Radiation Biology Flashcards

1
Q

Linear Quadratic equation

A

S = exp^(-aD-BD^2)

S=surviving fraction
a = alpha, coefficient
of radiation sensitivity for linear component
B = beta. coefficient
of radiation sensitivity for quadratic component
D = dose delivered as single fraction

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2
Q

EQD2 equation

and describe the concept

A

EQD2 allows us to work out an equivalent total dose as if it was in 2Gy per fraction for treatments that were given either hyper- or hypo- fractionated. To be able to calculate if it has reached tolerance dose for noral tissues when adding 2 or more treatments.

EQD2 = D x (a/B + d) / (a/B + 2)

a/B = this is the ratio for tissue/tumour under consideration with the unit Gy

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3
Q

Define Biologically Effective Dose (BED) of a given schedule, including the formula for its calculation

A

i. BED is a measure of the true biological dose delivered calculated using the a/B ratio for the tissue/tumour. BED is mainly used for stereotactic RTx now
ii. BED = nd x(1+(d/(a/B)) )

a/B ratio is for tissue/tumour, unit Gy

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4
Q

i. Outline the concept of forgotten dose and its clinical relevance to retreatment of spine

A

i. Forgotten dose is the concept that normal tissue may have some recovery of their tolerance dose following radiation exposure, such that a proportion of the radiation dose is “forgotten”
ii. For the spine TD5/5 is 50Gy,

Spinal Cord 
* Heavily studied in mice and primates 
* Definite evidence of repair 
	■ Mice 
		● Juvenile mice immediately regained tolerance and peaked at 2 months (~120% of total tolerance for cumulative dose) 
		● Adult mice began to regain tolerance at 2 months and peaked at 5 months (~140% of total tolerance for cumulative dose) 
	■ Primates (ED10 paralysis) 
		● More gradual recovery of tolerance (150% of total tolerance for cumulative dose at 1 year, 156% at 2 years and 167% at 3 years) 
* Small human series have published data 
	■ No myelopathy seen for cumulative EQD2 125-172% with interval between 4 months and 13 years 
	In humans  Nieder et al (2006) assessed 78 patients that received re-irradiation to spinal cord. Concluded that if more than 6 months between treatment courses and the BED for each course was < 98Gy(2), risk of myelopathy low if total cumulative BED was <135.5Gy. No case of myelopathy with BED <120Gy (with a/B 2Gy for cervical and thoracic and 4Gy for lumbar)
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5
Q

ii. List the factors that need to be taken into account when considering re-treatment in general

A

i. OARs
i. - Initial radiation dose to OARs and calculating the EQD2 if was not treated in 2Gy fractions.
ii. - Initial radiation volume and coverage of OARs and how this relates to the planned re-irradiation ?degree of overlap.
ii. Concurrent treatments (chemotherapy or immunotherapy) delivered at the time of initial RT which could affect tolerance of OARs
iii. Time interval between the initial treatment and the planned retreatment, is there a potential for recovery of normal tissue tolerance
iv. Patient factors –
i. function of tissue/organ being retreated, i.e. is the underlying organ function good or does the patient have underlying medical conditions such as emphysema and needing to retreat lung
ii. Expected life span of patient or QOL if left untreated: are they at risk of developing late toxicities, or do the benefits of retreating outweigh late toxicity risks
iii. Patient age and ability to tolerate RTx
v. Alternative treatment options – consider if surgery or supportive care would be better options

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6
Q

Briefly discuss the re-treatment tolerance for late effects for spinal cord. Include in your answer any relevant clinical and laboratory data where appropriate.

A

Tolerance dose for spinal cord = 50Gy for 0.02% risk of meylopathy
Late effects for spinal cord is myelopathy which is permanent and Lhermitte’s sign which is usually reversible. Retreatment tolerance for spinal cord can be increased with a time interval. There has been lab studies on rats and Rhesus monkeys that looked at spinal cord myelopathy with retreatment. ■ Primates (ED10 paralysis)
● More gradual recovery of tolerance (150% of total tolerance for cumulative dose at 1 year, 156% at 2 years and 167% at 3 years)

In small retrospective human studies it is estimated a 25% recovery in dose tolerance of the spinal cord has been suggested based on data that looked at a greater than 6 month period between two course of RT

Nieder et al (2006) assessed 78 patients that received re-irradiation to spinal cord. Concluded that if more than 6 months between treatment courses and the EQD2 for each course was < 48Gy, risk of myelopathy low if total EQD2 was 68Gy

NHS Christie guidelines are that it is safe to retreat as long as cumulative BED <120Gy and at least 6 months between #s

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7
Q

Outline the proposed mechanisms of late radiation injury to the spinal cord.

A

Late damage includes two principal syndromes. The first, occurring from about 6 to 18 months, involves demyelination and necrosis of the white matter from parenchymal cell loss; the second is mostly a vasculopathy and has a latency of 1 to 4 years.

  1. Parenchymal cell loss
    i. Occurs 6 months and over after radiation to spinal cord
    ii. Diffuse or focal demyelination and extensive necrosis, probably as a consequence of injury and loss of oligodendrocytes by killing of glial progenitor cells (astrocytes) which cause gliosis and eventuall fibrosis of the cord
  2. Vascular injury
    i. Occurs 6 months to years after radiation
    ii. Fibrin rich necrosis of white matter can occur and is thought to be caused by microvascular damage leading to increased capillary permeability and resultant slowing of blood flow to obliteration of vessels
    iii. Probable mechanism is endothelial cell damage, leading to eventual obliteration of small vessels
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8
Q

Define and discuss the concept of a serial functional subunit. Include in your answer a brief description of ‘threshold dose’.

A

In serial FSUs the function of the entire organ depends on the function of each individual FSU.
Serial FSU organ such as spinal cord are not significantly affected by volume effect. Function of the organ is dependent on dose (threshold dose); with a hotspot capable of causing a ‘break’ in the series structure if it reaches over a certain threshold dose.
Serial FSU = must not exceed max DOSE
Parallel FSU = must not exceed max VOLUME
No organ is completely serial or parallel.

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9
Q

ii. Below are two cell survival curves for low and high α/β cell lines. (1) Identify the axes and curves labelled A-D

A

A. Surviving fraction
B. Dose in Gy
C. high a/B ratio cell line
D. low a/B ratio cell line

The dose-responsiveness relationship for late responding tissues (low alpha/beta) is more curved than for early-responding tissues (high alpha/beta) . In the linear-quadratic formulation, this translates into a larger a/B ratio for early effects than late effects.

The a/ratio is the dose at which the linear (a) and the quadratic (B) components of cell killing are equal, that is, aD=BD^2 or D = a/B

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10
Q

Define the term α/β value and describe how it can be found from a cell survival curve. (1)

A

The ratio of the parameters alpha and B in the linear quadratic model; used to quantify the fractionation sensitivity of tissues.
From a cell survival curve the a/B can be found when the components of alpha cell killing are equal to B components of cell killing.

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11
Q

iii. List four potential limitations of the Linear Quadratic model in clinical practice. (2)

A

i. At very low doses per fraction<1Gy, the LQ model could underestimate the biological effect of a given dose, due to the low-dose hyper-radiosensitivity phenomenon.
ii. At very high doses per fraction >8Gy, the LQ model underestimates the biological effect due to factors such as vascular and stromal damage not being taken into account.
iii. The LQ model does not include a time factor and assumes sufficient time between fractions for repair of sublethal damage
iv. It does not take into account tissue /tumour repopulation over time
v. It has not been validated with concurrent chemotherapy

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12
Q

i. What is sublethal damage repair?

A

SLD is damage to DNA that is insufficient to kill cell given that there is intact DNA repair pathways and sufficient time between fractions.

SLD repair is the operational term for the increase in cell survival that is observed if a given radiation dose is split into two fractions separated by a time interval.

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13
Q

ii. Using a labelled diagram, outline the key differences in cell survival curves for a single fraction vs. multiple fraction course of radiation therapy. (2)

A

The concept of an “effective” survival curve for a multi fraction regimen is illustrated. If the radiation dose is delivered in a series of equal fractions separated by time interval sufficiently
long for the repair of sublethal damage to be complete between fractions, the shoulder of the curve is repeated many times. The effective dose-survival curve is an exponential function of dose, that is a straight line from the origin through a point on the single-dose survival curve corresponding to the daily dose fraction (e.g. 2Gy).
The dose resulting in one decade of cell killing (D10) is related to the D0 by the expression D10=2.3xD0

D0 of the effective survival curve (i.e. the reciprocal of the slope), defined to be the dose required to reduce the fraction of surviving cells to 37%, has a value close to 3Gy for cells of human origin

D10 is the dose required to kill 90% of the population.

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14
Q

iii. Describe the mechanism of acute skin reaction including how moist desquamation develops. (3)

A

Skin is an example of a H-type tissue from Michalowski’s classification (hierarchical)
H-type tissues have 3 groups of cells present: Stem cells, Maturing/partly differentiated and functional cells.
The skin is composed of the outer layer, the epidermis, which is the site of early radiation reactions, and the deeper layer, the dermis, which is the site of late radiation reactions
The epidermis is derived from a basal layer of actively proliferating cells, which is covered by several layers of nondividing differentiating cells to the surface, at which the most superficial keratinized cells are desquamated. It takes about 14 days from the time a newly formed cell leaves the basal layer to the time it is desquamated from the surface. The target cells for radiation damage are the dividing stem cells in the basal layer.
Early erythema develops in the second to third week of a fractionated regimen, similar to sunburn, which is caused by vasodilation, oedema, and loss of plasma constituents from capillaries. Reactions resulting from stem cell death take longer to develop and cause desquamation from depletion of the basal cell population.
At lower doses, islets of skin may regrow from surviving stem cells; at higher doses, at which there are no surviving stem cells within the area treated, moist desquamation is complete, and healing must occur by migration of cells from outside the treated area.

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15
Q

iv. Briefly describe consequential late effects and how they differ from late toxicities of radiation therapy. Give a clinical example. (2)

A

If intensive fractionation protocols deplete the stem cell population below levels needed for tissue restoration, an early reaction in a rapidly proliferating tissue may persist as a chronic injury. This has been termed a consequential late effect, that is, a late effect consequent to, or evolving out of, a persistent severe early effect. The earlier damage is most often attributable to an overlying acutely responding epithelial surface—for example, fibrosis or necrosis of skin consequent to desquamation and acute ulceration.

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16
Q

Name the structures labelled A to I in the diagram of an animal cell below:
(3)

A
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17
Q

Name the DNA-repair pathway that is involved during / immediately after DNA replication pertaining to incorrectly paired nucleotides. (1)

Microsatellite Instability (MSI) is caused by mutations in the genes responsible for the above pathway. Name at least one affected gene and name the associated syndrome. (1)

A

Proofreading / Mismatch repair. Proofreading, which corrects errors during DNA replication. Mismatch repair, which fixes mispaired bases right after DNA replication

Lynch syndrome (HNPCC) caused by mutation in the MLH1 gene

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18
Q

Define the term “doubling dose”.

A

Doubling dose is the dose required to double the incidence of spontaneous anomalies within a population.
Esitmated doubling dose fir humans is about 2 Sv from atomic bomb data.

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19
Q

Discuss the potential effects of significant ionising radiation exposure on the human embryo and foetus.

