad50), Ku70, Ku80, XRCC5 (Ku70/80 heterodimer), Rad50, Mre11, Xrs2 complex, XRCC4/Ligase IV, DNA-PK (and DNA-PKcs)
• V(D)J recombination is also an example of NHEJ!
• People with BRCA1/2 mutations rely on error prone NHEJ instead of HR.

Question 19

Homologous Recombination (HR)

Answer 19

Homologous Recombination (HR): Dependent on presence of homologous DNA (i.e. sister chromatid) - so this occurs mainly in the S/G₂ phase of the cell cycle!

• Defective HR leads to chromatid and chromosome aberrations, decreased proliferation, and ionizing radiation sensitivity
• High overall fidelity (error-free)
• Also important in repairing DNA damage at replication forks (replication-associated double-strand breaks) such as those caused by chemotherapy agents
• Key Actors: RAD51 (recombinase), RAD52, RAD54 (and probably RAD50), BRCA1/2 (BRCA1 is directly phosphorylated by ATM/ATR/CHK2; BRCA2 binds with RAD51 to form foci at sites of DNA breaks)

Question 20

Common XRT induced base changes

Answer 20

8-oxo-guanosine (can base pair with C or A; base paring with A leads to subsequent mutations during replication). MutT can dephosporylate 8-oxo-G and effectively remove it from the nucleotide ppol. MutB can remove 8-oxo-G from DNA. MutY removes the A that should not be paired with 8-oxo-G (so MutY removes a normal base paired to an abnormal base!)

5-hydroxymethyl uracil

Uracil

Thymine glycol

Question 21

Radiation Induced Transcription Factors

Answer 21

AP-1 (activator protein-1): Heterodimer of two different proteins (Jun/Fos)

*Jun Family: c-Jun, Jun B, Jun D

*Fos family: C-Fos, Fra-1, Fra-2, Fos B

NF_kB:

Question 22

Sterility and Dose

Answer 22

Male:

• Oligospermia : 0.15 Gy
• Azospermia (absence of living spermatozoa) : 0.5 Gy
• Permanent Sterility: 6 Gy in a single dose or 2-3 Gy in fractionated regimen
• Sterility does not change libido, hormone balance, or physical capability

Female:

• Permanent Sterility (pre-pubertal): 12 Gy
• Permanent Sterility (pre-menopausal): 2 Gy
• Sterility leads to pronounced hormonal changes comparable to natural menopause
• No latent period

Question 23

Carcinogenesis steps

Answer 23

Initiation: an irreversible genetic change that predisposes a cell to reproductive immortality and carcinogenic transformation

Promotion: a reversible series of growth stimulating events that causes the clonal outgrowth and amplification of an initiated cell

Progression: the accumulation of more genetic changes that lead to changes in the phenotypic characteristics of a immortalized cell population (increases the aggressive properties of a tumor (metastasis)

Question 24

4 R’s

Answer 24

Repair (minutes to hours) of sublethal damage: radiation damages a cell, but repair is insufficient; mutations are induced which immortalize the cell

Reassortment (several hours) of cells within the cell cycle: Remaining cells distribute to more radiosensitive parts of the cell cycle (G2/M)

Repopulation (hours to days): the death of surrounding cells due to radiation exposure (and the ability of radiation to stimulate growth) creates a volume for the clonal expansion of the initiated cell. Allows the repair of organ FSU’s

Reoxygenation (hours to days): Reoxygenation of the formerly hypoxic core as better oxygenated (and thus more radiosensitive) cells on the outer rings of the tumor are killed by radiation)

How are the 4R’s important to XRT?: Dividing a dose into several fractions spares normal tissues because of the repair of sublethal damage between dose fractions and cellular repopulation. At the same time, fractionation increases tumor damage because of reoxygenation and reassortment.

Question 25

Risk Models

Answer 25

Absolute risk model: assumes that radiation induces cancers over and above the natural incidence and is seperate from it (radiation makes its own “crop” of cancer seperate and unrelated to spontaneous tumors)

• Example: Leukemia (risk rises for a given period of time after radiation and then returns to the natural incidence rate)

Relative risk model: assumes that radiation increases the rate of naturally occurring cancers by a given factor (radiation induced tumors and the intrinsic rate at which cancer occurs are added together

• Example: the intrinsic cancer rate increases with age, predicting that irradiation at a younger age will drastically increase the rate of cancer in an older individual

***RR model is more conservative (predicts a higher rate of cancer)

• For Solid Tumors: excess cancer incidence and mortality are a linear function of dose up to about 2 Sv
• For Leukemia: excess incidence best fitted by linear-quadratic function of dose so that risk per unit of dose at 1 Sv is about three times that at 0.1 Sv.

Other facts to know:

• The dose to double the incidence of mutations in humans has been estimated at approximately 1-2 Sv.
  
  • the doubling-dose estimate for radiation-induced genetic mutations in humans is based on mouse data, coupled with estimates of the human spontaneous mutation rates.
• Radiation does not induce characteristic mutations, but only increases the incidence of mutations that occur spontaneously.
• A higher incidence of genetic abnormalities has not been found in the children of radiotherapy patients compared with children whose parents had not been irradiated prior to conception.
• The best estimates are that no more than 1-6% of spontaneous mutations in humans are due to exposure to background radiation.
• The absolute mutation rate for humans has been estimated at 0.1-0.6% per Sv

Question 26

Examples of functional endpoint tests

Answer 26

1. Pig Skin Test (dose given and acute/chronic reactions noted)
2. Lung Breathing rate
3. Spinal cord assays (observe for myelopathy)
4. LD₅₀ (basic life vs. death function!)

