Lecture 1: DNA damage & Repair 07/15/2019

DNA Damage – DNA is the critical target of ionizing radiation hence the reason why cells
die after irradiation

Ionization radiation methods

Direct
- involves ionization of DNA molecule itself leading to biological damage
- dominant process in High LET particles e.g. protons, a particles
- accounts for 1/3 of biological damage
- don’t require presence of O2 to ‘fix’ to damage – similar efficacy in hypoxic & oxic cells

Indirect Indirect radiation steps

- Involves ionization of water molecule (since 80% of cell 1. Primary P interacts to produce high
energy e
is water) creating free radicals (H2O+ and OH-) which 2. E interacts with water to produce free
interact with the critical target (DNA) and cause radicals
3. Free radicals produce changes in DNA
biological damage from breakage of chemical bonds
- Dominant process in Low LET - photons 4. Changes in chemical bonds result in
- Accounts for 2/3 of biological damage biological effects
- Requires O2 to ‘fix’ therefore hypoxic cells more
resistant to this type of radiation
- Step (1) physics (2) chemistry (3)(4) radiobiology

Damage Types
SSB – one strand of DNA molecule breaks
- Easy to repair

DSB – both strands of DNA molecule breaks thereby most

lethal to cell
- Harder to repair as it results in chromosome breakages
which are less likely to be repair before mitosis

Mechanisms
HR (homologous recombination) - requires undamaged DNA copy of sister chromatid
to function - only present after DNA replication has occurred: (1) S phase, G 2 phase and in early
mitosis
- Advantages: Good fidelity = more accurate as it uses undamaged DNA strand to replace
damage DNA
- Disadvantage: (1) slow/8-12 hrs. (2) can only occur after DNA has been replicated

NHEJ- does not require a sister chromatid to function therefore can occur in all phases
- Advantages: (1) fast (2) any time of cell cycle
- Disadvantage: Poor fidelity due to no template

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Cell fate after irradiation
Survival
a. with accurate DNA repair
b. with genetic changes

Death
a. Mitotic catastrophe due to lethal chromosomal aberrations (immediate or delayed)
b. Programmed cell death (apoptosis, autophagy)
c. Senescence: permanent cessation of cell division due to aging, differentiation or cellular
damage (c.f. necrosis: death with loss of cell membrane integrity)

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Lecture 2: DNA Damage sensor 07/15/2019

MRN complex is important for coordinating stages of the repair process of double-strand
DNA breaks
(1) initial detection of a lesion (2) halting of the cell cycle to allow for repair (3) selection
of a specific repair pathway

1. Initial detection is controlled by both

Nbs1 and MRE11
2. Cell cycle regulation is controlled by ATM
(phosphorylation) – dependent on Nbs1-
binds ATM to bring it to damaged DNA
3. Selection of repair pathway dependent by
MRE11
4. Rad50 tethers two linear DNA molecules
together while MRE11 fine-tunes the
alignment by binding to the ends of the broken chromosomes.

Pathway defects
Nijmegen Breakage Syndrome – absence of NSB1
Symptoms – (1)microcephaly (2)Immunodeficiency (3) radiation sensitivity (4) lymphoid
malignancy

Ataxia Telangiectasia Syndrome – mutation of ATM gene - therefore no activation of

downstream substrates + phosphorylation of H2AX
phosphorylates histone H2AX (protein associated with DNA) → changes chromatin
structure →allows other DNA repair proteins to access dsb
Symptoms – (1) lymphomas (2)radiosensitivity (3)cerebella ataxia (4)impaired immunity

Li-Fraumeni Syndrome – p53 activates genes that induce apoptosis and cell cycle delay
Mutations that make the protein more stable
associated with sarcomas, breast cancer, brain & adrenal tumors, leukemia’s

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Lecture 3: Clonogenic assays 07/15/2019

The effects of radiation on tissue due to dose are measured
with assays and the measurement results are given in the
form of cell survival curves or dose response curves
Clonogenic assays measures the reproductive integrity of the
Clonogenic stem cells (cells that can sustain proliferation) in
tissue and the measurement results in cell survival curves
2 components of cell killing
- One is proportional to dose (aD)
- One proportional to the square of
the dose (BD2).
- The dose at which the linear and
quadratic components are equal is
the ratio a/B

cell survival curve

- describes relationship between (1) surviving fraction of
cells that maintain reproductive integrity and (2) absorbed Limitations of LQ model
dose - Model predicts steadily increasing
slope, but experiments suggest a
- Fraction of surviving cells plotted on a logarithmic scale constant final slope therefore
against dose on a linear scale does not apply for large fractions
Benefits of LQ model
- Reasonably accurate in the range
Characteristics of Clonogenic assay data of commonly used fraction size
1) initial linear slope (1.8Gy – 4Gy)
- Simple to apply (only two
2) followed by a shoulder parameters to adjust a & B
3) the curve tends to become straight again @ higher doses

characteristics of linear-quadratic model

1) resultant cell survival curve is continuously bending; there
is no final straight portion so does not apply for large
fractions