A

Preimplantation
0-9 days (1st week)
* All or nothing response, either survives or dosesn’t after IR exposure
* Radiation causes embryonic death above 0.1Gy

Embryonic stage / organogenesis
10days – 6 weeks
* Theshold dose of 0.1Gy
* Radiation causes major structural abnormalities at this stage as it is organoogenesis
* Can also cause Severe growth restriction from cellular depletion - temporary
* Microcephaly
*
Early Foetal stage
6 weeks – 15 weeks
* Mental retardation, most severe if exposed between 8-15 weeks.
(40% per Sv), Result from failure of cells to migrate from proliferative zones. Threshold dose 0.3Gy for severe menatl retardation
* IQ decrease of 25 points per Gy
* Microcephaly
* Growth retardation - permanent
* Eye, skeletal and genital abnormalities

Late foetal stage ?third trimester
15 weeks – 40 weeks
- 15-25 weeks, Mental retardation Lower risk 10% per Sv
* Cancer following irradiation in utero
* Increases risk of childhood cancer by 40% above spontaneous level
* Excess absolute risk is 6% per Gy
* Growth retardation permanent

● Cancer following irradiation in utero
○ Data from obstetric x-rays, mostly in the third trimester
○ Increases the risk of childhood cancer by 40% above spontaneous level
○ Increases around 0.01 Gy
Excess absolute increase is 6% per Gy

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20
Q

Define the term “doubling dose”.
What is the estimated doubling dose for humans according to The United Nations Scientific Committee on the Effects of Atomic Radiation (UNSCEAR)?

A

Doubling dose is the dose required to double the incidence of spontaneous anomalies within a population.
Esitmated doubling dose for humans is about 2 Sv of absorbed radiation dose from atomic bomb data.

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21
Q

Regarding combinations of radiation therapy and systemic therapies:
- Define spatial co-operation and give one clinical example

A

Define spatial co-operation. (1)
i. Spatial co-operation refers to when the targets of radiation and chemotherapy are located at different anatomical sites, each modality acts independently of the other.

ii. Give one clinical example. (1)
i. Radiation used to treat primary site, and chemotherapy used for micrometastatic disease. Eg. Breast cancer
ii. Seclusion sites: brain, testes – where chemotherapy is the primary modality, and radiation is used for those sanctuary sites with inadequate chemotherapy penetration, such as PCI for small cell lung cancer. Or RT to testes and chemo for systemic treatment in testicular treatment

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22
Q

List classes of immunotherapy agents and give an example of an individual target in each

A
  1. Checkpoint Inhibitors:
    * Example: Pembrolizumab (Keytruda)
    * Target: Programmed Death 1 (PD-1) receptor
  2. Checkpoint Ligands:
    * Example: Atezolizumab (Tecentriq)
    * Target: Programmed Death Ligand 1 (PD-L1)
  3. CAR-T Cell Therapy:
    * Example: Kymriah (tisagenlecleucel)
    * Target: CD19 (Chimeric Antigen Receptor-T Cell Therapy targeting CD19)
  4. Cytokines:
    * Example: Interferon-alpha (various brand names)
    * Target: Various immune cells, including interferon receptors
  5. Oncolytic Viruses:
    * Example: Talimogene laherparepvec (T-VEC or Imlygic)
    * Target: Replication in cancer cells, leading to cell lysis and immune activation
  6. Monoclonal Antibodies:
    * Example: Rituximab (Rituxan)
    * Target: CD20 antigen on B cells in certain types of lymphoma and leukemia
    * Example: Trastuzumab (Herceptin)
    * Target: Human Epidermal Growth Factor Receptor 2 (HER2) in HER2-positive breast cancer
    * Example: Bevacizumab (Avastin)
    * Target: Vascular Endothelial Growth Factor (VEGF) to inhibit angiogenesis in various cancers
  7. Immune Checkpoint Ligand Blockers:
    * Example: Nivolumab (Opdivo)
    * Target: Programmed Death 1 (PD-1) receptor
    * Example: Ipilimumab (Yervoy)
    * Target: Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4)
  8. Vaccines:
    * Example: Sipuleucel-T (Provenge)
    * Target: Prostatic Acid Phosphatase (PAP) in metastatic prostate cancer
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23
Q

Describe the four main steps in the ‘abscopal’ effect. For each step, outline a key activity that takes place.

A

The ‘abscopal effect’ is a phenomenon in cancer treatment where localized radiation therapy not only affects the targeted tumor but also triggers an immune response that leads to the regression of non-irradiated tumors in distant parts of the body. This effect occurs due to the release of tumor-specific antigens and damage-associated molecular patterns (DAMPs) from the irradiated tumor, which activate the immune system to attack tumors outside the radiation field. The four main steps in the abscopal effect are as follows:

  1. Local Tumor Irradiation:
    Key Activity: The primary step involves delivering radiation therapy to the localized tumor. The radiation causes DNA damage and cell death in the targeted tumor cells.

2.Release of Tumor Antigens and DAMPs:
Key Activity: As a result of radiation-induced cell death, the irradiated tumor releases tumor-specific antigens and damage-associated molecular patterns (DAMPs). DAMPs are endogenous molecules released from dying cells that signal tissue damage to the immune system.

  1. Antigen Presentation and Immune Activation:
    Key Activity: Dendritic cells, which are specialized antigen-presenting cells, take up the released tumor antigens and present them to T cells. This process activates tumor-specific cytotoxic T cells, a type of immune cell capable of recognizing and attacking cancer cells.
  2. Systemic Immune Response:
    Key Activity: The activated cytotoxic T cells, along with other immune cells, enter the bloodstream and travel to distant sites in the body. These T cells recognize and attack cancer cells in non-irradiated tumors located far away from the original irradiated tumor. This systemic immune response is responsible for the regression of the non-irradiated tumors, resulting in the abscopal effect.

The abscopal effect is an essential mechanism in immunogenic cell death induced by radiation therapy. It highlights the potential of radiation therapy to not only directly kill tumor cells but also engage the immune system to target cancer cells throughout the body. While the abscopal effect is an exciting concept, it may not occur in all cases, and ongoing research is focused on understanding the factors that influence its occurrence and ways to enhance its effectiveness in cancer treatment.

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24
Q

What is the rationale for combining radiation therapy and immune system blockade? What are two mechanisms by which radiation can cause immune modulation?

A
  • The rationale for combining radiation therapy and immune system blockade lies in the potential synergistic effects of these treatments. Both radiation therapy and immune system blockade (e.g., checkpoint inhibitors) have shown promise in cancer treatment, and combining them can enhance their effectiveness in several ways:
  1. Abscopal Effect: Radiation therapy can cause localized tumor cell death within the treatment field. However, it can also stimulate the release of tumor-specific antigens and damage-associated molecular patterns (DAMPs) from dying cancer cells. This process, known as the abscopal effect, can trigger an immune response outside the irradiated area, leading to the recognition and destruction of distant, non-irradiated tumor cells by the immune system. Immune system blockade can further boost this systemic immune response, enhancing the abscopal effect and potentially improving overall tumor control.
  2. Immunogenic Cell Death: Radiation-induced cell death can be immunogenic, meaning it releases tumor antigens and inflammatory signals that activate the immune system. This can lead to the recruitment of immune cells, such as dendritic cells and cytotoxic T cells, to the tumor site, promoting a more robust anti-tumor immune response. Immune system blockade can prevent immune checkpoints from inhibiting these activated immune cells, allowing them to better recognize and attack cancer cells.
  • Two mechanisms by which radiation can cause immune modulation are:
    1. Inflammatory Cytokine Release: Radiation can induce the release of various inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), interleukin-1 (IL-1), and interleukin-6 (IL-6). These cytokines can recruit immune cells, promote antigen presentation, and enhance the immune response against cancer cells.
    2. Dendritic Cell Activation: Dendritic cells play a critical role in presenting tumor antigens to T cells, initiating an adaptive immune response. Radiation can cause the release of tumor antigens, which are then taken up by dendritic cells and presented to T cells, leading to the activation of tumor-specific cytotoxic T cells that can target cancer cells.
  • By combining radiation therapy and immune system blockade, clinicians aim to achieve better tumor control, improved response rates, and potential long-term benefits for patients.
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25
Q

An irradiated tumour has been described as an ‘immunogenic hub’, leading to local antigen formation as well as distant ‘abscopal’ effects.
Briefly describe the four main steps in the ‘abscopal’ effect. For each step, outline a key activity that take place. (4)

A

The ‘abscopal effect’ is a phenomenon in cancer treatment where localized radiation therapy not only affects the targeted tumor but also triggers an immune response that leads to the regression of non-irradiated tumors in distant parts of the body. This effect occurs due to the release of tumor-specific antigens and damage-associated molecular patterns (DAMPs) from the irradiated tumor, which activate the immune system to attack tumors outside the radiation field. The four main steps in the abscopal effect are as follows:
1. Local Tumor Irradiation:
* Key Activity: The primary step involves delivering radiation therapy to the localized tumor. The radiation causes DNA damage and cell death in the targeted tumor cells.
2. Release of Tumor Antigens and DAMPs:
* Key Activity: As a result of radiation-induced cell death, the irradiated tumor releases tumor-specific antigens and damage-associated molecular patterns (DAMPs). DAMPs are endogenous molecules released from dying cells that signal tissue damage to the immune system.
3. Antigen Presentation and Immune Activation:
* Key Activity: Dendritic cells, which are specialized antigen-presenting cells, take up the released tumor antigens and present them to T cells. This process activates tumor-specific cytotoxic T cells, a type of immune cell capable of recognizing and attacking cancer cells.
4. Systemic Immune Response:
* Key Activity: The activated cytotoxic T cells, along with other immune cells, enter the bloodstream and travel to distant sites in the body. These T cells recognize and attack cancer cells in non-irradiated tumors located far away from the original irradiated tumor. This systemic immune response is responsible for the regression of the non-irradiated tumors, resulting in the abscopal effect.
* The abscopal effect is an essential mechanism in immunogenic cell death induced by radiation therapy. It highlights the potential of radiation therapy to not only directly kill tumor cells but also engage the immune system to target cancer cells throughout the body. While the abscopal effect is an exciting concept, it may not occur in all cases, and ongoing research is focused on understanding the factors that influence its occurrence and ways to enhance its effectiveness in cancer treatment.

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26
Q
  1. Describe the steps involved in the activation of a “signal transduction pathway” and provide the name of a recognised pathway as an example. (2 marks)
A
  1. Receptor Activation
  2. Signal Amplification
  3. Intracellular Signal Transduction
  4. Activation of Effector Proteins
  5. Cellular Response

The Ras-MAPK Pathway

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27
Q
  1. Briefly describe the structure of the epidermal growth factor receptor (EGFR), how it is activated and its normal function.
A

Structure:
* EGFR is a single-chain glycoprotein that spans the cell membrane.
* It consists of an extracellular ligand-binding domain, a single hydrophobic transmembrane domain, and an intracellular domain with tyrosine kinase activity.

Activation:
* EGFR activation typically begins when a specific ligand, such as epidermal growth factor (EGF), binds to the extracellular domain of the receptor.
* Ligand binding induces a conformational change in EGFR, leading to the formation of receptor dimers (pairs of EGFR molecules).
* These dimers allow the intracellular tyrosine kinase domains to phosphorylate each other on specific tyrosine residues (autophosphorylation).
* Autophosphorylation activates the tyrosine kinase activity of EGFR, creating docking sites for downstream signaling molecules.

Normal Function:
* EGFR activation triggers several downstream signaling pathways, including the Ras-MAPK pathway and the PI3K-AKT -mTOR pathway
* The Ras-MAPK pathway regulates gene expression, leading to cell proliferation and differentiation.

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28
Q
  1. Describe three differences between a somatic and germline mutation. Is EGFR mutation somatic or germline? (2 marks)
A
  1. Timing of Mutation Occurrence:
    * Somatic Mutation: Somatic mutations occur in the non-reproductive (somatic) cells of an organism after conception.
    * Germline Mutation: Germline mutations occur in the reproductive (germline) cells, specifically in the sperm or egg cells, or in the early stages of embryonic development.
  2. Inheritance:
    * Somatic Mutation: Somatic mutations are not inherited.
    * Germline Mutation: Germline mutations are inherited.
  3. Role in Disease:
    * Somatic Mutation: Somatic mutations are often associated with the development of diseases like cancer. These mutations can lead to uncontrolled cell growth and the formation of tumors in specific tissues or organs.ie they will usually develop later in life
    * Germline Mutation: Germline mutations, when inherited, can lead to genetic disorders or predispositions to certain conditions that affect an individual throughout their life. And can result in conditions being present at birth or very early in their life These mutations are present in every cell of the individual’s body.