*These are good tests because they give us clinically relevant info and allowed us to figure out the tolerance doses for some tissues

Question 27

α/β ratio

Answer 27

α/β ratio: the dose at which the linear (α) and the quadratic (β) components of cell killing are equal (αD = βD² or D = α/β)

In-vivo, α/β ratios for normal tissues and tumors are derived from an analysis

Question 28

Isoeffect Plot

Answer 28

• A biological effect of a certain dose that is plotted against dose/fraction (doses/fractions that have equivalent biological effect in a given tissue)
• Isoeffect curves are often plotted with the log of the total dose on the y-axis and the log of the fraction size (from high to low) on the x-axis.
• Tissues with a greater repair capacity will show greater sparing with increasing fractionation (smaller fraction sizes) and therefore will have steeper isoeffect curves
• Tissues with steep isoeffect curves have low α/β ratios (a decrease in dose per fraction rapidly increases the total dose that a late responding tissue can tolerate)
• Increased proliferation will cause an increase in the slope of an isoeffect curve because it would take a higher total dose to kill the larger number of cells produced during the course of treatment.
• Reoxygenation decreases the slope of the isoeffect curve because it decreases the number of radioresistant hypoxic cells and hence reduces the total dose required to control the tumor, everything else being equal

Question 29

Early vs. Late dose response relationships

Answer 29

Early responding tissues:

• proliferate readily which sensitizes the cells, but they can also quickly replace cells killed by radiation (pseudo resistance and/or accelerated repopulation).
• exhibit radiation-induced injury during or shortly after a course of radiotherapy (short latency period)
• regeneration/repopulation of normal tissue can occur in acutely responding normal tissue during the course of a standard radiotherapy protocol
• latency period does not show much variation (it depends on the time it takes for cells to move from the stem cell compartment through the transit compartment, and finally to the terminally-differentiated, non-dividing parenchymal cell that is lost through normal wear and tear)
• Most affected by overall treatment time (shorter treatment time leads to increased severity; prolonged overall treatment time can spare early responding normal tissues)
• Shows more α component (linear) and less β component (quadratic)
• accelerated fractionation protocols can increase the reaction in early responding normal tissues

Late responding tissues:

• primarily composed of cells in G1 (resting) phase of the cell cycle, thus relatively radioresistant at least at small doses/fractions.
• exhibit radiation-induced injury months or years following the completion of radiotherapy (long latency period)
• length of time necessary before a late effect is observed clinically decreases with increasing dose
• Most affected by fraction size (many smaller doses cause LESS late effects than a few large doses; so dose/fraction is important); also somewhat increased by increasing total dose.
• Shows less α component (linear) and more β component (quadratic; curves toward the x-axis MORE!!)

Dosing Schemes:

• Hyperfractionation: can increase early reactions, can improve tumor control (or keep relatively equal), reduces late effects; thus increases therapeutic gain!
  
  • Therapeutic gain can be achieved only if the late-responding normal tissue has a lower α/β ratio than the tumor
  • RBEs for high LET forms of radiation are greater for late effects compared to early effects when hyperfractionation is used
• Accelerated: overall treatment time is reduced as is dose; aim is to reduce repopulation in rapidly proliferating tumors; can increase early effects to some degree (due to shorter treatment time)

Question 30

Russel Periods of development

Answer 30

1) Preimplantation: Extends from fertilization until embryo attaches to the uterine wall. Most sensitive stage to lethal effects of radiation (as low as 100mGy); decreases litter size but results in few abnormalities (so if an embryo survives it generally grows normally in-utero and afterward because there is functional redundancy (and few specialized cells) that allows the embryo to overcome some cell death)
- Mouse: Days 0-5
- Human: Days 0-9
2) Organogenesis: Period of major organ development. Radiation at this time produces an assortment of congenital anomalies in progeny (at doses as low as 1-2 Gy). Progeny that survive have reduced birth weight but by adulthood they catch up to normal weight, but have smaller heads (microcephaly and associated mental retardation). Exposure can also result in death, but only in neonates (not yet born).
- Mouse: Days 5-13
- Human: Days 10-41 (weeks 2 - 6)
3) Fetal Period: already present organs grow. Radiation at this time can affect organ system function. Small doses can reduce the fertility of progeny. A HIGH dose exposure is required to cause death during this period. Exposure during early fetal period causes PERMANENT growth reduction. Exposure after 30 weeks causes organ functional damage.
- Mouse: days 13-full term
- Human: days 41-full term (weeks 6 - full term)

Key Things to know about in-utero XRT exposure:

• Microcephaly: exposure to embryo younger than 15 weeks (weeks 0-7 without mental retardation, weeks 8-15 with mental retardation)
• Mental Retardation: exposure between weeks 8 - 25. (weeks 8-15 with microcephaly, weeks 16-25 without much microcephaly, highest risk weeks 8-15). Threshold dose of 12-20 rad. Probably due to disruption of cell migration within the brain.
• Low dose exposure in-utero: increases the spontaneous cancer incidence in the first 10-15 years of life by 40% (a factor of 1.4)
• Excess absolute risk: about 6% per Gy (similar to the risk estimates from the atomic bomb survivors for adult exposure)
• Once pregnancy is declared, max permissible dose to the fetus is 0.5 mSv per month

Question 31

Childhood cancer after in-utero XRT exposure

Answer 31

• Excess absolute risk increase is 6%/Gy
• An obstetric X-ray (10 mGy) results in a 40% increase in the risk of childhood cancer over the spontaneous level (another way of presenting this data is to say there is an increased incidence of leukema and childhood cancer by a factor of about 1.4)
• Exposure in the 3rd trimester increases the childhood malignancy risk the most

Question 32

Methods to Score Tumor Growth Experiments

Answer 32

1. Growth Delay: time taken for a tumor to regrow to the size it was at the time of radiation (only useful for tumors that shrink significantly after radiation therapy)
2. Time for tumor to reach a predetermined size: works best for tumors that show insignificant shrinking after radiation therapy.