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Lecture 5: Therapeutic Ratio 07/15/2019
Therapeutic ratio – refers to the ratio of TCP and NTCP at a specified dose level

Aim of RT schedules is to (1) maximize tumor control (2) minimize normal tissue damage
thereby improving the therapeutic ratio

Depiction of therapeutic ratio

- Curve A represents tumor control
probability
- Curve B represents normal tissue
complications probability
- The farther NTCP (B) is to the right of
TCP(A) – larger therapeutic index -
easier it is to achieve the therapeutic
goal

Ideally; RT schedule (1)

maximizes TCP >0.5 (2) Minimizes NTCP
<0.05

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Lecture 2: Linear Quadratic Equation 23/07/19 7:49 PM

Linear quadratic model is the method of fitting survival of cells following radiation to an
equation. Note this is for a single fraction + the a/b constants differ between different tissues
- Clinical used to calculate effectiveness of various RT dose schedules

Equation
S(D)=e−(αD+βD2)
o S = number of surviving cells
following a dose
o α & β =linear + quadratic parts of
the survival curve respectively
o The bigger the dose (D) – the
more B component dominates and
the smaller the dose the more the
a component dominates

Alpha/Beta ratio is the

dose/Gy when # of cells killed by linear
component = cell kill by quadratic
component (ad=Bd2
1. High αβ ratio = more linear cell
survival curve
2. Low αβ ratio = more curved cell
survival curve

Tissues:
Early responding tissues
o High αβ ratio
o Steeper initial curve at low
doses & Flatter curve at higher
doses
o Relatively more sensitive to
lower doses, but still able to
recover/repopulate at higher
dose
Late responding tissues
o Low αβ ratio
o Flatter initial curve at low doses
& Steeper curve at higher
doses
o Relatively more resistant to
lower doses, but unable to
recover/repopulate at higher
doses

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Lecture 6: Fractionation 07/15/2019
Fractionation – means RT is given over a period of weeks rather than a single session _
results in better therapeutic ratio

Conventional fractionation fractionation

- Fraction size of 1.8-2Gy, 1#/day, 5#/week dependent on five biological
factors
- Total dose varies for tumors. - Repair
- Often uses shrinking field technique: highest concentration of - Repopulation
- Redistribution
tumor cells receives additional dose (Boost)
- Reoxygenation
- Fractionation spares late reacting tissues
- Dividing of dose into multiple fractions spares normal tissue thru
repair of sublethal damage & repopulation of cells between
fractions. Concurrently fractionation increases tumor damage thru reoxygenation +
redistribution of tumor cells

Other fractionation schemes Intent is to

Hyper fractionation - same/better tumor control
- reduce late effects
- Regimen which uses more than 1#/day with smaller dose/fraction
- same/slightly increased
to reduce long term complications + allow delivery of higher total early effects
dose
- (1) fraction size is reduced (2) total dose is increased (3)
treatment time remains relatively unchanged
- Assumes – low α/β for late effects (generally true) – high α/β for tumor (may not hold for all
tumors)

Controlled clinical trial for H/N tmt Results

- hyperfractionated schedule of 80.5 Gy delivered in 70 fractions - Increased local control &
overall survival rates
(1.15 Gy twice per day) over a period of 7 weeks was compared - No increase in early/late
with a conventional regimen of 70 Gy delivered in 35 fractions of 2 side effects
Gy over 7 weeks. - Hyper fractionation is more
advantageous

Accelerated fractionation – tries to overcome accelerated repopulation (tumor cell

proliferation) by decreasing overall tmt time
Regimen in which (1) fraction size (2)total dose and (3) number
of fractions are unchanged (or somewhat reduced) Results
- Increased local control &
overall survival rates
Protocols - increase in early effects
Accelerated (w/concomitant boost) - late side effects
- 1.8 Gy/fraction/day, 5 days/week – and 1.5 Gy/fraction/day to a (unchanged)

boost field as a second daily treatment for the last 12 treatment days
given over 6 weeks

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- showed increased local control

Pure Accelerated
- 2# of 1.6 Gy/day,6 hours apart (for SLD repair) 5 days/week. Treatment was given over 7
weeks, including a 2 week gap to allow some recovery from early effects.
- gap in treatment, negating the benefit of the accelerated treatment – results in accelerated
repopulation of tumor