Be aware that germline mutations can also lead to cancer – Knudson’s hypothesis – and if there is a germline mutation predisposing to a certain cancer then it will occur earlier in life than would be expected for that cancer and can lead to cancer in all organs associated with that cancer risk e.g both breasts and both ovaries if an inherited BRCA mutation

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29
Q
  1. The DNA sequence of a gene can be altered in a number of ways. Describe five types of genetic mutations. (3 marks)
A
  1. Point Mutation: (sense and non-sense)
    * A point mutation involves the substitution of a single nucleotide (base) with another nucleotide in the DNA sequence.
  2. Insertion Mutation:
    * An insertion mutation involves the addition of one or more extra nucleotides into the DNA sequence. This can disrupt the reading frame, leading to significant changes in the protein’s amino acid sequence downstream of the insertion.
  3. Deletion Mutation:
    * Deletion mutations involve the removal of one or more nucleotides from the DNA sequence. Like insertions, deletions can shift the reading frame and result in a completely different amino acid sequence in the protein.
  4. Frameshift Mutation:
    * Frameshift mutations occur when the number of inserted or deleted nucleotides is not a multiple of three (the number of nucleotides in a codon). This leads to a shift in the reading frame of the gene, causing significant alterations in the amino acid sequence of the resulting protein.
  5. Duplication Mutation:
    * Duplication mutations involve the replication of a segment of DNA, leading to an extra copy (or copies) of that segment in the gene. This can result in the production of a longer-than-normal protein or changes in gene regulation.
  6. Inversion mutations:
    * Where a segment of DNA is flipped in orientation
  7. Translocation mutations:
    * Where segments of DNA from one chromosome move to another
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29
Q
  1. Name six hallmarks of cancer. Which of these concepts apply to the role of EGFR mutation in the development of human cancer? (5 marks)
A
  1. Sustaining Proliferative Signaling
  2. Evading Growth Suppressors
  3. Resisting Cell Death (Apoptosis
  4. Enabling Replicative Immortality
  5. Inducing Angiogenesis
  6. Activating Invasion and Metastasis
  7. Avoiding Immune Destruction
  8. Tumor-Promoting Inflammation
  9. Deregulating Cellular Energetics.
  10. Genome Instability and Mutation

EGFR mutations are primarily associated with the first hallmark, Sustaining Proliferative Signaling. When EGFR is mutated it can cause continuous signaling for cell growth and division. This uncontrolled signaling can lead to the development and progression of various types of cancer, particularly non-small cell lung cancer (NSCLC).
While EGFR mutations primarily relate to the first hallmark, EGFR mutations can indirectly lead to resistance to cell death, and genomic instability, by promoting cell survival through sustained growth signaling.

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30
Q
  1. Define the term “tolerance dose”. (1 mark)
A

The maximum radiation dose that is associated with an acceptable probability of developing clinically relevant signs and symptoms of late normal tissue damage.(or at a specific time interval in the future)

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31
Q

List six factors that may not be accounted for when determining the tolerance dose of an organ according to the QUANTEC papers. (2 marks)

A
  • Fractionation (and time treatment delivered in)
  • Total irradiated volume
  • Age of patient
  • Concomitant therapies
  • Health status of organ/patient
  • Previous radiation treatment to that area
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32
Q

c. Explain the concept of tissue architecture as described by Withers and how it is structured in a serial and parallel organ.
When considering the maximum tolerance dose of an organ at risk, how would this differ if you were irradiating a parallel organ as compared to a serial organ? ( 2 marks)

A

Serial Organs:
- In a serial organ, the functional subunits are arranged in a series or sequential fashion, meaning they are connected in a way that if one subunit is damaged or irradiated, the entire organ’s function may be compromised.
- Examples of serial organs include the spinal cord and small bowel

Parallel Organs:
- In a parallel organ, the functional subunits are organized in parallel, meaning they function independently of each other. Damage to one subunit does not necessarily affect the function of the remaining subunits.
- Damage to a portion of a parallel organ may lead to a reduction in overall organ function, but it doesn’t result in complete loss of function. Examples of parallel organs include the lungs and kidneys.

Differences in Irradiating Parallel and Serial Organs:
1. Parallel Organ:
* More susceptible to volume effect rather than a threshold dose. With increasing volume of FSUs irradiated in the organ, there is an increase of organ dysfunction.
* Threshold volume of organ irradiation before dysfunction is seen.

  1. Serial Organ:
    * In contrast, for a serial organ, the tolerance dose is relatively low because damage to any part of the organ can lead to complete loss of function, and this is considered unacceptable in clinical practice. Therefore there is a threshold dose.
    * Not significantly affected by volume of organ irradiated as long as under threshold dose.
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33
Q

d. How does the “volume effect” impact on clinical tolerance when irradiating a skin cancer and why does this happen? (2 marks)

A

Skin is mostly a “parallel FSU”. Therefore it’s clinical tolerance is more dependent on its total volume irradiated rather than total dose.
The smaller the volume of skin irradiated the more potential for un-irradiated clonogenic/ stem cells to migrate into the irradiated volume to help heal the irradiated volume. The larger the volume the harder it is for migration of outer stem cells.- this results in slow healing of the irradiated area which can lead to severe pain and infection and would be poorly tolerated by the patient and may lead to consequential late effects

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34
Q

What is the equation for EQD2

A

EQD2 = D x (a/b +d)/(a/b+2)

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35
Q
  1. Briefly discuss at least six factors that need to be considered when considering retreatment to an organ. (3 marks)
A
  1. Patient factors
  2. Expected life span of function of tissue/organ being retreated, i.e. is the underlying organ function good or does the patient have underlying medical conditions such as emphysema and needing to retreat lung
  3. patient or QOL if left untreated: are they at risk of developing late toxicities, or do the benefits of retreating outweigh late toxicity risks
  4. Patient age and ability to tolerate RTx – and what does the patient want!
  5. Tumour factors
  6. Alpha/beta ratio of tumour – might also consider tumour’s radiosensitivity
  7. Alternative treatment options – consider if surgery or supportive care would be better options
  8. Are the organs in the area being treated serial or parallel organs
  9. Treatment factors
  10. Proposed treatment dose and fractionation
  11. Time interval between the initial treatment and the planned retreatment, is there a potential for recovery of normal tissue tolerance
  12. Concurrent treatments (chemotherapy or immunotherapy) delivered at the time of initial RT which could affect tolerance of OARs
  13. Time interval between the initial treatment and the planned retreatment, is there a potential for recovery of normal tissue tolerance – this is a repeat of number 2, but is very important!
  14. Previous treatment dose, fractionation and type
  15. Volume of spinal cord previously treated – the question was generic rather than about the spinal cord specifically but what you are stating is correct and relates to the additional point I put in tumour factors
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36
Q
  1. Outline the concept of “forgotten dose” in relation to the tolerance dose of organs at risk. (1 mark)
A

Forgotten dose is the concept that organs at risk may have some recovery of their tolerance dose following radiation exposure, such that a proportion of the radiation dose that had been previously given is “forgotten”

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37
Q

What is the tolerance dose for spinal cord (QUANTEC)

A

tolerance dose for spinal cord is 50Gy (0.2% risk of myelopathy)
at 60Gy, 6% risk of myelopathy
at 69Gy, 50% risk of myelopathy

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38
Q

i. List at least 4 types of DNA damage that can be induced by ionising radiation. (1 mark)

A
  • Double-strand breaks
  • Single strand breaks
  • Base damage
  • Cross-linking
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39
Q

Use a table to compare and contrast the two double-strand break DNA repair pathways in relation to: (8marks)
- repair mechanism
need for a template
repair accuracy
time required for repair
predominance in phases of a cell cycle tissue type predominance
- used proteins / protein complexes
- associated genetic conditions

A
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39
Q

i. Define the terms sublethal damage and potentially lethal damage. (1 mark)

A

Sublethal damage = DNA damage which is insufficient to cause cell death, assuming intact DNA repair pathways.

Potentially lethal damage = DNA damage that can cause cell death unless favourable conditions are in place to allow repair such as cell environment or cell cycle position

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40
Q

Outline the purpose of pulsed-field gel electrophoresis (PFGE) in regards to DNA damage and the key steps in this assay.

A

PFGE is useful for studying DNA damage as it allows for separation and visualization of large DNA fragments.

Key Steps in PFGE:
1. DNA Sample Preparation:
* Isolate the DNA of interest, which may be from cells, tissues, or other sources.
* Treat the DNA, if necessary, to induce damage or generate specific fragments. This treatment might involve restriction enzymes to create specific cleavage points or other methods to induce DSBs.

  1. Embedding in Agarose Gel:
    * Mix the DNA sample with a molten agarose gel, which will immobilize the DNA within the gel matrix.
    * Pour the mixture into a mold to create a gel block containing the DNA.
  2. Electrophoresis Setup:
    * Place the agarose gel block in a specialized PFGE chamber that can generate a pulsed electric field.
    * Submerge the gel in an electrolyte buffer that facilitates DNA movement during electrophoresis.
  3. Electrophoresis with Pulsed Electric Field:
    * Apply an electric field across the gel, but unlike traditional gel electrophoresis, PFGE alternates the direction of the field in a pulsing manner.
    * The pulsing of the electric field changes the direction of DNA migration within the gel, allowing for the separation of large DNA fragments based on size.
  4. Visualization:
    * After electrophoresis, the gel is stained with a DNA-binding dye, typically ethidium bromide.
    * The DNA fragments are visualized under UV light, revealing a distinctive banding pattern of separated fragments.
  5. Analysis:
    * Analyze the PFGE gel to determine the size and distribution of DNA fragments.
    * The resulting pattern can provide information about the extent and nature of DNA damage.
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41
Q

Identify the main advantage of comet assay versus PFGE? (1 mark)

A

Higher sensitivity to subtle DNA damage and relatively simple and cost effective

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42
Q

List at least two lethal and two non-lethal chromosome aberrations

A

Lethal:
Ring
Acentric
Dicentric

Non-lethal:
Symmetric translocation
Small deletions
inversion

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43
Q

Which chromosome aberration assay is used to assess total body irradiation dose, and what is the lowest dose it can detect? (1 mark)

A

The dicentric chromosome assay (DCA). It is a biodosimetry technique that quantifies the number of dicentric chromosomes in peripheral blood lymphocytes to estimate the radiation dose received by an individual. Dicentric chromosomes are abnormal structures formed when two separate chromosomes become fused due to radiation-induced DNA damage resulting in two centromeres.

Can detect a recent total body exposure of as low as
0.25Gy

(Hall pg 32)

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44
Q
  1. State the formula for the linear quadratic model of cell kill and individually define each of the terms of the equation. (3 marks)
A

S= e^(-aD-BD^2)
S= surviving fraction
A and B are the co-efficiants of radiation sensitivity
D = dose delivered as single fraction

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45
Q
  1. Below are cell survival curves for low and high α/β ratio cell lines. (1 mark)
A
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46
Q

ii. Define the term α/β value and describe how it can be found from a cell survival curve. (1 mark)

A

a/B describes the bendiness of the curve and assumes 2 components of cell killing, one proportional to dose and one proportional to square of dose. Initial slope determined by a, B component causes the curve to bend at higher doses
a/B is found at the point of the curve when the a component = B component

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47
Q
  1. State two limitations of the linear quadratic model.
A
  • Treatment time not taken into account
  • Less reliable at extremes of dose
  • A/B are an estimation
  • May be heterogeneity of a/b in tumour and normal tissues
  • Does not account for other factors: concurrent chemo, radiosensitisers, oxygen etc
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48
Q

For both acute and late responding normal tissues, provide α/β value ranges. (1 mark)

A

High 8-10Gy
Low 2-3Gy

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49
Q

For both acute and late responding normal tissues, state the relative effects on normal tissue complication probability for the following four scenarios: (4 marks)
* increasing total dose
* hyperfractionation
* hypofractionation
* reducing overall treatment time (keeping total dose constant)

A

increasing total dose
- Acute – increased effect correct
- Late – increased effect not always as dose per fraction rather than total dose is important but there is a still a tolerance dose which might be exceeded if total dose increased

Hyperfractionation
- Acute – no change – if pure hyperfractionation then no change or may be slightly reduced as less dose delivered in a time period as less than 1.8 Gy per per day ie the time is not shortened – although the total dose is potentially still the same so ultimately it may be no change.

