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Radiation induced cell kill and damage
1. RADIATION INDUCED CELL
KILL AND DAMAGE
Moderator: Prof. R Kapoor
Dr. Namrata Das
Junior Resident
Dept. of Radiotherapy and Oncology
3.8.18
2. OUTLINE
• Introduction – importance of radiobiology
• DNA Damage by ionizing radiation
• LET
• Chromosome Damage and Repair
• Cell survival curves
• 5 Rs of Radiobiology
• Clinical Response of Normal Tissue – Early and Late Effects
• Summary
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3. INTRODUCTION
The purpose of radiotherapy is to maximize tumour kill and minimize normal
tissue damage.
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IONIZING RADIATION
DIRECT/INDIRECT ACTION
ON CRITICAL TARGET (DNA)
DNA DAMAGE
ONCOGENESIS,
MUTAGENESIS
CELL KILL REPAIR
DNA DAMAGE RESPONSE (DDR)
5. THERAPEUTIC INDEX
The ratio of the tumor response for
a fixed level of normal tissue
damage has been called either the
therapeutic ratio or the therapeutic
index.
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DOSE: Its is defined as the mean energy(ε) imparted by
ionizing radiation to a matter of mass m in a finite
volume V, D=dε/dm
SI unit of dose = Gray (Gy)
1 Gy = 1 Joule/kg
100 rad (older unit) = 1 Gy
6. DNA DAMAGE BY IONIZING RADIATION
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BIOLOGICAL EFFECTS OF RADIATION
DIRECT ACTION INDIRECT ACTION
ABSORPTION IN HUMAN BODY
DIRECTLY IONIZING INDIRECTLY IONIZING
TELETHERAPY SOURCE: IONIZING RADIATION
ELECTROMAGENTIC RADIATION PARTICULATE RADIATION
7. IONIZING RADIATION
Ionizing radiation: has sufficient energy to eject one or more orbital electrons
from the atom or molecule
Ionizing
Radiation
Electromagnetic
X Rays
(extranuclear)
Gamma Rays
(intranuclear)
Particulate
Charged Uncharged
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8. INDIRECTLY IONIZING RADIATION
DEPENDING UPON THE ENERGY OF THE PHOTON AND THE ATOMIC
COMPOSITION OF THE ABSORBING MATERIAL
ABSORPTION TAKES PLACE BY
PHOTOELECTRIC EFFECT COMPTON EFFECT PAIR PRODUCTION
PHOTON LOSES 0-80% OF ITS ENERGY
PRODUCTION OF SEVERAL FAST ELECTRONS (KE)
IONIZE OTHER ATOMS OF BREAK VITAL CHEMICAL
ABSORBER BONDS
INITIATE THE CHAIN OF EVENTS THAT
RESULTS IN BIOLOGICAL DAMAGE
9. Electromagnetic Radiation: Dual nature
• Wave nature
• Particle Nature
- stream of photons/ packets of energy
E (Energy) = hv,
λ (Angstrom) = 12.4/E )keV
h = Planck’s constant, v = frequency, λ = wavelength
• Size of individual quanta versus total energy
• E = 124 eV, λ = 10-6 cm – ionizing radiation
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10. 3.8.18
LET (LINEAR ENERGY TRANSFER)
Linear energy transfer (LET) is the energy transferred per unit length of the
track. The special unit usually used for this quantity is kiloelectron volt per
micrometer (keV/μm) of unit density material.
Ionizations and excitations are not distributed at random.
Localized along the tracks of individual charged particles.