Question 33

Thermal Enhancement Ratio (TER)

Answer 33

TER = D_noheat/D_heat

Question 34

Function of small tumor suppressors p16INK4a and p14ARF

Answer 34

p14ARF: binds to MDM2 thus preventing MDM2 from binding and ubiquinating p53 (thus p14ARF allows p53 tetramerize and act as a transcription factor to induce apoptosis

p16INK4a: binds to CyclinD/cdk4 complex, thus inhibiting it from phosphorylating RB (which is bound to E2F). Since RB does not get phosphorylated by CyclinD/cdk4 (and then CyclinE/cdk2) it stays bound to (and inhibits) E2F leading to G1 arrest!

Question 35

Methods of oncogene activation

Answer 35

1. Gene Amplification: More copies of gene made
2. Gene Mutation:
3. Chromosome rearrangements (novel regulatory sequences or fusion transcripts)

Question 36

NF_kB

Answer 36

Transcription factor that mediates response to several stimuli (generally exerts a pro-survival influence through interference with apoptotic signals)

• Heterodimer of p65/p50 (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)
• Exists in high levels in cell but is sequestered in cytoplasm by I_kB (Following formation of DNA double-strand breaks and reactive oxygen species in irradiated cells, kinases (including ATM) phosphorylate IκB, targeting it for ubiquitination and degradation, which allows NF-κB to translocate to the nucleus from the cytoplasm where it can act as a transcription factor)
• TNF liberates NF_kB from I_kB (RIP activates IKK1/IKK2 comple (aka NEMO),which phosphorylates I_kB), enabling NF_kB to enter the nucleus
• Active NF-κB induces transcription of a set of genes that encode the anti-apoptotic IAP’s (“inhibitors of apoptosis”). NF-κB 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
• Competition between p53 (TP53) and NF-κB for CBP/p300 may play an important role in determining the balance between apoptosis and cell cycle arrest following irradiation.

Question 37

Phosphatidylinositol 3-kinases (PI3K)

Answer 37

• Large family of kinases
• Phosphorylate many proteins (ATM, ATR, DNA-PK, PI3K, AKT, mTOR)
• Many cancers have altered PI3K signaling (many have a loss in PTEN function which undoes the phophorylating that PI3K does

Question 38

Mismatch Repair

Answer 38

• counters Microsatellite instability (MIN)
  - Example of MIN is HNPCC
• Key Actors: MMR

-

Question 39

Epigenetic Silencing

Answer 39

Often via DNA methylation at Cytosine (5-Me-Cytosine) at CpG di-nucleotides

Question 40

Histone Modifications

Answer 40

1) Increased acetylation (via acetyltransferases): increased gene expression
2) Increased methylation (via methyltransferases): increased gene expression

Question 41

Therapeutic Gain

Answer 41

Therapeutic Gain = Biological effect ChemoRT_tumor / Biological effect ChemoRT_normaltissue

-We would like to see a synergism, however sometimes we just increase normal tissue/organ damage

Question 42

Expected lifetime of the products of ionization

Answer 42

1. The initial ionization process takes approximately 10^-15 second.
2. The primary radicals produced by the ejection of an electron typically have a lifetime of 10^-10 second.
3. The resulting hydroxyl radical has a lifetime of approximately 10^-9 second.
4. The DNA radicals subsequently produced have a lifetime of approximately 10^-5 second.

Question 43

Important Assays to know

Answer 43

• Alkaline elution: measures single-strand breaks and some base damages
• Neutral comet assay: measures DNA double-strand breaks
• Annexin V-labeling: detects apoptosis
• Western Blot: detection of proteins
• Northern Blot: detection of RNA
• Southern Blot: detection of DNA
• TUNEL assay: used to identify apoptotic cells by detecting DNA fragmentation

Question 44

Human genetic diseases characterized by sensitivity to ionizing radiation

Answer 44

1. Nijmegen breakage syndrome (NBS1 mutation; NBS is a direct phosphorylation target of ATM and complexes with Rad50 and MRE11 to make the MRN complex; defective cells lack an S phase checkpoint)- Lymphoma
2. familial breast cancer (BRCA1)
3. ataxia telangiectasia (ATM) - Leukemia/Lymphoma
4. ataxia telangiectasia-like disorder (MRE11 mutation; MRE11 normally complexes with Rad50 and NBS)
5. LIG4 syndrome (deficient in the DNA ligase IV enzyme (LIG4))
6. Seckel Syndrome (ATR deficiency (low quantity of ATR but functional)
7. Fanconi’s anemia - unclear mechanism (FANCD2 is a target of ATM kinase; FANCD1 is also known as BRCA2!); also sensitive to crosslinking agents - Leukemia
8. Homologues of RecQ-Bloom syndrome, Wernder syndrome, Rothmund-Thompson syndrome (defect in DNA helicases required to unwind DNA 3’ to 5’ and have 3’ to 5’ exonuclease activity; monitor DNA replication fork for damage, acting as intermediaries between replicaiton and recombination)

***XPC (Xeroderma Pigmentosa) is involved in DNA repair of UV induced damage (pyrimidine dimers aka thymidine dimers) and is NOT radiation sensitive!!!!

Question 45

Linear Energy Transfer (LET)

Answer 45

LET: (L = dE/dl where dE is the average energy localy imparted to the medium by a charged particle of specified energy in transversing a distance of dl): average energy deposited per unit path length (keV/µm). Linear energy transfer (LET) is the average amount of energy a particular radiation imparts to the local medium per unit length; ie: Energy per Length. For radiotherapy, we are normally concerned about small amounts of energy over small distances, so the units we use are keV/μm.