  • Late – less late effects for normal tissues

Hypofractionation
- Acute – if more total dose given in a shorter period which is the usual effect of hypofractionation then acute effects could increase
- Late – More late effects yes as sensitive to dose per fraction

reducing overall treatment time (keeping total dose constant)
- Acute likely to increase as delivering a higher total dose in a time period compared to standard fractionation – so acute effects will develop earlier and likely to be more intense
- Late – no change as long as time between fractions ok, late effects impacted by dose per fraction not time

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50
Q

Define the term hyperfractionation. (0.5 marks)

A

hyperfractionation is the delivery of radiation at a dose of less than 1.8 – 2 Gray per fraction over a conventional overall treatment time, employing multiple fractions per day.

A therapeutic advantage is thought to derive from the more rapid increase in tolerance with decreasing dose per fraction for late responding (low a/B) normal tissues than for tumpurs

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51
Q

Discuss the impact of hyperfractionated treatment schedules on tumour control and normal tissue effects.

A

Hyperfractionation if delivered as a once daily fractionation will extend treatment time and therefore has the potential to enable accelerated repopulation in the tumour which can worsen tumour control. As such hyperfractionation is usually delivered in combination with acceleration so that more than one fraction is delivered per day. This prevents an extended treatment course and may even result in a shortened treatment course compared to standard fractionation – this reduces the risk of repopulation and can improve tumour control.

Hyperfractionation on normal tissues has the potential to reduce effects in late responding tissues as the tissues are sensitive to dose per fraction. Acute responding tissues may have no change in effects if total dose the same and treatment deilivered as a once per day fractionation. If hyperfractionation is combined with acceleration (ie more than one fraction per day) then there should be no increase in the effects on late responding tissues as long as there is adequate time between fractions for repair. This may make it possible to give a higher total dose which would not change the effects on late responding tissues but would improve tumour control. If hyperfractionation is combined with acceleration this could increase the effects on early responding tissues as there will be more dose delivered in a shorter time period and will also increase effects in early responding tissue if the total dose is increased.

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52
Q

What are the hallmarks of cancer

A
  1. Sustained proliferative signalling
  2. Evading Growth Suppressors
  3. Avoiding immune destruction
  4. Resisting cell death
  5. Tumour-promoting inflammation
  6. Inducing angiogenesis
  7. Activating invasion and metastasis
  8. Genomic instability and mutation
  9. Deregulating cellular energetics
  10. Replicative immortality
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53
Q

List five types of cell death following ionizing radiation.

A
  1. Mitotic catastrophe
  2. Apoptosis (accept programmed cell death or interphase death)
  3. Radiation induced senescence
  4. Necrotic cell death
  5. Autophagy
54
Q

Describe the mechanism of cell death for mitotic catastrophe and apoptosis, following ionizing radiation.

A

Mitotic catastrophe
* Occurs during or shortly after a failed mitosis.
* Cell attempts to divide without proper repair of DNA damage, there are stops in
metaphase with aberrant mitosis and multinucleated giant cells.
* Mitotic catastrophe can also serve as a trigger for other cell death pathways.

Apoptosis (accept programmed cell death or interphase death)
* Occurs via intrinsic and extrinsic pathways. Radiation activates the intrinsic pathway and relies on p53.
* Apoptosis can be a result of both early or late cell death. Late apoptosis may be activated by mitotic catastrophe.
* DNA damage elicits downstream signaling to either block cell cycle progression to allow DNA repair, or progression to cell death when DNA damage overwhelming.
* Defined by morphological criteria (rounding up of the cells, nuclear pyknosis, karyohexis and phagocytosis of the apoptotic body by adjacent cells), the requirement for active participation of the dying cell and DNA laddering on gel electrophoresis.

55
Q

Recommended Dose Limits (ICRP)
- Occupational worker
- Public

A

Occupational worker: 20mSV/y averaged over 5y, max 50mSV/y

Public exposure (annual) 1mSV/y

56
Q

Recommended Dose Limits (ICRP)
- Occupational worker
- Public

A

Occupational worker: 20mSV/y averaged over 5y, max 50mSV/y

Public exposure (annual) 1mSV/y

57
Q

Medical diagnostics estimated doses
- CXR
- CT chest
- CT abdominal
- Full body CT

A

CXR 0.14mGy
CT chest 20-30mGy
CT abdo 22-60mGy
Full body CT 50-100mGy

58
Q

Properties of protooncogenes and Tumour suppressor genes

A

Protooncogenes:
- One mutational event required to contribute to the cancer
- Gain of function mutant allele, acts in a dominant fashion

Tumour Suppressor Gene:
- Two mutational events required to contribute to the cancer
- Funtion of the mutant allele: Loss of function, acts in a recessive fashion

59
Q

Ways protooncogene can become activated

A
  • Retroviral integration
  • Deletion / point mutation
  • Gene amplification
  • Chromosome rearrangement
60
Q

Facts about p53

A
  • p53 is a tumour suppressor gene that is lost or inactivated in over half of human cancers
  • Wtp53 is activated by damage to DNA
  • Damage to DNA during G1 can lead to blocking cell cycle progression (G1 arrest). Damage to DNA during G2 can lead to apoptosis
  • p53 binds to promoters p21, mdm2, gadd45, PCNA, BAX, NOXA, PUMA
  • Most human cancer cells either have mutations that inactivate p53 directly or have mutations in other loci that lead to loss of p53
  • Some viruses lead to p53 inactivation by binding to p53. These include E6 of HPV, Adenovirus E1B
  • p53 is destabilized by mdm2, which targets it for degradation and also directly inhibits its transactivation activity.
61
Q

Roles of specific cytokines:
- PDGF
- TGF-beta
-TNF
-IL1
- Interferon
- IL6

A
  • PDGF - increases damage to vasculature
  • TGF-beta - increases inflammation (pneumonitis), decreases growth of connective tissue and epithelial cells; causes fibrosis and vascular changes, requires two different types of receptors for signal transduction
  • TNF - inflammatory response, cytotoxic for tumour cells, regulated by PKC pathway, associated with radiation-induced complications, induces apoptosis, systemic administration can cause septic-shock like symptoms
  • IL-1 - fever, inflammatory responses, produced by fibroblasts and inflammatory cells
  • Interferon - inhibits cell proliferation in general, but can cause immune cell activation
  • IL-6 potent pro-inflammatory cytokines
62
Q

Tumour Cure Probability

A
  • Based on Poisson statistics
  • P= e^-n
  • P is probability of cure, n is average number of surviving clonogenic tumour cells
  • TCD37 would leave about 1 cell/tumour
  • TCD90 would leave about 10^-1 cell/tumour
  • TCD99 would leave about 10^-2 cells/tumour
63
Q

Chemotherapy
- Alkylating agents
- Natural products (antibiotics, plant alkaloids)
- Antimetabolites

A
  • Alkylating agents: inhibit DNA/RNA synthesis; cell cycle non-specific; cisPT, BCNU, procarbazine
  • Natural products (antibiotics, plant alkaloids): interfere with DNA, inhibit Topo enzymes, mitosis inhibitors, inhibit protein synthesis; cell cycle non-specific; bleomycin, Taxol, Doxorubicin, vincrisitine
  • Antimetabolites: interfere with DNA/RNA, inhibit enzymes for synthesis, cell cycle dependent; 5FU, HU, MTX
64
Q

DNA damage assays

A
  • Comet assay: sensitivity 5cGy for alkaline, method: cells lysed, nuclei electrophoresed. Fragmented DNA moves away from the nucleus to form the comet tail
  • Pulse-Field gel electrophoresis: DSB >5-10ZGy, DSB quantified by relative migration in the gel
  • Y-H2AX assay: Most sensitive assay, detects DNA repair, the gamma-H2AX assay provides a sensitive and rapid method for detecting early DNA damage.

Chromosome aberration assay
- the DCA (dicentric chromosome assay) is the gold standard for assessing acute phase radiation exposure, by scorring aberrations in lymphocytes from peripheral blood. May be used to estimate total body doses in humans accidentally irradiated. The lowest single dose that can be detected readily is 0.25Gy

  • Plasmid -based assays: ● Plasmids are supercoiled DNA
    ● SSB converts plasmids into open circle form whereas DSBs convert it into linear form
    These can then be differentiated using gel electrophoresis despite having the same base pairs
  • Micronucleus assays
  • ● Assay detects level of chromatin damage and development of micronuclei
    ● Irradiated cells dropped onto microscope slide and airdried
    ● Fixed with methanol and stained with 5% Giemsain buffered saline
    Depending on cell type, 500 to 3000 cells analysed to score frequency of micronuclei.
65
Q

DNA damage assays

A
  • Comet assay: sensitivity 5cGy for alkaline, method: cells lysed, nuclei electrophoresed. Fragmented DNA moves away from the nucleus to form the comet tail
  • Pulse-Field gel electrophoresis: DSB >5-10ZGy, DSB quantified by relative migration in the gel
  • Y-H2AX assay: Most sensitive assay, detects DNA repair,
  • Plasmid-based assay: DNA plasmids are circular areas of DNA Can contain dna repair proteins
    Stain for these proteins (fluoresence)
  • Micronucleus assay: Micronuclei occur when DNA fragments are not attached to mitotic spindle (dsDNA break)
  • Chromosomal aberration assays
66
Q

How to measure tumour response in Vivo?

A
  • Small intestine crypt stem cells: Mice given total-body radiation are sacrificed and the jejunum sectioned after 3 days. Number of regenerating crypts/circumference of the jejunum is scored. Crypt survival does not reflect cell survival unless dose sufficient to reduce the stem-cell survivors to <1/crypt.
67
Q

What are IN VIVO techniques to generate survival curves

A

N VIVO ASSAYS:

Spleen colony assay:
Donor gets various test dose radiation. Donor BM cells get intravenously injected to recipient that got
whole body radiation. Surviving colonies form in spleen which derived from the donor stem cells. The
surviving colony numbers are counted from recipients spleen.

Lung colony assay:
Similar to above. Tumour cells readily form colonies in the lungs after intravenous injection of single cell
suspension.

Limiting dilution assay:
Non-cloning assay. Suspension of tumour cells is made. Large number of subcut injections into animals
with a range of inoculum sizes. Take-rate is plotted against inoculum size. Experiment between control vs
radiated cells.

68
Q

What is PARP?

A

Poly adipose-Ribose Protein
Involved in SSB repair. PARP is needed for repair, and inhibitors of enzyme potentiate the lethal effects of toxic agents. The enzyme is induced by SSB.

116K protein that uses NAD as a substrate to catalyze the covalent transfer of ADP-ribose to a variety of nuclear protein acceptors and. Then transfers an additional 60-80 ADP-ribose units to the initial protein.

Post-translational modification of proteins.

Depletion of PARP increases the frequency recombination, gene amplification, sister chromatid exchanges, and micronuclei formation when cells are exposed to genotoxic stresses, suggesting a role for PARP in genomic stability

69
Q

What is gamma-H2Ax

A

H2Ax is a histone protein that becomes phosphorylation at the gamma position when cells have been exposed to DNA-damaging agents like ionizing radiation (gamma-H2Ax)
- Antibodies to y-H2Ax are used to detect the phosphorylation protein
- THe phosphorylation protein y-H2Ax localizes to sites of DNA damage.
- Some repair proteins that co-localizes with y-H2Ax include ATM, DNA-PKCs, p53, NBS1, Ku70/Ku80, MRE11 and others
- Presence of y-H2Ax has become an important tool in identifying foci of DNA repair in nuclei of irradiated cells.
- It is a sensitive assay for detecting exposure to ionizing radiation

70
Q

What are the assumptions of the Linear Quadratic Model?