Function of:
1. Charge on ionizing radiation: increasing charge -> increasing LET
2. Velocity: velocity decreases -> LET increases
11. 3.8.18
LET (LINEAR ENERGY TRANSFER)
SPARSELY IONIZING DENSELY IONIZING
X Ray/ ϒ Ray α rays/ recoil protons
Unit electrical charge and small mass Greater mass
12. 3.8.18
10 MeV proton (recoil proton from high energy
neutrons): intermediate ionization density
500 keV proton(produced by lower energy neutrons
e.g., from fission spectrum): high ionization density
1-MeV electron (produced, by cobalt-60 γ-rays): particle
is very sparsely ionizing
5-keV electron (secondary electrons produced by x-rays of
diagnostic quality): sparsely ionizing but a little denser than the
higher energy electron
13. SPURS BLOBS
100eV 100-500eV
3 ION PAIRS (OH) 12 ION PAIRS (OH)
4 nm DIAMETER 7 nm DIAMETER
95% OF ENERGY
DEPOSITION EVENTS IN
CASE OF X-RAYS AND
GAMMA RAYS
NEUTRONS, ALPHA
PARTICLES, GREATER
PROPORTION OF BLOBS
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• Clustered lesion/ Multiple
damaged site
• Coined by John Ward
14. 3.8.18
In 1962, the International Commission on Radiological Units defined this quantity
as follows:
L=dE/dl
The LET (L) of charged particles in medium is the quotient of dE/dl, where dE is
the average energy locally imparted to the medium by a charged particle of
specified energy in traversing a distance of dl.
LET (LINEAR ENERGY TRANSFER)
15. 3.8.18
LET (LINEAR ENERGY TRANSFER)
Co-60 gamma rays 0.2
250 kV X rays 2
10 MeV photons 4.7
150 MeV proton 0.5
2.5 MeV alpha particles 166
16. OPIMAL LET
• 100 keV/μm: At this density of
ionization, the average
separation between ionizing
events just about coincides with
the diameter of the DNA double
helix (20 Å or 2 nm)
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17. DNA DAMAGE BY IONIZING RADIATION
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BIOLOGICAL EFFECTS OF RADIATION
DIRECT ACTION INDIRECT ACTION
ABSORPTION IN HUMAN BODY
DIRECTLY IONIZING INDIRECTLY IONIZING
TELETHERAPY SOURCE: IONIZING RADIATION
ELECTROMAGENTIC RADIATION PARTICULATE RADIATION
18. ABSORPTION OF X-RAYS
I. DIRECTLY IONIZING
- Charged particles
- High kinetic energy
- Direct disruption of atomic
structure
II. INDIRECTLY IONIZING
- X rays, Gamma rays
- Compton effect predominates
- Fast electron mediated damage:
ionize other atoms, break chemical
bonds, initiate chain of events
leading to damage
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19. DNA DAMAGE BY IONIZING RADIATION
3.8.18
BIOLOGICAL EFFECTS OF RADIATION
DIRECT ACTION INDIRECT ACTION
ABSORPTION IN HUMAN BODY
DIRECTLY IONIZING INDIRECTLY IONIZING
TELETHERAPY SOURCE: IONIZING RADIATION
ELECTROMAGENTIC RADIATION PARTICULATE RADIATION
20. ACTION OF RADIATION
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I. DIRECT ACTION
Critical target: DNA
• Radiation (electromagnetic pr charged
particles) interacts directly with the critical
target in the DNA
• Atoms of target ionized/ excited initiating chain
of events
• Dominant process with radiations of high LET
21. II. INDIRECT ACTION
Free radical mediated damage by
interaction of radiation with water
molecules
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NUCLEUS
DNA
CYTOPLASM:
WATER
ACTION OF RADIATION
23. Chromosomes and cell division
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INTERPHASE
I. G1 (Gap1) : Metabolic events continue profusely
II. S (Synthesis): DNA duplicated, number of
chromatins doubled
III. G2 (Gap2): Enzymes related to division are
synthesized
IV. G0: Cells transiently stop their metabolic activities
CELL CYCLE
MITOSIS
I. Prophase
II. Metaphase
III. Anaphase
IV. Telophase
24. CHROMOSOME DAMAGE AND REPAIR