Examples:

1. Orthovoltage Photons (250keV): 2.0 keV/μm
2. Cobalt-60 Photons (1.17 - 1.33 MeV) 0.2 keV/μm
3. Linac based Photons (3 MeV): 0.3 keV/μm
4. Alpha-particle (2.5 MeV): 166keV/μm
5. Neutrons (14 MeV) Track Average: 12 keV/μm
6. Neutrons (14 MeV) Energy Average: 100 keV/μm
7. Protons (10 MeV)
   
   1. Average: 4.7 keV/μm
   2. On Entering Phantom: 0.5 keV/μm
   3. At Bragg Peak: 100 keV/μm
8. Proton (150 MeV): 0.5 keV/μm
9. Fe ions/heavy space particles (2 GeV): 1000keV/μm

Key Facts about LET:

1. LET is proportional to the charge density of a medium
2. LET is proportional to the charge (squared) on the particle moving through a medium
3. LET is inversely proportional to the speed (squared) of the particle
4. LET is related to teh density of ionization along the particle’s track
5. Maximum cell killing occurs at an LET of approximately 100 keV/μm (because DNA double helix is ~this diameter (20A or 2nm)
6. RBE decreases with increasing LET above about 100 keV/μm (“overkill effect”; essentially there is alot of energy wasted because ionizing events are too close together!!)
7. RBE shows the greatest changes for LET values between roughly 20 and 100 keV/μm
8. OER decreases slowly with increasing LET for low LET values, but falls rapidly after LET exceeds about 60 keV/μm

Question 46

Caspases

Answer 46

Caspases: intracellular cysteine proteases

Initiator caspases: first to be activated, cleave and activate the effector/executioner caspases; these include caspases 2, 8 (extrinsic), 9 (intrinsic) and 10.

Effector/executioner caspases: including caspases 3, 6, and 7, which then cleave, degrade or activate other cellular proteins.

Activation of caspases: regulated by members of the BCL2 family and by the inhibitors of apoptotic protein (IAP) family.

• BAX is one of a series of pro-apoptotic members of the BCL2 family. These pro-apoptotic BCL2 family members regulate the release of cytochrome c from mitochondria and elicit the subsequent activation of caspases.
• Another important function of p53 is that it causes upregulation of pro-apoptotic PUMA.
• X-linked IAP (XIAP) inhibits the activity of caspases directly (anti-apoptotic).
• DIABLO is a pro-apoptotic protein that prevents IAPs from inhibiting caspases.

BAX and p53 are required for some forms of DNA damage-induced apoptosis

Question 47

Examples of Radiation-Induced Aberrations

Answer 47

Lethal

• Dicentric chromosome (“unstable”)
• Ring chromosome
• Anaphase bridge (chromatid aberation)

Non-Lethal

• Symmetric translocation - break in two pre-replicative chromosomes with broken ends being exchanged (difficult to see except with FISH (aka chromosome painting)
• Small interstitial deletion - two breaks in same arm of the same chromosome, leading to loss of the genetic information between the two breaks

Question 48

Sublethal Damage Repair vs. Potentially Lethal Damage Repair

Answer 48

Sublethal damage repair: 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, as opposed to a single exposure.

Potentially lethal damage repair: operationally defined as an increase in cell survival after delivery of a large, single radiation dose under environmental conditions not conducive to progression of cells through the cell cycle for several hours after irradiation. 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. PLD is believed to be complex DSBs that are repaired slowly as compared to simple DSBs. Therefore, cells that are left in stationary phase after irradiation (for 6 hours or more) display enhanced survival as they have time to repair complex DSBs before resuming progression through the cell cycle.

Repair of DNA damage and rejoining of chromosome breaks presumably underlie both the sublethal and potentially lethal damage repair.

Question 49

ATM

Answer 49

ATM (Ataxia Telangictasis Mutated):

• Exists as a inactive dimer until there is ionizing radiation induced DNA DSB –> dissociates into active monomer and phosphorylates targets involved in DNA repair (H2AX, SMC1, BRCA1, p53BP1) and cell cycle control (p53, CHK2, CHK1, NBS1)
• Phosphoinositol 3-kinase like kinase (PIKK)
• Serves as the central orchestrator of the signal transduction response to DSB’s
• Cells deficient in ATM activity display cell cycle checkpoint defects and IR sensitivity.
• Activated ATM phosphorylates multiple, distinct target proteins, including histone H2AX (to γ-H2AX, leading to DNA repair machinery recruitment), p53 (tumor suppressor), BRCA1 (tumor suppressor), SMC1.
• CHK2 and MDM2 are involved in control of the G1–S phase transition. Upon phosphorylation by CHK2, p53 is stabilized, causing cell cycle arrest in G1. ATM also phosphorylates MDM2, which reduces the ability of MDM2 to negatively regulate p53.
• NBS1 and CHK2 are implicated in S phase progression. PUMA (“p53-upregulated modulator of apoptosis”) is a pro-apoptotic gene that can induce cell death via a p53-dependent pathway.
• G2 checkpoint regulation by ATM is thought to occur through 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.

Question 50

Angiogenesis

Answer 50

• Vascular endothelial growth factor (VEGF) is induced under hypoxic conditions through the action of hypoxia-inducible transcription factors that bind to the VEGF promoter to stimulate its transcription and, as a consequence, to stimulate angiogenesis (thus we use VEGF-inhibitor’s such as Avastin)
• 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, 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
• In the absence of angiogenesis, tumors would only be expected to reach a diameter of about 2 mm
• Microvessel density, a measure of angiogenesis, has been correlated positively with metastatic spread for most tumor types.
• Angiostatin and endostatin are inhibitors of angiogenesis
• basic fibroblast growth factor is a positive regulator of angiogenesis
• An increased apoptotic index is often observed in hypoxic regions of tumors.
• 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. 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. Under aerobic conditions, HIF-1α is hydroxylated by HIF prolyl hydroxylases. Hydroxylation at two prolyl residues targets HIF-1α to the von Hippel-Lindau E3 ubiquitin ligase resulting in HIF-1α ubiquitination and subsequent proteosomal degradation, thereby limiting upregulation of target genes. Because the hydroxylation catalyzed by prolyl hydroxylases requires molecular oxygen, HIF escapes inactivation under hypoxic conditions.