A
  • DNA hits, events that damage DNA, are random with probability proportional to dose.
    Double strand breaks are required for cell sterilization (i.e. death or, equivalently, inability to reproduce)
  • There are two methods of producing a double strand break
  • One quanta of radiation damages both DNA strands (αD). This is referred to as a “double hit” because it damages two strands with one hit.
  • Two quanta of radiation each breaking a single strand, produce a double strand break (βD2). These are referred to as “single hits” because one strand is damaged with each hit.
71
Q

Common Fractionation Schemes

A

Standard Fractionation

1.8 - 2Gy per fraction
1 treatment per day, Monday - Friday
Assuming 109 tumor cells and an expected kill ratio of 50% per 2Gy fraction, 30 fractions is sufficient to reduce the number of expected surviving cells to less than 1.

BID (Hyperfractionation)
2 fractions delivered per day (separated by at least 6 hours)
1.2Gy per fraction
Reduced late effects such as those to the central nervous system
Early effects, such as those to the skin or GI tract, are unchanged

HypoFractionation
>2Gy per fraction
Up to 1 fraction per day
Increases late effected
Decreases early effects

Radiosurgery
1-5 fractions with doses ranging between 8 and 90 Gy per fraction
Such high dose fractions potentially invalidate the linear quadratic model and are currently not well understood.
Radiosurgery focuses on avoiding dose to normal tissue rather than on improving therapeutic ratio. As a result, it is commonly used only for small lesions and special cases such as trigeminal neuralgia.

72
Q

Factors in Fractionated Radiotherapy (The Five Rs)

A
  1. Repair

Sublethal damage is repaired in both tumors and healthy cells. Differences in repair rate may be exploited.

  1. Repopulation

Cell division and population growth occurs, albeit to an inhibited degree, between fractions.

  1. Reoxygenation

Tumors often have poor vasculature and, as a result, are anoxic. This lack of oxygen makes tumor cells more radioresistant. Fractionation allows time for some tumor cells to die which improves oxygenation of the remaining cells. This effect increases radiosensitivity during subsequent fractions.

  1. Redistributions

The distribution of cells in a given cell cycle stage changes with fractionation.

  1. Radiosensitivity

Cells differ in their intrinsic radiosensitivity. Radiosensitive cells include haematological cells and epithelial cells as well as haematological tumor cells. Radioresistant cells include neurons and myocytes as well as melanoma and sarcoma tumor cells.

73
Q

Describe BED and give formula

A

Biologically effective dose allows for simple assessment of the biological effect of a particular dose and fractionation scheme, given the alpha/beta ratio of the tissue in question. Two fractionation schemes are equally effective when their BED values are equal.

BED = nd x (1 + (d/alphabeta))

n is the number of fractions delivered
d is the dose per fraction
α/β is the alpha over beta value derived from the linear quadratic model

74
Q

Describe Equivalent Dose (EQD) and give formula

A

Equivalent dose, often notated EQDx where x is the reference dose per fraction, is used to find an equivalent fractionation scheme to a reference scheme. Because standard 2Gy per fraction is most common, EQD2 is most often used.

EQDx= nd x ( d+ aB / x + aB)

n is the number of fractions delivered
d is the dose per fraction
α/β is the alpha over beta value derived from the linear quadratic model

75
Q

Describe Relative Biological Effectiveness and give formula
and factors affecting RBE

A

Relative biological effectiveness (RBE) is the ratio of absorbed dose required to produce an effect under reference conditions to the absorbed dose required to produce the same effect under another set of conditions. RBE is a useful concept because the effect of a given dose is determined not just by the absorbed dose but also by the type of radiation and the circumstances under which the radiation is delivered.

RBE = Dref/Deval

Deval is the dose which is being evaluated
Dref is the reference dose
NOTE: Doses here are isoeffective dose

Key Point: A positive RBE indicates that the dose under investigation is more effective than the reference dose.
High LET particles, such as protons and alpha particles, produce more radiation damage per unit path length. This increases the probability of double strand breaks for a given absorbed dose in increases RBE.

Factors influencing RBE
Linear Energy Transfer (LET)
Radiation quality
Fractionation
Dose rate
Biological system in question
Biological conditions

76
Q

What is oxygen enhancement ratio and what is the formula.

A

Oxygen is a radiosensitizer which improves the RBE compared to anoxic conditions. Oxygen enhancement ratio is a special care of BRE measuring the impact of oxygenation on radiosensitivity.

OER = Danoxic /Doxic

OER for photons, X-rays and gamma rays, is typically between 2.5 and 3
Neutrons have a low OER, typically around 1.5

77
Q

How does radiation damage cells?

A

Radiation damages cells by causing breaks within the structure of DNA. There are two primary mechanisms by which this damage occurs.

Direct Radiation Damage (10%)
- Radiation impacts sufficient energy to DNA bonds to directly cause breaks.

Indirect Radiation Damage (90%
- Indirect damage occurs when radiation interactions outside of the DNA to produce free radicals which in turn damage DNA.

A free radical is an atom or molecule carrying an unpaired electron in an outer shell.
Hydroxide (OH-) is the most common free radical produced by radiation within the body. Hydroxide is produced in the body through interaction of water (H2O).

H2O + gamma = H+ + OH-

Ionising radiation ionises water molecules (H20 → O· + OH·)

Highly reactive free radicals break chemical bonds

These react with DNA molecules and form further free radicals within the DNA structure (R·)

The outcome of this depends on oxygenation (in a process termed fixation)

If well oxygenated, R· + O2 → RO2· → ROOH

DNA repair pathways detect damage and invoke enzymatic processing

Fixed damage is irreversible

If hypoxic, R· reacts with H+ and is restored to original form

DNA damage is reversed

78
Q

What is the Law of Bergonie and Tribondeau?

A

The law of Bergonie and Tribondeau states: The radiosensitivity of a cell is directly proportional to reproductive rate and is inversely proportional to its degree of differentiation.

This means that radiosensitivity increases with:
Increased rate of cell division
Low degree of specialization (stem cells are very radiosensitive)
Higher metabolic rate
Increased oxygenation
Increased length of time they are actively proliferating

79
Q

List five types of cell death following ionizing radiation.

A
  1. Mitotic catastrophe
  2. Apoptosis (accept programmed cell death or interphase death)
  3. Radiation induced senescence
  4. Necrotic cell death
  5. Autophagy

2.5 marks = correctly identifying all 5 mechanisms
* 1.5 marks = correctly identifying 4 mechanisms
* 0.5 marks = correctly identifying 3 mechanisms
* 0 marks = 2 or less mechanisms

80
Q

Describe the mechanism of cell death for mitotic catastrophe and apoptosis,
following ionizing radiation.

A

Mitotic catastrophe
* Occurs during or shortly after a failed mitosis.
* Cell attempts to divide without proper repair of DNA damage, there are stops in
metaphase with aberrant mitosis and multinucleated giant cells.
* Mitotic catastrophe can also serve as a trigger for other cell death pathways.

Apoptosis (accept programmed cell death or interphase death)
* Occurs via intrinsic and extrinsic pathways. Radiation activates the intrinsic
pathway and relies on p53.
* Apoptosis can be a result of both early or late cell death. Late apoptosis may be
activated by mitotic catastrophe.
* DNA damage elicits downstream signaling to either block cell cycle progression
to allow DNA repair, or progression to cell death when DNA damage
overwhelming.
* Defined by morphological criteria (rounding up of the cells, nuclear pyknosis,
karyohexis and phagocytosis of the apoptotic body by adjacent cells), the
requirement for active participation of the dying cell and DNA laddering on gel
electrophoresis.

1.5 marks each max for Mitotic catastrophe and Apoptosis
* 0.5 marks = per correct point up to a maximum of 1.5 marks

81
Q

Name the two key pathways of DNA double stranded repair and list the key
steps and the key proteins involved in each pathway.

A

Non-homologous end joining (NHEJ acceptable):
modifies broken DNA ends and ligates them together without the need of a template and minimal regard for
homology.

Steps
i. Double stranded break recognition
ii. End binding and tethering with Ku 70/80 forming a complex at site of DNA damage.
iii. End processing by ARTEMIS -removal of damaged or mismatched nucleotides by nucleases and resynthesis by DNA polymerases.
iv. Strand invasion, DNA synthesis and resolution.
v. Ligation by DNA ligase IV and XRCC4

Key Proteins
* Ku 70, Ku 80, Artemis, DNA dependent protein kinase, DNA ligase IV and XRCC4

Homologous recombination – requires a homologous strand (typically sister
chromatid), used as a template for DNA repair.

○ Recognition of DNA double stranded break by ATM-MRN complex, and H2AX is phosphorylated. 
○ Single stranded breaks are made around the area of DSB (by Ctlp) 
○ The single-stranded filaments are then coated with replication protein A (RPA) which is displaced by the protein RAD51. This step is regulated by BRCA1/2. 
○ Strand exchange - The RAD51 coated DNA searches and invades sister DNA. Forms a DNA crossover or Holliday junction.
○ Helicases enlarge this crossover region and DNA synthesis is carried out by DNA polymerases. 
○ At the end, the crossover points are cut by resolvases and ligated probably with ligase 1. 
○ This process is error- free, and can take several hours to complete.  

Key Proteins
* RAD 51, CtIP, BRCA1, BRCA2, RAD52, RAD54

82
Q

G1/S checkpoint
(also known as the restriction point)

A

The transition from G1 to S phase is controlled by the activation of the E2F transcription factor.

E2F is kept inactive during G1 as bound to Rb protein
During the normal cell cycle, Rb becomes phosphorylated by cyclinD-CDK4,6 and cyclinE-CDK2.
Phosphorylation of Rb releases E2F.
E2F then acts as a transcription factor and allows entry to S phase.

If cells irradiated in G1 phase:
ATM is activated by DSBs and phosphorylates both mdm2 and p53 which then induces genes that can promote apoptosis (Bax, Puma) and induce cell-cycle checkpoints by upregulation of p21 (p21 is a CDK inhibitor)
p21 inhibits the G1 cyclin/CDK compexes, thereby preventing the phosphorylation of Rb and entry into the S phase.

Primary signalling proteins: ATM, p53, p21

83
Q

S phase checkpoint

A

S phase checkpoint slows progression and initiates repair.
CyclinA-CDK2 complex initiates DNA replication

If DSBs: ATM/ATR are activated by DSBs and phosphorylate the Chk1/2 kinases.
Chk1/2 kinases then phosphorylate and
inactivate CDC25A and CDC25C.

CDC25A/C are required for progression through S phase by activating cyclin/CDK complexes.

BRCA1 and BRCA 2 also involved at this checkpoint

Primary signalling proteins: ATM, Chk1/2, CDC25A/C, BRCA1,2

84
Q

‘Late’ G2 checkpoint

A

Late G2 phase checkpoint applies to cells irradiated in all phases of cell cycle.
- it applies to cells that may have already been previously irradiated and may have had delays in G1 or S checkpoints, but when they arrive in the G2 phase they experience a second delay prior to entry to mitosis.
Results in accumulation of cells in G2

Cyclin B-CDK1 complex

ATR dependent (no ATM)

ATR is activated which then phosphorylates Chk1 kinase, which then phosphorylates and inactivates CDC25A/C.

Primary signalling proteins: ATR, Chk1, CDC25A/C

85
Q

‘Early’ G2 checkpoint

A

Early G2 checkpoint applies to cells irradiated in G2, it is called early as it rapidly blocks their movement into mitosis.
This checkpoint is activated by relatively low doses of radiation (however <1gy could fail to activate checkpoint and lead to low-dose hyper -radiosensitivity)

Cyclin B-CDK1 complex

If DSBs: ATM is activated by DSBs and phosphorylate the Chk1/2 kinases.
Chk1/2 kinases then phosphorylate and
inactivate CDC25A and CDC25C.

