Radiotherapy- Radio-biology basis, cell survival curves

1. CELL SURVIVAL
CURVE Dr Savio James
DNB resident
Radiation Oncology
MCS Patna

2. What is cell
survival curve
? What is
survival?
◦ A cell survival curve describes the relationship between the
radiation dose and the proportion of cells that survive.
◦ For differentiated cells that do not proliferate, such as nerve,
muscle, or secretory cells, death is loss of specific function
◦ For proliferating cells, such as stem cells in the hematopoietic
system or the intestinal epithelium, loss of the capacity for
sustained proliferation is cell death- ( reproductive death )
◦ So all those cells who maintained the clonogenic potential is said to
be survived
◦ For a tumor to be eradicated, it is only necessary that cells be
“killed” in the sense that they are rendered unable to divide and
cause further growth and spread of the malignancy.

3. EFFECT OF RADIATION ON CELL
1. DIRECT ACTION:
◦ Radiation interacts directly with critical targets in cell
◦ Atom of target itself may be ionized leading to the chain of
physical and chemical events ,which may eventually produce
biological damage
◦ Dominant process in the interaction of high LET particles with
biologic material
◦ Cannot be modified by sensitizers or protectors

4. EFFECT OF RADIATION
ON CELL
2. INDIRECT ACTION :
◦ Radiation interacts with other molecules (
mainly water ) within the cells to produce free
radicals , which through diffusion in the cells
damage critical organ within cell
◦ About 2/3 of the biological damage by low LET
radiation (sparsely ionizing radiation ) is due to
indirect action
◦ Indirect action can be modified by chemical
sensitizer or radiation protectors

5. MODES OF
CELL
DEATH
1. MITOTIC
2. APOPTOTIC
◦ Mitotic death is dominant mode following
irradiation

6. MITOTIC
DEATH
◦ Cells die while attempting to divide because of damaged
chromosomes.
◦ Death may occur in the first or a subsequent division following
irradiation.
◦ Asymmetric exchange-type aberrations (rings, dicentrics &
anaphase bridge) are the principle mechanisms for radiation-
induced mitotic death in mammalian cells.
◦ These aberrations require two chromosome breaks

7. APOPTOTIC
DEATH
◦ First described by Kerr and colleagues
◦ In Greek means “falling off,” like petals from flowers or
leaves from trees
◦ Programmed cell death, with a stereotyped sequence of
morphologic events
◦ Can also be induced in some normal tissues and in some
tumors by radiation.
◦ Highly cell-type dependent. Hemopoietic and lymphoid
cells are particularly prone.
◦ gene-controlled : p53 is proapoptotic; bcl-2 is
suppressor

8. How survival curves are plotted
Tissue from tumor
or normal
proliferating tissue
Chopped into
small pieces
Prepared into a
single cell
suspension
(trypsin used )
Cultured at body
temperature in
complex medium
Such grown
cultured cells form
- Established cell
lines
They are used for
all radiobiological
studies in vitro

9. In vitro
culture
& Plating
efficiency
Cells from actively dividing stock culture are prepared into
suspension
Number of cells in suspension counted by hemocytometer
Seeded into dish ( eg: 100 )
Incubate for 1-2 weeks
Colony will be formed from each cells ( not all if 100
seeded 50-90 colonies)
Plating efficiency indicates the percentage of cells seeded
that grow into colonies

10. Surviving
fraction
These culture dish if exposed to a dose of 8Gy of X
ray and then incubated , following changes will be seen .
Some seeded single cell still may be single
Some cells may show nuclear deterioration as they die of
apoptosis
Some cells may form tiny abortive colonies ( one or two
mitosis )
Some cells have grown into large colonies – they are
considered to have survived

11. What is cell
survival curve
?
◦ A cell survival curve is a graphical representation of
fraction of cells surviving after giving a dose
of radiation.
◦ The graph is obtained by plotting the dose along the
linear X axis and the surviving fraction along the
logarithmic Y axis

12. Shape of Cell Survival curve
◦ For sparsely ionizing (low LET) radiations such as x-
rays :
◦ At low dose, survival curve starts out straight on the
log-linear plot (i.e) the surviving fraction is an
exponential function of dose
◦ At higher doses, the curve bends
◦ At very high doses, the survival curve often tends to
straighten again.
◦ For densely ionizing (high-LET) radiations, such as
alpha particles or neutrons
◦ the cell survival curve is a straight line from the origin
: that is, survival is an exponential function of dose.
Dose plotted on a linear scale and
surviving fraction on a logarithmic
scale

13. Shape of Cell Survival curve
◦ Shoulder Region
◦ Shows accumulation of SUB-LETHAL DAMAGE.
◦ The larger the shoulder region, the more dose will initially be needed to kill the same
proportion of cells, so less radiosensitive
◦ Shape of cell survival curve tells us the radiosensitivity, repair and recovering ability of
the cell. The shape of the curve gets altered if the conditions are changed.