• DNA is the critical target of the cell
• Both direct and indirect effects of radiation leads to single stranded or
double stranded DNA breaks
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Double stranded DNA break
Single stranded DNA break
25. Number of DNA lesions per cell immediately after a dose of 1 Gy of X- Rays:
• Double stranded break (DSB): 40
• Single stranded break (SSB): 1000
• Base damage >2000
• DNA-DNA crosslinks: 30
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Cell killing, carcinogenesis,
mutation
31. CLASSIFICATION OF RADIATION INDUCED DAMAGE
SUBLETHAL DAMAGE POTENTIALLY LETHAL
DAMAGE
LETHAL DAMAGE
CAN BE REPAIRED UNDER NORMAL
CIRCUMSTANCES
SUBLETHAL DAMAGE REPAIR:
TIME INTERVAL BETWEEN TWO DOSES
(0→2 HOURS)
1. ENZYME THEORY
2. CHEMICAL POOL THEORY
CAN BE MODIFIED BY POST
RADIATION ENVIRONMENETAL
CONDITIONS
IF MADE SUB-OPTIMAL FOR
GROWTH→DO NOT ALLOW
DAMAGED CELLS TO ATTEMPT
MITOSIS
IF MITOSIS IS DELAYED, DNA DAMAGE
CAN BE REPAIRED
RADIORESISTANT TUMOURS HAVE
THE ABILITY TO REPAIR PLD
IRREVERSIBLE AND
IRREPAIRABLE
LEADS TO CELL DEATH
35. Cell death after irradiation
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Lethal aberrations lead to cell death in the following ways:
• Apoptosis
• Mitosis
• Senescence
• Instant death
BYSTANDER EFFECT
• Defined as the induction of biological effects (damage, cell death) in cells not directly
traversed by a charged particle.
• Effect pronounced when gap junctions exist with irradiated cells
37. CELL SURVIVAL CURVE
•Relationship between radiation dose and the proportion
of cells that survive
•Cell death:
1. Proliferating cells: loss of reproductive capacity
2. Non proliferating cells: loss of specific function
38. 3.8.18
X Axis: Surviving fraction (linear scale)
X Axis: Surviving fraction (log scale)
CELL SURVIVAL
CURVES
39. Cell culture technique
SF (SURVIVING FRACTION):
COLONIES COUNTED
CELLS SEEDED X (PE/100)
PLATING EFFICIENCY=
% OF CELLS SEEDED THAT
GROW INTO A COLONY
40. TARGET THEORY
This theory is an attempt to explain how radiation causes cell kill.
In this theory, the idea is that the cell contain specific regions in the
DNA that are important to maintain the reproductive integrity and
hence for radiation damage.
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SINGLE TARGET – SINGLE HIT MULTI-TARGET SINGLE HIT
TWO VERSIONS
41. D10: DOSE AT WHICH SF IS
REDUCED TO 10%
D0: MEAN LETHAL DOSE
DOSE CAPABLE OF
PRODUCING ONE LETHAL
EVENT PER CELL
REDUCES THE SF TO 37%
D10 = 2.3D0
Survival curve is exponential
(i.e. a straight line in a semi-logarithmic plot of
cell survival against dose)
SINGLE TARGET – SINGLE HIT
42. 3.8.18
MULTI-TARGET SINGLE HIT
For mammalian cells in general, their response to radiation is
usually described by ‘shouldered’ survival curves.
One hit by radiation on each of n sensitive targets in the cell is
required for death of the cell.
n = number of targets
Dq= D0loge n → Quasithreshold dose
44. I. So far the specific radiation targets have not been identified for
mammalian cells
II. Rather, what has emerged is the key role of DNA strand breaks and their
repair, with sites for such DNA damage being generally dispersed
throughout the cell nucleus
III. Multi-target model: predicts a response that is flat for very low radiation
doses. This is not supported by experimental data: there is overwhelming
evidence for significant cell killing at low doses and for cell survival
curves that have a finite initial slope.
DRAWBACKS
45. D1—DOSE REQUIRED IN THE LOW DOSE
REGION TO REDUCE THE SURVIVAL FROM 1 TO
0.37
CORRECTLY PREDICTS FINITE CELL KILLING IN
THE LOW DOSE REGION
BUT CHANGE IN SURVIVAL OVER THE RANGE 0
TO Dq OCCURS ALMOST LINEARLY.