CDC25A/C are required for progression through G2 to M phase by activating cyclin/CDK complexes.

BRCA1 and BRCA 2 also involved at this checkpoint

Primary signalling proteins: ATM, Chk1/2, CDC25A/C, BRCA1,2

86
Q

Define Tumour doubling time TD
and Potential doubling time (Tpot)

A

TD = Time required to double tumour size in vivo
- Accounts for cell loss, hypoxia, microenvironment factors etc
- Usually volume doubling time (VDT) due to ease of measurement. Other ways to measure i.e. biochemical measures, clonogenic doubling times.

Potential doubling time (Tpot)
- Tumour doubling time
- Does not take into account cell loss etc.

87
Q

What are the determinants of cell growth rate

A
  • Cell cycle time (Tc) - time required to complete cell cycle.
  • Growth fraction (GF) - proportion of cells that are actively dividing. Ki67 can be used
  • Cell loss factor (CLF) - proportion of cells lost from the GF - Ischaemia/necrosis
    CLF = 1 - (Tpot/VDT)
88
Q

What makes up the tumour microenvironment?

A
  • Vasculature (VEGF, HIF-1)
  • Fibroblasts (responsible for stromal inflammatory response and generated from malignant cells)
  • Immune cells
  • ECM
  • Hormones
89
Q

What is accelerated repopulation?

A

Net clonogenic doubling time during or shortly following irradiation which exceeds that observed prior to radiotherapy.

Time of onset termed “kick off”

90
Q

Mechanisms for accelerated repopulation

A
  1. Hypoxia
    - Radiation preferentially kills oxygenated cells (oxygen fixation hypothesis)
    - Previously ischaemic cells then have more access to nutrients and proliferate more rapidly.
    - Cell loss decreases accompanied by a propensity for repopulation.
  2. Asymmetry loss
    - Excess stem cell generation to counteract cell loss from radiation damage
  3. Acceleration of stem cell proliferation
    - Acceleration of Tc (cell cycle time)
91
Q

What are the 3 acute radiaiton syndromes and dose thresholds for each

A

Haematopoietic Syndrome
● >0.7 Gy
● Bone marrow failure.
● Latent phase 1-6 weeks
● Death in a few months from infection and haemorrhage.

Gastrointestinal Syndrome
● >10 Gy
● Depopulation of the gastrointestinal epithelium
● Latent stage of a few days
● Death within 2 weeks from infection, dehydration and electrolyte imbalance

Cerebrovascular Syndrome
● >50 Gy
● Onset within minutes
● Uncontrolled intracranial hypertension and circulatory collapse, likely due to damage of microvasculature
● death within 3 days

92
Q

What are the mechanisms of post radiation regeneration of normal tissues

A

Regeneration of early-responding normal tissues occurs via same mechanisms as tumour repopulation
1. Asymmetry loss
2. Accelerated cell cycling time
3. Abortive divisions

93
Q

What is the MOA of cisplatin? What are common side effects

A

Cisplatin is a platinum based chemotherapy. Cisplatin interferes with DNA replication, which kills the fastest proliferating cells.
Cisplatin crosslinks DNA in several different ways, interfering with cell division by mitosis. The damaged DNA elicits DNA repair mechanisms, which in turn activate apoptosis when repair proves impossible.

SE’s
- Nephrotoxicity
- Hearing loss
- Peripheral neuropathy
- N+V
- Electrolyte disturbance
- Haemolytic anaemia

94
Q

Define the term “tolerance dose”

A

The dose which achieves an acceptable probability of a defined side effect in an organ at a particular timeframe.

95
Q

Define the Oxygen Enhancement Ratio (OER)

A

Ratio of dose in hypoxic conditions to dose in oxic conditions to achieve the same biological effect.

Dose for hypoxic conditions/ dose for oxic conditions

OER is 2.5-3 for low LET radiation, close to 1 for high LET radiation

96
Q

The normoxic tumour receives a dose of 4Gy. On a labelled axis, draw a cell survival graph illustrating the difference between oxygenated cells and hypoxic cells with an OER of 3.

A

hypoxia line be 3x higher than normoxia line at 4Gy, eg. At 0.1 survival rate, normoxia dose would be at 4Gy, and hypoxia line would be at 12Gy for an OER of 3.
Example of a curve

97
Q

Explain how fractionated radiation therapy helps overcome tumour hypoxia

A

Hypoxia = chronic hypoxia secondary to tumour cells being outside of the diffusion limit of oxygen and acute hypoxia due to aberrant closing of blood vessels causing normally oxic tissues to be transiently hypoxic
Acute hpoxia = acute hypoxia is accounted for by the fractionated schedule due to variable blood flow in tumours and hopefully being able to capture the tumour in a more oxygenated state during treatment
Chronic hypoxia = outer cells close to the blood supply and therefore well oxygenated are killed off by radiation, the tumour size shrinks and the more central cells in the tumour aare now closer to the blood supply and start to be better oxygenated and therefore more sensitive to radiation

98
Q

Explain how the following drugs work to overcome hypoxic radioresistance:
* Tirapazamine
* Nimorazole
* Nicotinamide and Carbogen

A
  1. Tirapazamine:
    * Tirapazamine is a hypoxia-selective cytotoxin used as a radiosensitizer in radiation therapy.
    * It works by exploiting the differential sensitivity of oxygenated and hypoxic cells to radiation. Tirapazamine is activated in hypoxic (oxygen-deprived) regions of the tumor.
    * Once activated, tirapazamine generates toxic radicals that can cause DNA damage in hypoxic tumor cells, making them more susceptible to radiation-induced cell death.
  2. Nimorazole:
    * Nimorazole is a radiosensitizer that mimics oxygen. It is not metabolised by tumour cells and diffuses further than o2 and therefore can penetrate into hypoxic areas of tumours and perform the same role as oxygen. This results in sensitisation of hypoxic cells and increased cell kill.
  3. Nicotinamide and Carbogen:
    * Nicotinamide and carbogen are used in combination as radiosensitizers to overcome hypoxic radioresistance.
    * Nicotinamide is a vasodilator that helps increase blood flow to the tumor, potentially improving oxygen supply to previously hypoxic regions. Addresses acute hypoxia.
    * Carbogen is a mixture of carbon dioxide and oxygen that further enhances oxygenation of the tumor. Addresses chronic hypoxia.
    * The combination of nicotinamide and carbogen increases the oxygen availability within the tumor, making previously hypoxic areas more radiosensitive.
99
Q

methods attempted to overcome radioresistance due to hypoxia

A
  • Fractionation
  • Tirapazamine
  • Nimorazole
  • Nicotinamide and carbogen breathing
  • Hyperbaric oxygen chamber
  • Hyperthermia
  • High LET radiation
  • Blood transfusion / Correcting anaemia
100
Q

Explain the effects of HIF-1 on:
- tumour vasculature
- tumour metabolism
- tumour metastasis

A
  1. Tumor Vasculature:
    - HIF-1 promotes angiogenesis.
    - In response to hypoxic conditions within the tumour, HIF-1 activates the expression of various angiogenic factors, such as vascular endothelial growth factor (VEGF).
  2. Tumor Metabolism:
    - HIF-1 induces changes in tumour cell metabolism, shifting it towards a more glycolytic and less oxygen-dependent pathway. By promoting glycolysis through up regulation of GLUT1 and GLUT3, HIF-1 allows tumour cells to generate energy and essential metabolites under low-oxygen conditions. Warburg effect.

3 Tumor Metastasis:
- HIF-1 is involved in the regulation of genes associated with tumor metastasis.
- Upregulate MMP and suppresses E-cadherin to promote cell invasion and metastasis

101
Q

Define “relative biological effectiveness”

A

Dose of reference radiation / dose of test radiation for the same biological effect.

Reference radiation usually 250keV.

If the RBE is greater than 1, it indicates that the test radiation is more biologically effective at producing the specified effect than the reference radiation. Conversely, if the RBE is less than 1, it suggests that the test radiation is less effective.

102
Q

Describe the process of vascularisation of tumours and why their growth rate depends on this.

A

Tumours growth rate depends on vascularization as this helps in supplying essential nutrient and oxygen supply and waste removal to maintain optimal microenvironement.
These are delivered to cells through blood vessels before diffusing across a gradient to reach the cell. The diffusion distance is short so blood vessels are required to take nutrients/o2 the majority of the distance.
Otherwise tumour would become necrotic and die. Also helps with allowing tumour to metastasise.

Process of vascularisation of tumours:
1. Hypoxic environment / accumulation of HIF-1:
* Tumour angiogenesis is initiated when the growing tumor reaches a size where it becomes oxygen and nutrient deprived due to inadequate blood supply from existing vessels.
* In an hypoxic microenvironment there is an accumulation of HIF-1a in the cell.
2. Angiogenic Factors upregulated /angiogenic switch:
* HIF-1 is activated in a hypoxic environment and then upregulates proangiogenic factors including VEGF, PDGFs and FGFs, TNF-alpha
* Angiogenic switch is when there is an unbalance of pro and anti angiogenic factors.
3. Proteolytic degradation of basement membrane
* Pro-angiogenic factors also promote secretion of MMPs which is a proteolytic enzyme which degrades the basement membrane allowing endothelial cells to detach.
4. Endothelial Cell Migration and Proliferation:
* Endothelial cells respond to angiogenic factors by migrating toward the tumour and proliferating.
* These cells then organize into tube-like structures, forming new blood vessels that penetrate the tumour mass.
5. Establishment of Tumour Vasculature:
* These newly formed vessels connect to the existing vasculature, supplying the tumor with oxygen and nutrients, thus supporting its growth and progression
* Tumour vasularization does not have a resolution phase and is usually leaky and immature leading to variable blood flow..

103
Q

Several key proteins such as Rb protein, p53 and p21 prevent progression through the cell cycle at a checkpoint. Discuss in detail the mechanism by which these proteins act to regulate the cell cycle at this checkpoint.

A

Rb, p53, and p21 regulate the cell cycle at the G1/S checkpoint.

Rb inhibits the activation of genes required for DNA replication by binding to E2F. If conditions are suitable for entry into S phase Rb becomes phosphorylated by cyclinD-CDK4 and cyclinE-CDK2. Phosphorylation of Rb releases E2F which then acts as a a transcription factor and allows entry into S phase.

p53 monitors DNA integrity and activates p21 in response to stress or damage. In response to DNA damage ATM is activated by DSBs and phosphorylates both MDM2 and p53. This activates p53 which then can promote apoptosis (Bax,Puma) and increase p21 concentrations.

p21 inhibits CDK-cyclin complexes, this therefore inihibits cyclinD-CDK4 and cyclinE-CDK2 thereby preventing Rb phosphorylation therefore Rb remains bound to E2F and promoting cell cycle arrest as E2F unable to be active.

This coordinated regulation ensures that cells do not progress to the S phase unless the conditions are suitable for DNA replication.

104
Q

Describe the multistep carcinogenesis process

A

This multi-step process is modelled in the initiation-promotion model

  1. Initiation (initial mutation/DNA change) ->
  2. promotion (clonal expansion of mutated cells driven by chemical/epigenetic factors e.g. oestrogen) ->
  3. malignant conversion (further mutations results in neoplasia) ->
  4. progression (tumour grows/invades and can develop further malignant characteristics)
105
Q

What is the vogelstein model of carcinogenesis?