14. MODELS OF
DESCRIPTION OF THE
CELL SURVIVAL
CURVE

15. Historical
context

16. The most important R
of Radiobiology -Repair
◦ Single strand breaks are usually considered repairable
◦ Double strand base are usually not repairable if the
breaks are close together ( complex or clustered
DSBs- 15 to 20 base pair damage within about 1
helical turn of DNA – they are more lethal than
normal DSBs )
◦ At low does both DNA stands are unlikely to be hit –
so SSBs common at low does
◦ At high doses DSBs will be more common , so repair
will be low.
◦ Consequently if we say if there are low repair – the
survival curve will be steeper- that is more cell death.

17. Survival Curves for
Normal vs Cancer cells
◦ Cancer cells already have a damaged (mutated)
DNA repair mechanisms so even at low doses
the repair in cancer cells are lower than normal
cells.
◦ So for cancer cells the curve will be straighter
◦ There is a window of opportunity at low doses
where the survival of normal tissue exceeds that
of cancer.

18. ◦ So from this we have understood the we have a windows of
opportunity at around 2Gy per fraction ,
◦ But this was not found initially by radiobiological experiments
but was found by clinical practice in radiotherapy and were
later explained by cell survival curve.
◦ Assuming dose to normal tissue and cancer cells same .

19. Mechanistic
models of cell
survival
◦ We needed a mathematical model that describes the effect of
radiotherapy on tumor cells and normal tissues.
◦ The most used two models are
1. Linear quadratic model
◦ Dual radiation action
◦ First component proportional to dose
◦ Second proportional to dose squared
2. Multi target model
◦ Based on probability of hitting the target
◦ Widely used for many years still has merit
The initial model that was proposed was single target model

20. Single Target Model
◦ According to Single hit model single hit on a
single target within the cell leads to cell death.
◦ This generates an exponential cell survival
curve which appears as a straight line on a semi-
logarithmic scale.
◦ This model is useful for highly sensitive human
tissues, if high LET radiation is used.
◦ For mammalian cells in general, their response
to radiation is more usually described by
‘shouldered’ survival curves.

21. Multi-target
single-hit
◦ To model shouldered type of response, a more general version of
target theory can be used called multi-target single-hit
inactivation
◦ In this extended target idea, the cell has ‘n’ sensitive targets rather
than just a single target, and just one hit by radiation on each and
every one of those n targets is required for death of the cell
◦ An obvious shortcoming of the basic multi-target model is that, it
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
◦ To take this into account two component model were proposed

22. Multi- target model
◦ Multi target model was widely used for many years.
It still have merit
◦ The initial slope D1 - singe event cell killing
◦ Final slope D0- multiple event cell killing
 A dose of radiation that introduces an average of
one lethal event per cell leaves 37% still viable is
called D0 The smaller the dose, D0 the greater
the radio sensitivity.
◦ Cell killing follows exponential relationship. A
dose which reduces cell survival to 50 % will if
repeated reduce survival to 25% and similarly to
12.5% on third exposure .
◦ So surviving fraction never become zero.
◦ The width of shoulder - Extrapolation number 'n'
or Quasithreshould dose Dq

23. Linear
Quadratic
model
◦ Linear Component :
◦ A double strand breaks caused by the passage of single charged particle
(densely ionizing radiation High LET) Eg: alpha particle , protons
, neutrons, heavy ions
◦ Quadratic component:
◦ Two separate single strand break caused by two different charged
particles ( sparsely ionizing low LET)
◦ Eg : X rays , Gama rays
◦ The equation for LQ model is based on Poisson statistics which deal
with rare event, since this probability that any specific DNA molecule
will be damaged is low.

24. Single
particle
events
◦ For single particle events , P is a linear function of dose , D
◦ So the mean number of lethal events per DNA molecule can be
expressed as αD and Po represents the probability that there are no
single particle lethal events , ie it is the surviving fraction of cells , (
S)
◦ For a single particle to damage both arms of the DNA at the same
time it has to be densely ionizing radiation.
◦ Hence single particle events are caused primarily by the high LET
component of the radiation.
◦ For photon and electron beams , it’s the very low energy secondary
ionizing radiation that are high LET ( slow electrons )
◦ And hence give rise to α events .