Question 51

Mitotic Index (MI)

Answer 51

MI = λT_M/T_C (where MI is the mitotic index, T_M is the length of mitosis and T_C is the total cell cycle time)

Example: MI of a cell line is 5%, growth fraction is 100%, cell cycle time is 14 hours, correction factor λ is 0.7: How long is mitosis?

T_M = (MI)(T_C/λ) = (0.05)(14 hours)/0.7 = 1 hour.

Note: Even without performing this calculation, it should be noted that the duration of mitosis for most mammalian cells is typically ~1 hour.

Question 52

Cell Loss Factor

Answer 52

Cell Loss Factor = 1- (T_pot/T_D).

• mean T_C (cell cycle time) 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 (T_S), using the equation Tpot = λTS/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.

Question 53

T_pot

Answer 53

**T_pot = λT_S/LI ** (where LI is the labeling index, T_S is the duration of the S phase, and λ is a constant ranging from about 0.6 to 1.0)

Growth factor is taken into account in T_pot

Question 54

Absorbed, Equivalent and Effective Dose

Answer 54

Absorbed Dose (in Gray, formerly in rad): Energy per unit mass

• 1 Gy of X-rays < 1 Gy of Neutrons

Equivalent Dose (in Sievert, formerly in rem) = Absorbed dose * radiation weighting factor (W_R)

• 1 Sv of X-rays = 1 Sv of Neutrons

Radiation Weighting Factor (W_R):

• Photon: 1
• Electron: 1
• Proton: 2
• Charged Pion: 2
• Alpha Particle: 20
• Fision fragments/heavy ions: 20
• Neutrons (10 keV): 5
• Neutrons (10 keV to 20 MeV): 10 to 20
• Neutrons (>20 MeV): 5

Effective dose (in Sievert) = Sum of Absorbed Dose * W_R * W_T

Tissue Weighting Factor (WT):

• Lung/Stomach/Colon/Bone Marrow/Breast/remaining tissues: 0.12 each
• Gonads: 0.08
• Thyroid/Esophagus/Bladder/Liver: 0.04 each
• Bone Surface/Skin/Brain/Salivary Glands: 0.01 each

Committed Equivalent dose (in Sievert): Equivalent dose integrated over 50 years (relevant to incorporated radionuclides)

Committed Effective dose (in Sievert): Effective dose integrated over 50 years (relevant to incorporated radionuclides)

Doses important for Populations:

Collective Effective dose (in Person-Sievert): Product of the average effective dose and the number of individuals exposed

Collective committed effective dose (in Person-Sievert): Integration of the collective dose over 50 years (relevant to incorporated radionuclides)

Question 55

Dose Reduction Factor (DRF)

Answer 55

DRF = Dose of radiation in the prescence of the drug/ Dose of radiation in the absence of the drug

Question 56

Deterministic vs. Stochastic Effects

Answer 56

Deterministic Effects: loss of tissue function due to loss of a number of cells, which has a threshold in dose, and the severity of the effect is dose related. Example: Radiation-induced cataracts, late tissue fibrosis

• severity increases with dose; practical threshold; probability of occurrence increases with dose (e.g. cataract)
  
  • 2 Gy to the lens is threshold for single acute exposure
  • 5-8 Gy to the lens is threshold for protracted exposure
  • latent period of ~8years for cataract formation
• Cardiovascular disease and other non-neoplastic diseases seem to go up at doses of more than about 0.5 Sv

Stochastic Effects: Irradiated cells are viable after irradiation but are modified (mutated) and the probability of of cancer increases with dose, probably with no threshhold, but the severity of the cancer is NOT dose related (a cancer induced by 1 Gy is no worse than one induced by 0.1 Gy, but the probability of induction is greater with increased dose). NO DOSE is SAFE!! Example: Radiation Carcinogenesis, Heritable effects

• severity independent of dose; no threshold; probability of occurence increases with dose (e.g. cancer)

Question 57

NCRP Radiation Exposure Limits (and key ICRP differences)

Answer 57

Effective Dose: Sum of Absorbed dose * W_R * W_T

(W_R = radiation weighting factor; W_T = tissue weighting factor)

Deterministic Effects:

1. 150 mSv/year (15 rem/year) for the lens of the eye
2. 500 mSv/year (50 rem/year) for localized areas of the skin, the hands, the feet

Stochastic Effects:

1. No occupational exposure until 18 years of age
2. Effective dose in any year should not exceed 50mSv (5 rem)/year
3. Individual worker’s lifetime effective dose should not exceed age in years * 10mSv (1 rem * age in years)
   
   1. ICRP is 20 mSv/y averaged over 5 years

Embryo/Fetus Exposure (once pregnancy declared):

• 0.5 mSv/month (0.05 rem/month)
  
  • ICRP is a total of 1mSv to abdomen surface)

Public Exposure (annual): Does not include medical imaging dose as this is assumed to confer a “benefit” to the person

• Effective dose limit: continuous or frequent exposure: 1 mSv/year (0.1 rem/year)
• Effective dose limit (infrequent exposure): 5 mSv/year (0.5 rem/year)
  
  • ICRP is still 1mSv/year…no infrequent vs. frequent designation!!!
• Dose equivalent limits - Lens of eye: 15mSv/year
• Skin/extremities: 50 mSv/year

Education/Training Exposure (annual to even <18 year olds): (ICRP has no statement on these dose constraints!!)