A

The Vogelstein model of molecular carcinogenesis

Model tumour is colorectal carcinoma

Key aspects

Incorporates the two hit model (each gene needs both alleles to lose function)

Requires multiple separate genes to be involved

Pathway

Normal epithelium
First hit of a tumour suppressor gene (APC)

Initiation (Mucosa at risk)
Second hit of TSG via methylation or mutation (APC)

Promotion (Hyperplasia/early adenoma)
Mutations of proto-oncogene (KRAS)

Progression (Late adenoma)
Homozygous loss of other tumour suppressor genes (E.g p53)

Invasion (Carcinoma)
Gross chromosomal abnormalities (E.g. Telomerase)

Carcinoma and unlimited proliferation

In the presence of wildtype p53, benign tumour enters senescence and does not become invasive

i.e. Adenoma/polyp

106
Q

a) Briefly outline the late effects that occur (at a morphological level) in normal blood vessels after radiation therapy

A
  • Endothelial damage leads to thromboses and capillary necrosis
  • Smooth muscle cells diminish over several years.
  • In arterioles replaced by collagen fibres, lose elasticity resulting in stenosis.
  • In larger capillaries and veins contribute to telangiectasia
  • Subintimal lipid deposition leads to atherosclerosis
  • Arterial damage > 50 Gy, capillary damage >40 Gy
  • Veins are less sensitive than arteries.
107
Q

Describe the four phases of acute radiation syndrome and the specific side effects that will result from the body being exposed to increasing levels of radiation dose.

A

Four phases of ARS are prodromal phase, latent phase, manifest illness, and recovery phase:
1. prodromal phase is the initial period of symptoms before organ dysfunction
a. neuromuscular (fatigue, headache) and gastrointestinal symptoms (anorexia, nausea, and vomiting)
b. at high doses, added symptoms of fever and diarrhoea, and earlier onset
2. latent phase is period when the patient feels well before manifest illness, during which tissue cells are dying
3. manifest illness (critical phase) is the phase of illness caused by organ failure after radiation
4. recovery phase when the body/organ is able to heal by repopulating damaged organ to preserve function, otherwise death can occur

  • Haematopoietic Syndrome
  • Threshold dose > 0.7Gy
  • prodromal phase onset within 2 days; nausea, vomiting, malaise; lasts minutes to days
  • latent phase lasting 1-6 weeks
  • manifest illness results from bone marrow failure. Symptoms of anorexia, fever, malaise,
    bleeding. Death within weeks to months
  • recovery phase when marrow is repopulated from stem cells, up to 2 years
  • Gastrointestinal Syndrome
  • threshold dose > 10Gy
  • prodromal phase onset within hours; nausea, vomiting, diarrhoea; lasts a few days
  • latent phase of less than a week as the GI tract epithelium dies
  • manifest illness of dehydration, malaise, anorexia, diarrhoea, electrolyte imbalances
  • Death within 2 weeks
  • Cerebrovascular Syndrome
  • threshold dose > 50Gy
  • onset of prodromal phase within minutes symptoms of confusion, nausea, vomiting,
    diarrhoea
  • latent phase often short, a few hours
  • manifest illness of convulsion and coma due to circulatory collapse, intracranial
    hypertension.
  • Death within 3 days
108
Q

What is the reirradiation tolerance for spinal cord?

A

Approximately 40-50% repair at >12 months

Generally safe if cumulative EQD2 <60Gy

Note that clinical considerations (life-expectancy, risk of myelopathy due to cord compression) may lead to accepting higher risks

Heavily studied in mice and primates

Definite evidence of repair

Mice
Juvenile mice immediately regained tolerance and peaked at 2 months (~120% of total tolerance for cumulative dose)
Adult mice began to regain tolerance at 2 months and peaked at 5 months (~140% of total tolerance for cumulative dose)

Primates (ED10 paralysis)
More gradual recovery of tolerance (150% of total tolerance for cumulative dose at 1 year, 156% at 2 years and 167% at 3 years)

Small human series have published data
No myelopathy seen for cumulative EQD2 125-172% with interval between 4 months and 13 years

109
Q

Define the term Relative Biological Effectiveness (RBE). Discuss how RBE changes as a function of LET

A

Relative biological effectiveness is the difference in biological effect of a test radiation compared to a reference radiation (typically 250KeV photons)

RBE = Dose of reference radiation / dose of test radiation to achieve the same biological effect

RBE increases as LET increases up until LET = 100KeV. The initial increase from LET 1 – LET 10 is slow but rapidly increases until LET reaches 100. From this point onwards LET begins to reduce at a similar rate to it’s rapid increase. The rationale for this is higher LET radiation is more effective at cell killing up to a point where the density of ionisations is equal to the width of the DNA helix (2nm). This corresponds with LET of 100 Kev. Past this point further increase in LET does not cause more cell kill but rather overkill and therefore the RBE reduces.

110
Q

Describe the activation pathways for apoptosis. Include named examples of specific factors or proteins involved in these pathways.

A

Intrinsic vs extrinsic

Intrinsic pathway (seen in response to radiation)
DNA damage -> sensing/activation of ATM-MRN complex -> activation of p53 -> increase in pro-apoptotic proteins (BAX, PUMA) -> activation of caspase 9 -> caspase 3 activity -> initiation of apoptosis

Extrinsic
Extracellular factors bind to cell membrane receptors – FAS L to FAS, TNF alpha etc. This causes activation of caspase 8 which then results in activation of caspase 3 and apoptosis.

111
Q

List the morphological features in the cell associated with apoptotic cell death.

A

Nucleus = DNA fragmentation and laddering, chromatin condensation.
Cytoplasm – formation of apoptotic bodies
Cell membrane – blebbing, cell shrinkage

112
Q

Briefly describe the growth of a tumour over time and how this is impacted by the tumour microenvironment.

A

Tumour growth follows the Gompertzian pattern/model, with a sigmoid-shaped growth curve in the 3 parts

Part 1 of the curve
- exponential growth
- small number of tumour cells with adequate nutrition/blood supply - growth fraction is near 100%

Part 2 of the curve
- Linear cell growth
- Microenvironment becomes harsher with areas of restricted blood supply for tumour cells
furthest from vessels
- Cells close to vessel have sufficient oxygen/nutrients for proliferation
- Growth fraction falls, and cell loss increases

Part 3 of the curve
- Plateauing of tumour growth
- tumour microenvironment unfavourable with only a small fraction of cells sufficiently
supplied with oxygen and growth factors to allow proliferation
- cell loss factor becomes equal to growth fraction
- clinically a tumour mass, likely with areas of necrosis

113
Q

Describe the step by step cell culture technique and process of generating a clonogenic cell survival curve.

A

Cells from actively growing stock culture are prepared into suspension by use of trypsin- which causes cells to detach from the surface of the culture vessel.

Number of cells per unit volume is counted using an electronic counter

Cells are seeded onto a dish and incubated for 1-2 weeks

Colonies are counted at the end of incubation

The plating efficiency is counted: PE=Number of colonies Counted/Number of cells Seededx 100

Parallel dishes are seeded with a number of cells calculated based on estimated surviving fraction at each dose increment. 

The plates are exposed to increasing, specified dose of x-rays and incubated for 1-2 weeks before being fixed, stained and colonies counted. Some of the following observations can be made:
- Some of the cells are single, with some showing apoptotic death
- Some have formed tiny abortive colonies with limited rounds of divisions
- Some have formed large colonies similar to unirradiated controls. These cells are said to have survived.

The surviving fraction is thus:  SF=Colonies Counted/(Cells Seeded × PE)×100
SF is plotted against the relevant dose level
114
Q

Describe the processes by which a DNA sequence is converted to a protein product.

A

Transcription – 3 steps
Initiation
* RNA polymerase binds to promotor sequence.
* RNA polymerase separates the DNA strands to allow exposure of a single strand of DNA.
Elongation
* RNA polymerase reads off one strand (the template strand) and adds a complementary nucleotide to create an RNA chain. This occurs in a 5’ to 3’ direction.
Termination
* Sequences called terminators exist in the DNA sequence. When they are transcribed they cause the RNA chain to be released from the RNA polymerase.

Post transcription modification
* End modification – cap on the 5’ end, 3’ poly A tail.
* Introns spliced out. Exons combined again.
* mRNA transported to ribosomes

Translation
Initiation
* Ribosome forms around mRNA and tRNA with methionine binds to the start codon.
Elongation
* mRNA is read one codon at a time – a new tRNA binds to each respective codon. This causes the protein chain to increase in size.
Termination
* Stop codon enters the ribosome.
* The protein chain is terminated and exits the ribosome

115
Q

For each of the following terms, provide a definition and two (2) factors influencing tumour:
i. radiosensitivity
ii. radioresponsiveness
iii. radiocurability (3 marks)

A

Radiosensitivity is the tendency of tumour cells to undergo cell death when exposed to ionising radiation. Degree of tumour kill following dose of radiation, often measured as surviving fraction. Factors are cell origin, cell cycle position and oxygen conditions (oxic vs hypoxic) and cell cycle time

II. Radioresponsiveness Overall level/amount of clinical response following radiotherapy (e.g. reduction in pain, visible shrinkage of tumour)
- Factors include radiosensitivity of tumour, and total dose delivered
Factors are radiosensitivity and cell origin (carcinomas are more radioresponsive than sarcomas).

III. Radiocurability is the tendency of a tumour to be sterilised (cured) by ionising radiaton. Factors are (similar to radiosensitivity) differentiation, growth fraction, cell cycle time. Also clonogen number (tumour size)

116
Q

How do HPV viral oncoproteins promote the malignant phenotype?

A

 - Protein E5; activates EGF pathway (sustained proliferative signalling), and downregulates MHC1 (avoid immune destruction)
 - Protein E6; binds and inactivates p53 which impairs G1/S checkpoint and apoptotic pathway (evading growth inhibitors and resisting cell death). E6 also prevents telomere shortening (replicative immortality)
 - Protein E7; binds to Rb protein which allows release of E2F and progression to S phase (sustained proliferative signalling). It also inactivates p21 and p27 which are involved in regulating G1/S checkpoint (evading growth inhibition)

117
Q

Pathophysiology of late radiation-induced skin injury

A
  • RT causes damage to vessel endothelial cells which may take years to die through mitotic death (long latency)
  • Loss of endothelial cells leads to vessel dysfunction and impaired perfusion to epidermis which can cause chronic ulceration
  • Loss of vessel smooth muscle cells can lead to telangiectasia
  • RT also activates fibroblasts to fibrocytes via activation of cytokines (TGF-B and PDGF) and subsequent deposition of collagen. This collagen deposition causes fibrotic skin. Latency of 3 years.
118
Q

Principles of RTOG toxicity scoring systems, why they are useful in clinical radiotherapy

A
  • System to objectively and simply quantify the severity of acute/late side effects of radiotherapy
  • The scoring criteria are organ specific with specific endpoints for each score/level
  • International standards mean that comparisons can be easily made between institutions
  • System useful both for clinical decision making and in regards to clinical research when comparing treatment regimes/modalities etc
  • 0 = no effect; 1 = mild, reversible; 2 = moderate, able to self care; 3 = severe, likely hospital care; 4 = life-threatening; 5 = death
119
Q

For skin RT, smaller areas are treated with hypofractionated schedules whereas larger treatment areas treated with conventional 2Gy/# RT. Explain radiobiological basis for this.

A
  • When treating smaller areas, the potential for repopulation by stem cell migration from surrounding tissue is greater than for larger areas where distance of migration is too far (in central region)
  • Therefore larger skin fields have reduced repopulation potential - relying on local repopulation by stem cell asymmetric loss and cycle acceleration
  • Larger skin fields therefore use a conventional fractionation schedule to reduce the amount of normal tissue stem cell kill (early responding tissue) with each fraction and allowing more time for repopulation of epithelium.
120
Q

Describe how synergistic lethality can be used with DNA repair processes and explain how the addition of PARP inhibitors to radiation therapy has the potential to increase tumour cell kill without increasing damage to normal cells

A

Synergistic lethality with DNA repair can be achieved when tumours with a deficiency in one DNA repair pathway are treated with concomitant inhibition of another pathway(s). The therapeutic ratio is improved as the tumour experiences the loss of multiple DNA repair pathways, an event that is known to have high lethality. The inhibition of repair pathways in the adjacent normal tissues should be well tolerated as they have a wider range of alternative pathways still available.