No sparing of damage should occur as dose
per fraction is reduced below 2Gy - not found
to be the case either experimentally or in
clinical radiotherapy
ADJUSTED MULTI- TARGET MODEL
46. L-Q (LINEAR-QUADRATIC) MODEL
1972, developed by Dougler and
Fowler
Cell death due to ionizing
radiation has two components:
1. Linear Component:
Directly proportional to dose, D
2. Quadratic Component:
Directly proportional to the
square of the dose, D2
47. • SINGLE-TRACK EVENTS
• α—PROBABILITY OF INACTIVATING A
TARGET DIRECTLY BY SINGLE HIT EVENT
• LOW DOSE REGION
• TWO-TRACK EVENTS
• β– PROBABILITY OF INACTIVATING A
TARGET DIRECTLY BY MHE
• HIGH DOSE REGION
48. p1 : Effect with one radiation hit P2: Effect with two radiation hits
p1 = α P2=β
Corresponds to the cells that cannot repair
themselves after one radiation hit.
Corresponds to cells that stop dividing after
more than one radiation hit, but can repair the
damage caused by the radiation
Shows the intrinsic cell radiosensitivity Corresponds to cells that stop dividing after
more than one radiation hit, but can repair the
damage caused by the radiation.
Reflects
Important for high-LET radiation.
Apoptotic and mitotic death are dominant.
Important for low-LET radiation.
Mitotic death is dominant.
49. The components of cell
killing that are proportional
to dose and to the square of
the dose are equal if:
α/B RATIO
50. LATE REACTING TISSUE EARLY REACTING
TISSUE
α/β = 3 Gy α/β = 10 Gy
spinal cord, bladder,
kidney
skin, mucosa
MHE surpasses SHE
early, at low doses
Broad shoulder Narrow shoulder
51. • As dose/# is increased, the surviving fraction reduces quicker in late
reacting tissue than early reacting tissue or more cell killing in late reacting
tissue than early reacting tissue.
• So increase in dose/# will damage LRT more than early reacting tissue.
• Total dose is to be calculated by α/β value of organ at risk should always be
late reacting tissue.
• If calculated by α/β value of early reacting tissue then it leads to more
damage to late reacting tissue.
CLINICAL SIGNIFICANCE
52. The linear-quadratic approach in clinical practice
•To formulate equivalent fractionation schemes.
•To calculate additional doses after breaks from
radiotherapy.
•To get information on acute and late responses.
53. Universal cell survival curve
• The USC was constructed by hybridizing two classic radiobiologic
models: the LQ model and the multi-target model.
• For ablative doses beyond the shoulder, the survival curve is
better described as a straight line as predicted by the multitarget
model.
• The USC smoothly interpolates from a parabola predicted by the
LQ model to the terminal asymptote of the multitarget model in
the high-dose region. From the USC, were derived two
equivalence functions, the biologically effective dose and the
single fraction equivalent dose for both CFRT and SBRT.
57. The slope of the survival
fraction (SF) curve (1/D ) is
large for high-LET
radiation.
Small for low-LET
radiation.
2. LET
58. As oxygen tension decreases the terminal
portion of the curve gets shallower.
So to get the same SF, dose of RT has to be
increased
Applicable to the terminal portion of CSC,
where all the cell killing is by MHE (indirect
action)
At low dose levels, all the cell killing is by SHE.
No SLD: oxygen not required to fix the damage
3. OXYGENATION
59. OER (OXYGEN ENHANCEMENT RATIO)
OER = Required dose under hypoxic conditions
Required dose under oxygenated conditions
to produce the same level of cell killing
Oxygenation can modify the indirect effect of free radicals:
LOW LET RADIATIONS HAVE A HIGHER OER (2.5-3.5)
However, the OER plays no role in the direct effect of high-LET
radiation; OER is 1 in this case.
60. • The oxygen fixation
hypothesis:
• About two-thirds of the
biologic damage produced
by x-rays is by indirect
action mediated by free
radicals.
• The damage produced by
free radicals in DNA can be
repaired under hypoxia
but may be “fixed” (made
permanent and
irreparable) if molecular
oxygen is available.