• Effective dose limit: 1 mSv/year (0.1 rem/year)
• Dose equivalent limits - Lens of eye: 15mSv/year (1.5 rem/year)
• Skin/extremities: 50 mSv/year (5 rem/year)

Negligible Individual Dose (annual): the dose below which further expenditure to improve radiation protection is unwarranted: 0.01 mSv/year (0.001 rem/year)

Lifesaving Events: exposure up to 0.5 Sv (choose older workers if possible. Exposure over 0.5 counsel regarding short/long term consequences

Average Annual equivalent dose to radiation workers: 2 mSv (total detriment of 1/10000; this is equal to the risk of a fatal accident in a safe industry)

Question 58

Oncogene and Tumor Suppressors

Answer 58

Oncogenes are gain-of-function mutations, so only one copy needs to be activated, so they act in a DOMINANT fashion

Examples of Oncogenes: H-ras, K-ras, N-ras, c-myc, L-myc, N-myc, neu, EGFR, brc-abl, bcl-2, src, ras

Oncogene Activation Mechanisms:

• Retroviral integration
• Point Mutation
• Chromosome rearrangement (translocation)
• Gene Amplification

Tumor Suppressor genes involve loss-of-function mutations, so that both copies must be lost; so they can be said to act in a RECESSIVE fashion)

Examples of Tumor Suppressors: p53, Rb, p14ARF, p16INK4A, VHL, PTEN, PTCH, MEN1, APC, E-CAD, BRCA1, BRCA2, WT1, NF1, NF1, NF2,

Question 59

Common Chemotherapy Agents

Answer 59

• Alkylating Agents: cell cycle nonspecific
  
  • Nitrogen Mustard derivatives
    
    • cyclophosphamide
    • chlorambucil
    • melphalan
  • Ethylenimine derivatives
    
    • thiotepa
  • Alkyl sulfonates
    
    • busulfan
  • Triazine derivatives
    
    • Dacarbazine
  • Nitrosoureas
    
    • BCNU (carmustine)
    • CCNU (lomustine)
  • Temodar (oral prodrug with blood-brain barrier penetration)
    
    • DNA alkylator
    • effective when the promoter for MGMT is methylated (silenced)
• Antibiotics: cell cycle nonspecific
  
  • Doxorubicin (Adriamycin) (anthracycline) - more toxic to hypoxic cells (key toxicity is cardiac and recall reactions!)
  • Daunorubicin (anthracycline)
  • Dactinomycin (more toxic to aerobic cells)
  • Bleomycin (most toxic to aerobic cells because of free radical cell killing)
  • Mitomycin C (most toxic to hypoxic cells because of bioreduction)
• Antimetabolites:
  
  • Methotrexate (folic acid antagonist that binds dihydrofolate reductase decreasing synthesis of thymidine and purine nucleotides); more toxic to hypoxic cells
  • 5-FU (structural analog to thymine; irreversibly inhibits thymidylate synthetase)
  • Gemzar (inhibits ribonucleotide reductase to block DNA synthesisby preventing the de novo biosynthesis of deoxyribonucleoside triphosphate precursors; pyrimidine analog
• Nucleoside Analogues: cell cycle specific
  
  • Cytarabine (cytosine analog)
  • 5-azacytidine
• Vinca Alkaloids:* (G₂ phase specific)*(microtubule polymerization inhibitors)
  
  • Vincristine (Oncovin)
  • Vinblastine (Velban)
  • Vinorelbine (Navelbine)
• Taxanes: (M-phase specific) -microtubule stabilizing agents
  
  • Paclitaxel (Taxol)
    
    • peripheral neuropathy
  • Docetaxel (Taxotere)
    
    • peripheral neuropathy
• **Miscellaneous Agents: **
  
  • Procarbazine (more toxic to aerobic cells)
  • Hydroxyurea - (**S phase specific) **- ribonucleotide reductase inhibitor
  • Cis-Platinum (cell cycle nonspecific) - both interstrand and intrastrand DNA crosslinking thus inhibiting DSB repair. Causes ototoxicity and renal toxicity.
• **Topoisomerase Inhibitors: **
  
  • Eotposide - Top II inhibitor
  • Topotecan - Top I inhibitor
  • Irinotecan - Top I inhibitor
• **Targeted Therapy: **
  
  • Cetuximab (Erbitux): anti-EGFR monoclonal antibody (grade 2 or >acneform rash with radiation is a good sign!)
    
    • Panitumumab is a similar new drug!
  • Erlotinib (Tarceva): EGFR small molecule tyrosine kinase inhibitor
  • Bevacizumab (Avastin): anti-VEGF monoclonal antibody
  • Imatinib (Gleevec): small molecule Tyrosine kinase inhibitor; blocks the ATP-binding site of the p210 tyrosine kinase domain of the BCR-ABL fusion protein in CML (also against c-kit and PDGF-R to lesser extent)
  • Rituximab (Rituxan): monoclonal antibody against B-cell surface antigen CD20 (induces lysis and apoptosis)
  • Crizotinib: small molecular inhibitor of ALK and ROS1 kinases
  • Trastuzumab (Herceptin): anti-HER2/neu antibody
  • PARP-inhibitors: inhibit base excision repair; seem to super sensitize BRCA mutated cells; seems to sensitize cells to XRT
  • Sorafenib: small molecule multi-kinase inhibitor that targets RAF1, KIT, FLT3, VEGFR (KDR) and PDGFR. (RAF1 is a component of the RAS signaling cascade)
  • Gefitinib: small molecule tyrosine kinase inhibitor
  • Sunitinib: tyrosine kinase inhibitor with multiple targets including EGFR, FLT3, VEGFR and KIT
  • Sirolimus: binds to the FKBP12 complex and inhibits mTOR (FRAP1), a downstream target of the PI3K/AKT pro-survival signaling pathway that is activated by radiation exposure
    