PARP inhibitors inhibit the poly ADP ribose polymerase pathway of single strand DNA repair. Cells treated with PARP inhbitors accumulate single strand DNA breaks. When these cells undergo DNA replication in the S phase of the cell cycle each single strand break becomes a double strand break. These can then be efficiently repaired if the cell still retains normal DNA DSB repair capability, which should be the case in the normal tissues. The cancer cells that do not have a functioning copy of BRCA2 will have deficient Homologous Recombination, the preferred pathway for repairing this sort of DNA damage. This increases the probability of tumour cell death from inadequate DNA DSB repair.

This pathway can be made more potent by using the PARP inhibitor in combination with radiation therapy. Radiation therapy creates a very large number of difficult to repair lethal lesions in the tumour cells

PARP-i = Olaparib, rucaparib

121
Q

Effects of radiation on the testes including spermatogenesis and endocrine function

A

Tolerance doses;
oligospermia = 0.15Gy,
Azoospermia and temporary sterility = 0.5Gy,
2Gy can cause one year of sterility
permanent azoospermia >6Gy in single dose
- 2.5-3 gy in fractionated dose over 2-4 weeks also causes permanent sterility
- Testes is more sensitive to low dose rate (often better to give single dose than fractionated dose) unlike other body tissues.

Leydig cells are more radioresistant and have a longer cell cycle time. Effects of testosterone is a later effect than reduced sperm count.
The threshold dose for reduced endocrine function is 20Gy (prepubertal) 30Gy (adult)

122
Q

Describe the in vitro cell culture technique to generate cell survival curves

A
  • Cells from active culture are suspended in a medium using trypsin
  • Number of cells per unit volume measured
  • Cells are then plated, exposed to varying doses of radiation, and control plate
  • Plates are then incubated for 1-2 weeks in identical conditions
  • Number of colonies grown on each plate measured and used to generate cell survival data with plating efficiency values

Plating efficiency - number of colonies grown / total number of cells plated x 100

Surviving fraction - proportion of cells surviving after irradiation, corrected for plating efficiency
SF = Colonies counted/ (cells seeded x PE) x 100

123
Q

Steps in apoptotic pathway in response to ionising radiation

A

DNA damage -> sensing/activation of ATM-MRN complex -> activation of p53 -> increase in pro-apoptotic proteins (BAX, Puma) -> activation of caspase 9 (intrinsic pathway) -> caspase 3 activity -> initiation of apoptosis

124
Q

Define the following terms:
- Protooncogene
- Oncogene
- Oncoprotein
- Tumour suppressor gene

A
  • Proto-oncogene
  • A gene which when functioning normally serves a role in normal cellular functioning. When mutated, he gene acts as an oncogene
  • Oncogene
  • A gene which when activated , leads to the production of proteins and pathways that stimulate unregulated (malignant) growth via transcription of oncoprotein
  • Oncoprotein
  • The protein produced/coded by an oncogene that stimulates unregulated growth
  • Tumour suppressor gene
  • gene that checks and regulates cell growth/repair to prevent proliferation of mutated or damaged cells
125
Q

Pathogenesis of moist desquamation

How does re-epithelisation occur? Why is a more fractionated course recommended for large skin treatment areas?

A
  • Moist desquamation occurs due to loss of epidermis resulting in exposure of underlying dermis and ulcer formation
  • Squamous epithelium generally has a high turnover and proliferation rate, therefore, as these cells are lost, new stem cells differentiate to replace them as functional tissue
  • Stem cells are killed acutely by IR, reducing potential to repopulate the epidermis and therefore causing desquamation
  • Exudative response occurs to cover ulcerated area with fibrin and cellular remnants.
    Latency period between start of treatment and development of moist desquamation (for conventional RT) is approximately 4 weeks
  • Re-epithelisation occurs by migration of stem cells from outside treatment field into area of stem cell loss, and also from changes in local stem cell kinetics including loss of asymmetry, accelerated divisions, and abortive divisions.
  • With large treatment areas, there is reduced potential for migration of stem cells from nearby tissue. Therefore, a more fractionated course will reduce the dose per fraction (and increase surviving fraction of stem cells), as well as prolong treatment time to allow more repopulation to occur from local stem cells rather than relying on migration of stem cells from surrounding skin.
126
Q

a)Define in-situ hybridisation.
b) Describe the basic principles and general uses of in-situ hybridisation,
including methods of visualisation.
c) Give a specific example of the use of in-situ hybridisation

A

a) Define in-situ hybridisation: In situ hybridisation is a molecular cytogenetic technique that uses a complementary DNA/ RNA strand (probe) to mark specified DNA sequences in the genome/chromosomes.

B) Basic principles and general uses of in situ hybridisation:

DNA/RNA probes are constructed, labelled with fluorescent dye
DNA/RNA probe applied with chromosomes on a glass slide
Sample is incubated and probe undergoes hybridisation with target DNA
Sample is washed and then viewed under fluorescent microscope
This technique is used generally to detect the presence or absence of specific DNA sequences on chromosomes
c) specific example: test for amount of HER2 gene copies on breast cancer tissue (HER2 status)

127
Q

Methods of quantification of cancer associated genes:
- Comparative genomic hybridisation
- in situ hybridisation
- Spectral keryotyping

A

Comparative Genetic Hybridisation

Allows detection of chromosomal count in cells

Detection of unbalanced copy number abnormalities of chromosomes or chromosomal segments

  1. Microarray is created using segments of DNA from a normal diploid cell (the probe)
  2. Segmented to create 1000s of targets
  3. Sample DNA specimens are generated and fluoro-tagged
    - DNA from tumour is labelled one colour (green)
    - DNA from another diploid control is labelled a different colour (red)
  4. The sample DNAx2 are introduced into the microarray and compete to bind with the probes
  5. If equal copies, the DNA will glow yellow
    - If there are extra tumour copies, the DNA will glow green
    - If the tumour has lost segments, the DNA will glow red

Detects unbalanced copy differences

If the error is balanced, will not be evident

In-situ Hybridisation (ISH)

Allows detection of gene expression (e.g. Amplification) . Used in HER-2

  1. A probe is created of the gene of interest (complimentary sequence)
  2. Probe is tagged with a fluorescence (FISH) or chromophore (CISH) moiety
  3. The Sample DNA is denatured (allowing single stranded) and introduced with the probe
  4. The probe binds to the complementary sequence of the sample DNA
  5. Remainder of probe is washed away after incubation
  6. By fluorescence or colour analysis, the number of copies per cell of the gene of interest can be seen

Spectral Karyotyping

Similar to ISH, but allows more detailed analysis

  1. Similar process to ISH, however multiple probes used (one for each chromosome)
  2. Each probe uses a different colour
  3. Colour analysis is performed after incubation
  4. Able to detect DNA for each chromosome
    Including translocations

Thus provides a karyotype and basic translocation information

128
Q

List and define the tumour kinetic factors influencing the tumour growth rate

A
  • Cell cycle time (Tc): average time for a tumour cell to progress through cell cycle
  • Growth fraction (GF): ratio of proliferating cells to total cells (near 100% for early tumours)
  • Cell loss factor: ratio of cell loss rate to cell proliferation rate
  • These tumour kinetic factors influence:
  • Tumour doubling time (Td): time taken for tumour to double its volume
  • Potential doubling time (Tpot): theoretical doubling time of tumour in the absence of cell loss
129
Q

Describe structure of histone proteins and chromatids

A

Achromatid, is one half of a duplicated chromosome. Made up of tightly coiled DNA around histones, like beads on a string to form nucleosomes.
- Histones are basic proteins, and their positive charges allow them to associate with DNA, which is negatively charged. Under the microscope in its extended form, chromatin looks like beads on a string. The beads are called nucleosomes. Each nucleosome is made of DNA wrapped around eight histone proteins that function like a spool and are called a histone octamer. Each histone octamer is composed of two copies each of the histone proteins H2A, H2B, H3, and H4. The chain of nucleosomes is then wrapped into a 30 nm spiral called a solenoid, where additional H1 histone proteins are associated with each nucleosome to maintain the chromosome structure.
- The amino acids on histone tails are prone to modifications like acetylation, methylation, phosphorylation
- Acetyl groups can add or remove functional groups around histones (modifying transcription)
- Acetyl decreases the bind of H1 histones
- Acetylated histones are more relaxed and can undergo increased transcription
- Cancer has overall increased acetylation of histones
- Note that decreased cytosine methylation results in histone acetylation

130
Q

4 chemotherapy examples that effect specific organs that will reduce tolerance and which organs these are

A
  • Lungs: Bleomycin can cause lung toxicity resulting in fibrosis
    - Heart: Doxorubicin can cause cardiotoxicity which lowers heart tolerance
    - Kidneys: Cisplatin causes nephrotoxicity
    - Peripheral neuropathy: Vincrisitine and cisplatin
    - Liver: Methotrexate can cause hepatotoxicity
    Ototoxicity: Cisplatin can affect hearing
131
Q

How amifostine works as radioprotector

A

Amifostine has Sulfhydryl (-SH) compounds that helps protect tissue from radiation by scavenging of free
Radicals.
Amifostine is a prodrug and only becomes active when it is dephosphorylated by alkaline phosphatase. Levels of alkaline phosphatase are higher in normal tissues (than tumour tissues) and blood vessels and therefore it
would be hoped to have a selective action of reducing the biological effect of radiation only in normal cells. But this selectivity is not 100% guaranteed and it is not widely used in clinical practice due to the concern that it is also potentially protecting tumour cells.
Tissue variability – good for protecting haematopoietic system, GI, salivary glands etc. Not good for CNS (doesn’t cross BBB).
Clinically used rarely (e.g. protecting salivary gland for HN cancers) or by astronauts going to lunar missions.

132
Q

Describe the structure and role of the ECM

A
  • This comprises the connective tissue, collagen and hormones/growth factors surrounding the tumour
  • Tumours can generate excessive growth factors to fuel rapid growth
  • They can also generate cytokines which help in immune localisation (with resultant inflammation)
  • ECM and the associated hormones can drive cellular differentiation leading to variable phenotypes
  • In addition, migration through the ECM is a defining part of tumour metastasis
  • The ECM can be prone or resistant to permeability
  • Cancers can release enzymes (collagenase, matrix metalloprotease - MMP) to promote metastasis
  • ECM plays an important role in tissue development, repair, support and homeostasis.
  • In tumours , the Ecm plays an important role in shaping the tumour microenvironment and influences cancer progression, metastasis, and therapeutic response. This process is called ECM remodelling and is characterised by changes in protein content and enzymatic activity which influences signal transduction and cell-matrix alterations. ECM remodelling involves dynamic alterations in ECM composition, organization, and biomechanical properties. ECM remodelling is induced by factors such as hypoxia, acidosis, inflammatory cells or proteases secreted by tumour or stromal cells.
133
Q

Combination of radiation therapy with immunotherapy, including mechanisms by which radiation may enhance anti-tumour immunity

A

** 1. Exposure of Tumor-Specific Antigens: **Radiation therapy damages cancer cells, causing them to release tumor-specific antigens. These antigens become visible to the immune system, promoting the priming and activation of cytotoxic T cells.

** 2. Modulation of the Tumor Microenvironment: **Radiation can alter the tumor microenvironment, making it more conducive to immune cell infiltration and activity. This includes increasing the expression of molecules that attract immune cells to the tumor site.

** 3. Upregulation of Immunogenic Mutations: **Radiation can upregulate the expression of genes involved in the response to DNA damage and cellular stress, potentially exposing immunogenic mutations to the immune system.

** 4. Enhancement of Immune Signaling Pathways:** Radiation can regulate the expression of interferons and affect signaling pathways such as *cGas-STING *pathway, which are crucial for enhancing immune responsiveness.

**5. Conversion of ‘Cold’ Tumors to ‘Hot’ Tumors: **Radiation can convert immunologically ‘cold’ tumors, which are not easily recognized by the immune system, into ‘hot’ tumors that are more immunogenic and responsive to immunotherapy.