61. I. DURING FRACTIONATION:
Sub lethal damage repair:
reappearnace of shoulder after
every fraction
Hence, to get the same SF:
Total dose of radiation has to be
increased
4. DOSE FRACTIONATION
62. 2. EFFECT OF DOSE PER FRACTION:
• AS THE DOSE PER FRACTION
INCREASES, THE SF REDUCES QUICKER
IN LRT THAN IN ERT
• INCREASE IN DOSE/FRACTION
DAMAGES THE LRT MORE THAN ERT
63. • Cell survival is greater
for a delivered
radiation dose if the
dose rate is decreased
• Due to the
proliferation of
undamaged living cells
and SLD repair during
the dose intervals
5. DOSE RATE
64. CLINICAL SIGNIFICANCE
1. Low dose rate continuous RT as used in conventional brachytherapy may be
considered as very large number of very small dose/ Fc.
2. While shifting from LDR to MDR brachytherapy, the total dose to get the s
same effect is to be decreased.
When dose rate is reduced beyond 100 cGy/hr.
No change in total dose.
Because all cell killing is due to SHE.
65. Relative Biological Effectiveness (RBE)
The National Bureau of Standards in 1954 defined relative biologic
effectiveness (RBE) as follows:
The RBE of some test radiation (r) compared with x-rays is defined by
the ratio D250/Dr, where D250 and Dr are, respectively, the doses of x-
rays and the test radiation required for equal biologic effect.
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66. 5 Rs of RADIOBIOLOGY
These are the biological factors that affect the responses of normal and
tumour tissue in fractionated radiotherapy.
1. REPAIR OF SUBLETHAL DAMAGE
2. REPOPULATION
3. REASSORTMENT
4. REOXYGENATION
- Withers, 1975
5. RADIOSENSITIVITY
- Fertil, 1981
67. 1. REPAIR OF SUBLETHAL DAMAGE
SLD can be repaired within hours under normal conditions unless an
additional radiation dose is given (inducing further SLD).
• This generally occurs due to the indirect effect of radiation.
• Single strand breakage in DNA (+).
Occurs in mammalian cells at low doses, and this damage is repaired during
interfraction intervals.
Results in therapeutic gain because it occurs in both tumour and normal
tissue
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68. • Occurs if interval between the split doses is 10-12 hours (exceeds the
length of the cell cycle for rapidly growing cells)
• Occurs in both tumour and normal cells
• DEPENDS UPON THE TYPE OF TISSUE AND VARIES DURING
RADIOTHERAPY (speeds up after the first doses of radiation therapy
“accelerated repopulation”)
• Therapeutic gain can only be obtained if the normal tissue divides faster
than the tumour
2. REPOPULATION
69. • The radiosensitivities of cells vary with the phase of the cell cycle.
• Cells in resistant phases of the cell cycle may progress into a sensitive phase during
the next dose fraction.
• Probability that tumor cells will be exposed to radiation during a sensitive phase
increases, and this probability will continue to increase over the course of the
treatment, and so the benefit of the radiation will also increase.
3. REASSORTMENT
70. The damage produced by free radicals in DNA can be repaired under hypoxia but may
be “fixed” (made permanent and irreparable) if molecular oxygen is available.
• If the radiation is given in a series of fractions separated in time sufficient for
reoxygenation to occur
• TUMOUR REOXYGENATES IN BETWEEN THE FRACTIONS
• INCREASES TUMOUR RADIOCURABILITY
4. REOXYGENATION
71. • If the radiation is given in a series
of fractions separated in time
sufficient for reoxygenation to
occur
• TUMOUR REOXYGENATES IN
BETWEEN THE FRACTIONS
• The presence of hypoxic cells
does not greatly influence the
response of the tumor.
• Conversely, when a large SF is
used, hypoxic cells limit
radiocurability
• TIME SEQUENCE OF
REOXYGENATION = 6 HOURS
72.
73. • Involves multiple components (INTRINSIC OR ENVIRONMENTAL)
• The term “radiosensitivity” was first defined by Bergonie and Tribendau in 1907;
they suggested that radiosensitivity was directly proportional to mitosis and
inversely proportional to differentiation.
• Since radiosensitivity may be affected by external conditions, the term SF2 was
introduced by Fertil in 1981.
• SF2 = surviving cell fraction after a radiation dose of 2 Gy.
As SF2 increases, radiosensitivity decreases.