    • Rapamycin, everolimus, and temsirolimus are similar
  • Bortezomib: inhibits the chymotrypsin-like activity of the 26S proteasome and inhibits the degradation of many proteins that undergo ubiquitin-mediated degradation (like IκB, thus indirectly keeping NF-κB inactive)
  • Tipifarnib: inhibits farnesyl transferase, an enzyme that is required for the sustained activity of the RAS signaling cascade
  • Ipilimumab: antibody against the immune checkpoint molecule CTLA-4.
• Hormonal:
  
  • Gosarelin (Zoladex) - GnRH agonist
  • Leuprolide (Lurpon): GnRH agonist
• Anti-Androgen:
  
  • Casodex: binds/inhibits androgen receptors
  • Tamoxifen: Estrogen receptor binding
  • Arimidex: aromatase inhibitor (blocks estrogen production)

Question 60

Categories of Mammalian Cell Sensitivity

Answer 60

1. Vegetative intermitotic cells: stem cells (erythroblasts, intenstinal crypt cells, germinal cells of the epidermis)
   
   1. divide regularly; no differentiation
   2. high sensitivity to XRT
2. Differentiating intermitotic cells:(Myelocytes)
   
   1. Divide regularly; some differentiation between division
   2. intermediate sensitivity to XRT
3. Connective Tissue/Blood vessels Cells: (in between 2 and 4)
4. Reverting Postmitotic cells:(Liver)
   
   1. Do not divide regularly; variably differentiated
5. Fixed Postmitotic cells: (Nerve cells, Muscle cells)
   
   1. Do not divide; highly differentiated
   2. Low sensitivity to XRT

Question 61

Labeling Index (LI)

Answer 61

LI = λT_S/T_C

LI = the fraction of cells that take up tritiated thymidine (i.e. the fraction of cells in S)

Question 62

Accelerated Repopulation

Answer 62

Accelerated Repopulation: the triggering of surviving cells (clonogens/stem cells) to divide more rapidly as a tumor shrinks after irradiation or treatment with any cytotoxic agent

• starts in head & neck cancer in about 4 weeks after initiation of fractionated radiotherapy
• ~0.6 Gy per day is needed to compensate for this repopulation
• it may be better to delay the start of treatment than to introduce interruptions during treatment
• Local tumor control is decreased by ~1.4%/day for head&neck cancer for each day overall treatment time is prolonged; in cervix carcinoma ~0.5%/day local control reduction

Question 63

Biological Effective Dose (BED)

Answer 63

BED = total dose * relative effectiveness

BED = E/α = (nd) * (1 + d/(α/β)) where n = # of fractions, d = dose per fraction

Question 64

Hypoxic Cell Radiosensitizers

Answer 64

• Nitroimidazoles: (Misonidazole, Nimorazole, Etanidazole) which have NO2 groups at the 2nd positiion
  
  • mimics oxygen by “fixing” damage produced by free radicals
  • on meta-analysis of H&N cancer trials, some subgroups have shown improved LC and OS
• Carbogen Breathing: (15% carbon dioxide + oxygen) given to patient to breathe to overcome chronic hypoxia
• Nicotinamide: to overcome acute hypoxia

Question 65

Hypoxic Cytotoxins

Answer 65

1. Quinone Antibiotics
   
   1. Mitomycin C
      
      1. bioreductive qualities
2. Nitroaromatic compounds (toxic!!)
3. Benzotriazine di-N-oxides
   
   1. Tirapazamine
      
      1. Also known as SR-4233
      2. Uptake is equal in hypoxic and aerobic conditions
      3. If it loses one electron in hypoxic conditions, it becomes cytotoxic
      4. When two electrons are extracted in aerobic conditions, it becomes non-toxic
      5. In aerobic conditions, it can also sensitize cells to radiation
      6. The potency of some chemotherapy agents can be enhanced by the presence of this cytotoxin
      7. aromatic heterocycle di-N-oxide,its full chemical name is 3-amino-1,2,4-benzotriazine-1,4 dioxide
4. Dinitrobenzamide modified nitrogen mustard
   
   1. TH-302
5. 2-nitroimidazole attached to dibromo iso-phosphoramide

Question 66

Hypoxia-Inducible Factor

Answer 66

Hypoxia-Inducible Factors:

• transcription factors that facilitae both oxygen delivery and adaptation to oxygen deprivation by regulating gene expression
  
  • angiogenesis
    
    • Î VEGF-A
  • metabolism
    
    • shift to glycolysis
  • metastasis
    
    • alters regulation of cell adhesion, ECM formation, and cell migration (E-cadherin, lysyl oxidase, CXCR4)
• contain α (oxygen sensitive) and β (constitutively expressed) subunits
• HIF-α subunits are oxygen regulated through hydroxylation; binding to VHL and degradation in the proteosome
  
  • under hypoxic conditions, hydroxylation decreases and HIF-α dimerizes with β subunit (aka ARNT), binds DNA at a 5’-RCGTG-3’ sequence, and initiates gene transcription!
• three HIF’s have been identified (HIF-1, HIF-2, and HIF-3)
  
  • HIF-1 is the “global regulator” of hypoxia-inducible gene expression
    
    • binds EPO enhancer
  • HIF-2 is more restricted in cell type expression
  • HIF-3 is not well understood
• Loss of the tumor suppressor genes VHL and PTEN can cause HIF to become active under normoxic conditions (HIF is able to stabilize)
• HIF-1 stabilization promotes radioresistance via release of proangiogenic cytokines such as VEGF
  
  • HIF-1 deficient tumors are more sensitive to radiation compared to wild-type tumors
• HIF-1 can also increase tumor radiosensitivity through the induction of apoptosis

Question 67

Drug resistant tumors

Answer 67

1. Multi-drug resistance : mdr p-glycoprotein expressed in the cell membrane
   
   1. shown to be important for Doxorubicin resistance
   2. Cross resistance to many related and unrelated drug types
   3. resistance produced can be of several orders of magnitude in survival for a given drug dose
   4. permanent change in cell phenotype (not transient)
   5. does not carry over to radiation resistance
   6. can be blocked with calcium channel blockers (verapami)
2. Increase in Glutathione production
   
   1. can be downregulated with buthionine sulfoximine (shown to reduce resistance to Melphalan and cis-platinum; but its toxic to kidneys)
   2. can cause small increase in radioresistance!!
3. Increased DNA repair
   
   1. can cause small increase in radioresistance!!