CHART Continuous Hyperfractionated Accelerated Results

Radiotherapy - Improved local control
54Gy / 36# / 12 days  3x daily, 6h apart, no weekend - Increased early toxicity that
only reached Grade III
break toxicity 
- Similar or reduced late
toxicity, exception of spinal
cord toxicity which
was increased

Split course treatment – planned gap of at least 7 days during a course of tmt
To account for tumor repopulation, extra dose need to be given – may be up to 0.6Gy/day

Advantages
1. Allow time for normal tissue healing to occur to reduce early side effects

Disadvantages

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1. Tumor repopulation occurs in the interval between treatments
2. Patient has a longer time between start and finish of treatment

Hypo fractionation
- Decreasing number of fractions = larger dose/fraction >2Gy
- Effective for tumors with low alpha/beta ratio (slow growing tumors)
- Slow growing tumors are resistant to small fraction sizes and would benefit from fewer large
fraction Advantages
- Seen for palliative pts.: allows large dose to be delivered 1. Shorter treatment length
2. Increased effect on low alpha/beta ratio
without inconveniencing the patient greatly. tumors such as prostate cancer
3. Palliative patients are unlikely to live for
long enough to experience the increased
late tissue toxicity
Perils of hypfractionation Disadvantages
1. Increased rate of late effects in normal
RAGE explored 44Gy/ 11#
tissues
Severe late effects (1)bone rib fracture (2) nerve – arm 2. Less chance of oxygenation and
pain/weakness/paralysis (3) subcutaneous fibrosis redistribution to occur

-lymphedema

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Lecture 7: OER 07/15/2019
The oxygen effect - biological affect of ionization radiation can be influenced by the
presence/absence of O2 within a cell. The larger the O2 the larger the Biological effect (cell killing)
particularly for Low LET (sparsely ionizing) radiation

OER figures:
Oxygen enhancement ratio – ratio of doses without and with - 2.5-3 for γ rays
- 1.0 for high LET
oxygen (hypoxic vs. well oxygenated cells) to produce same biological effect.
1.6 for neutrons

Importance of Oxygen – oxygen enhancement hypothesis

- Oxygen enhances DNA damage induced by free radicals, thereby
facilitating the indirect action of IR
- Combines with free electron on free radical creating a peroxide which is more stable
- c.f. it fix or makes the damage permanent cause by free radical
- In hypoxic conditions – Damage may repair itself

Many tumors are hypoxic due to:

- Chronic hypoxia from poor vascular supply
- Acute hypoxia due to temporary occlusion of tumor vessels

Overcoming hypoxia 1. LDR brachytherapy

fractionation - most commonly used in the modern setting. 2. Carbogen/nicotinamide –
(1)overcome chronic hypoxia
- Fractionation of dose kills oxygenated cells first, allowing
(2)nicotinamide to prevent vessel
the hypoxic population to migrate closer to the vascular closure and acute hypoxia
network and become oxygenated.
- The remaining fractionated dose then kills these previously
hypoxic cells.

Other methods
1. Hypoxic cell sensitizer – mimics role of oxygen in fixing radiation damage to DNA
(1)misonidazole
2. Hypoxic cell cytotoxic – selectively targets hypoxic cells & kills them (1)Tirapazamine
3. Hyperbaric oxygen therapy – increase oxygen pressure in tumor cells
4. Increase BED using (1)High LET particles – no oxygen needed to fix damage (2) dose
escalation- used in brachy for cervix
5. Increase RBC formation (1)EPO –worse tumor control X
6. Blood transfusion X

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Lecture 8: Repopulation 07/15/2019
Repopulation:
- (W)During an extended course of RT – cells that survive (clonogens) may divide + proliferate
thus increase number of cells that need killing
- (W)Applies to most tumors and early responding tissues
- Planned tmt breaks may be used to allow repopulation of normal tissue if rt reactions are
limiting (severe) – otherwise pt will have severe acute side effects
- Planned breaks reduce local control/cure rates

Accelerated repopulation (applies for tumors)

- Some studies which show tmt with chemo, surgery + RT can trigger surviving cells in a tumor
to divide & increase in number more rapidly than before tmt @ 4-5 weeks onwards especially
for H/N tmt
- Dose increment of 0.6Gy/day required to compensate for the repopulation [seen accelerated
repopulation with [concomitant boost]

Clinical relevance [H/N tmt]

- Better to delay initiation of tmt than to introduce delays during tmt because this extends
overall tmt time
- (W)If overall tmt time is too long – effectiveness of later dose fractions is compromised
because surviving clonogens in tumor have been triggered into rapid population

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