• Radiosensitizers are used to decrease SF2 .
5. RADIOSENSITIVITY
74. 3.8.18
CLINICAL RESPONSE OF NORMAL TISSUE
Radiation induced cell kill and damage
Early Effects Late Effects (>90 days)
75. 3.8.18
1. CASARETT’S CLASSIFICATION OF
TISSUE RADIOSENSITIVITY
2. MICHALOWSKI’s H- AND F-TYPE
POPULATIONS
• H-Type (Hierarchial model):
Continuously divide, such as stem
cells and intestinal epithelium.
Respond acutely to radiation
• F – type (Flexible): Tissue not
divided into compartments
containing different cells. Late
responding tissue. E.g. liver,
thyroid cells
76. 3.8.18
Functional Subunits (FSU): discrete anatomically delineated structures whose
relationship to tissue function is clear, e.g. nephron of kidney, acinus of lung,
lobule of liver
FUNCTIONAL SUBUNITS
(b) Serial: Failure of only one FSU results in a loss
of function of the entire organ.
e.g. lungs, liver, myocardium
(a) Parallel : a critical number of functional
subunits must be damaged before a clinical
response (i.e. loss of function) becomes
manifest (threshold volume), although
structural damage may be diagnosed in
individual FSUs.
e.g. spinal cord, rectum, esophagus
77. 3.8.18
VOLUME EFFECT IN RADIOTHERAPY
Tolerance dose is the dose that produces an acceptable probability of treatment
complications.
TD 5/5: This defines the minimum tolerance dose, which is the complication rate
if less than 5% over 5 years
TD 50/5: This defines the maximum tolerance dose, which is the dose that yiels a
complication rate of 50% over 5 yars.
79. QUANTEC – QUANTITATIVE ANALYSIS OF
NORMAL TISSUE EFFECTS IN THE CLINIC
• Summary of the association between dosimetric parameters and normal tissue
outcomes
• Important with the development of sophisticated three dimensional treatment
planning system
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80. 3.8.18
ASSESSMENT OF LATE EFFECTS OF
NORMAL TISSUE IN THE CLINIC
• S: Subjective
• O: Objective
• M: Management Criteria
• A: Analytic Laboratory and Imaging criteria
Organ Volume
Segmented
Irradiation
Type
End point Dose (Gy) or
Dose/Volume
Parameters
Rate(%) Notes on
Dose/Volume
Parameters
Spinal cord Partial organ 3D-CRT Myelopathy Dmax 50 0.2 Including full
cord cross
section
Parotid Bilateral
whole gland
3D-CRT Long term
salivary function
reduced to <25%
of pre-RT level
Mean dose <25 <20% For combined
parotid glands
81. THERAPEUTIC INDEX
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Efficacy of radiotherapy treatment
is evaluated by the locoregional
TCP (Tumour Control Probability)
and NTCP (Normal Tissue
Complication Probability)
82. SUMMARY OF FACTORS INFLUENCING BOTH
Tumoral factors:
• Intrinsic radio sensitivity
• Location, size of tumour
• Cellular type of tumour
• Effect of oxygen
Treatment factors:
• Dose-time fractionation
• Radiation quality (LET)
• Technique (small field size)
• Modality (Brachytherapy, conformal RT)
• Combination of RT with
surgery/chemotherapy
Factors related to organ tissue:
• Tissue radio sensitivity
• Volume of organ tissue within
radiotherapy portal
• Organ type – series, parallel
Treatment Factors:
• Dose-time fractionation
• Radiation quality (LET)
• Technique (small field size)
• Modality (Brachytherapy, conformal RT)
• Combination of RT with
surgery/chemotherapy
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TCP NTCP
83. SUMMARY
• Radiation induced cell kill and damage are important for both tumour
and normal tissue.
• The principle is to have maximum tumour cell kill and minimum
normal tissue damage.
• The processes by why which this is achieved are dependant upon the
various factors enumerated.
• There are methods of modifying radiation induced cell kill and
prevention of normal tissue damage by various newer modalities of
radiotherapy.
• Hence it is of paramount importance to understand radiobiology and
the factors influencing cell kill and damage.
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