Pleiotropic resistance: resistance to one drug results in cross-resistance to other drugs with a different mechanism of action.

Radioresistance and chemoresistance: may occur together, but radiation rarely induces chemoresistance and vice versa.

Drugs that penetrate blood-brain barrier: BCNU, Temodar, Cytosine arabinoside, hydroxyurea, methotrexate (at high IV dose)

Tests for resistance to a chemo agent: in-vitro clonogenic assays, xenograft tumors in nude mice, presence of micronuclei in treated cells

Question 68

Common Oncogenes and associated malignancies

Answer 68

• H-Ras (point mutation on 11): Colon
• K-Ras (point mutation on 12): Pancreas
• N-Ras (point mutation on 1): Melanoma
• EGFR (gene amplification on 7): Squamous cell carcinoma
• neu (gene amplification on 17): Breast, Neuroblastoma
• C-myc (8-14;2-8;8-22): Burkitt Lymphoma
• L-myc (gene amplificiation on 8): Lung
• N-myc (gene amplification on 8): Neuroblastoma
• brc-abl (9-22): CML
• bcl-2 (14-18):
• VHL: Kidney
• PTCH: Medulloblastoma, Basal Cell Carcinoma
• RET (gain of function mutation): Thyroid
• BRAF: Melanoma

Question 69

p53

Answer 69

• p53 is a tumor suppressor gene
• 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.
• p53 is modified post-translationally by phosphorylation or by acetylation in response to DNA damage
  
  • DNA damage causes p53 to become stabilized and active
  • Irradiation of cells activates ATM to add phosphate groups to p53.
  • MDM2 binding to p53 stimulates degradation of p53
• p53 is inactivated in more than half of all human cancers.
  
  • p53 is inactivated by loss of heterozygosity/loss of function
• p53 increases expression of GADD45A, p21 (encoded by WAF1/CIP1) and PCNA.
• p53 stimulates the activity of BAX and BID in irradiated cells, resulting in apoptosis.
• Following irradiation, p53 inhibits CDC25C, leading to inhibition of the G2 to M phase transition.
• Viruses that contain proteins that inactivate p53 include human papilloma virus, SV40 and adenovirus, but not EBV.

Question 70

Photodynamic therapy (PDT)

Answer 70

Photodynamic therapy (PDT):

• Requires a photosensitizer, oxygen and light to produce the active, toxic species, singlet oxygen.
• PDT has been used to treat both superficial tumors as well as more deep-seated tumors that can be accessed endoscopically and exposed to light using fiberoptic probes.
• Although direct tumor cell killing may occur, particularly when there is a long drug-light exposure that allows free diffusion of the photosensitizer into tumor tissue, in most instances, the main photosensitizing effect occurs while the drug is confined to the tumor vasculature and result in damage to cells therein, which leads to the indirect killing of tumor cells as a result of the vascular damage.
• Because oxygen is required for the PDT reaction, PDT is ineffective in hypoxia.

Question 71

Amifostine

Answer 71

Amifostine:

• is a pro-drug that is metabolized by alkaline phosphatase to the free thiol metabolite that acts as the direct radioprotective agent
• Amifostine must be administered intravenously 15-30 minutes before radiotherapy for maximal efficacy
• does not readily cross the blood brain barrier and therefore affords little radioprotection to tissues in the CNS
• Hypotension, nausea/vomiting, fatigue and fever/rash are the main toxicities associated with amifostine.

Question 72

Late Effects of Normal Tissue (LENT)

Answer 72

Late Effects of Normal Tissue (LENT)

• conference held in 1992
• led to introduction of the SOMA classification for late toxicity

Question 73

SOMA Scoring System

Answer 73

SOMA Classification for LATE TOXICITY

• Subjective: injury as perceived by the patient (usually by questionnaire or diary)
  
  • Example: Headache
• Objective: injury as assessed by clinical examination (may detect signs of tissue dysfunction that are still below the threshold that will give the patient symptoms, but that indicate how close to tissue tolerance the treatment is or that may be early indicators of more serious problems that are developing and will be expressed later)
  
  • Example: Neurological Deficit
• Management: the active steps that may be taken in an attempt to ameliorate the symptoms
  
  • Example: Anticonvulsives
• Analytic: involving tools by which tissue function can be assessed even more objectively or with more biologic insight than by simple clinical examination. Invasiveness and cost of any tool must be reasonable and proportional to the severity of the symptoms and the possible therapeutic consequences
  
  • Example: MRI

SOMA Scoring (Scale of 1 to 4)

Subjective:

• Grade 1: Occasional and minimal
• Grade 2: Intermittent and tolerable
• Grade 3: Persistent and intense
• Grade 4: Refractory and excruciating

Objective:

• Grade 1: Barely detectable
• Grade 2: Easily detectable
• Grade 3: Focal motor signs, vision disturbances, etc
• Grade 4: Hemiplegia, hemisensory deficit

Management:

• Grade 1: Occasional non-narcotic
• Grade 2: Regular non-narcotic
• Grade 3: Regular narcotic
• Grade 4: Surgical intervention

Analytic:

• Findings that are quantifiable via testing
  
  • MRI, PET, CT, lab tests, etc