25. Two particle
events
◦ With two particle events the probability that one arm of the DNA
molecule will be damaged is a linear function of dose , D.
◦ The probability of damage in the adjacent arm is also a linear function
of dose , D.
◦ Hence the probability that both arms will be damaged by two different
single particle events will be a function of D2
◦ So surviving fraction of cells is given by

26. L-Q model
Equation
◦ Hence when we consider together both events
◦ Where α represents the probability of lethal single-particle ( α -
type ) damage and β represents the probability that independent
two particle (β) - type ) events have combined to produce lethal
damage.
◦ The problem with above equation is it have some many variables ,
so it was difficult to use in clinical set-up.
◦ So by dividing the survival fraction by α to give the biological
effective dose(BED) equation.

27. α/β
◦ The most common assumptions are for tumors and
acute reactions α/β = 10 Gy
◦ For late reacting normal tissue α/β = 2-3 Gy
◦ When the dose become equal to α/β, the linear and
quadratic contribution to cell killing will be equal.
◦ The response of tissues or organs to radiation varies
markedly , depending on two factors - Inherent
sensitivity of individual cells and Kinetics of the
population.
◦ With regards to response time two types of tissue are
known -
◦ Early responding ( skin, mucosa, intestinal epithelium)
and Late responding ( spinal cord )
◦ For early effect the α/β is large : for late effects it is
small.
◦ For early effects α dominates at low doses
◦ For late effects β has the influence at doses lower than
for early responding tissues.

28. LIMITATIONS OF
L-Q MODEL:
◦ No provision for the influence of cell cycle,
proliferative or microenvironmental effects in the
overall dose, response and relationship.
◦ No way to account for differences in repair rates
b/w different tissues
◦ Uncertainty surrounding the model's applicability
for extremes of fractionation
◦ Limited understanding of how to apply the model in
patients receiving multimodality therapy

29. ◦ The LQ model has
taken over as the
model of choice to
describe survival
curves .

30. Factors
affecting cell
survival curve
Mechanism of cell death
LET
Fractionation
Dose rate effect
Intrinsic radio sensitivity
Oxygen
Cell cycle

31. Mechanism
of cell death
◦ Mitotic death results (principally) from exchange-
type chromosomal aberration.
- survival is linear and quadratic function of dose
- Survival curve is log-linear plot with broad shoulder
- Characterized by dose-rate effect
◦ Apoptotic death result from programmed death.
- straight line on log-linear plot.
- Characterized by exponential function of dose.
- Survival curve is straight & shoulderless
- little or no dose-rate effect.

32. LET
◦ Low- LET radiations :
Low dose region
Shoulder region appears
High dose region
Survival curve becomes linear and surviving fraction is
an exponential function of dose
Surviving fraction is a dual exponential
◦ High LET radiations
Survival curve is linear
Surviving fraction is a pure exponential function of dose

33. ◦ So repair decreases as LET increases cell survival
curve becomes more straight
◦ The OER decrease as LET increases – so hypoxic
tumors can be better treated with high LET
radiation
◦ The cell cycle effect decreases as LET increases- so
for treatment of cancers that have cells trapped in a
resistant cell phase of the cell cycle – high LET
radiation can be used.

34. Fractionation
◦ If the dose is delivered as fractions with
sufficient time , repair of sublethal damage
occurs
◦ Elkind & Sutton showed that when two
exposure were given few hours apart the
shoulder reappeared .
◦ If the dose is delivered as
equal fractions with sufficient time between for
repair of the sub-lethal (non-killing)damage,
the shoulder of the survival curve is
repeated many times.
◦ Dose required to produce the same reduction
in surviving fraction increases.

35. Dose- rate effect
Dose rate determines biological impact
◦ Reduction in dose rate causes reduced
cell killing ,due to repair of SLD.
◦ Reduction in dose rate generally reduces
survival-curve slope (DO increases).

36. Intrinsic radio
sensitivity
Mammalian cells are significantly more radio-
sensitive than microorganisms:
Due to differences in DNA content
Represents bigger target for radiation damage
Sterilizing radiation dose for bacteria is 20,000 Gy
Cells of human origin, both normal and malignant
has also been studied
Normal cells in general show a narrow range of
radio sensitivities if many hundred people are
studied.
But human tumor cells show very broad range of
D0 values.

37. The Oxygen
effect
◦ Oxygen modifies the biological effect of ionizing radiation .
◦ Oxygen is a powerful radiation sensitizer , so tumors that are
hypoxic tend to be resistant.
◦ The degree of sensitization is expressed in terms of oxygen
enhancement ratio - OER
◦ Hypoxic tumors can reoxygenate during course of treatment and
become more sensitive.
◦ Oxygen reacts with broken end of DNS molecule to make the
damage permanent that’s how it enhances radiation
effect (oxygen fixation )

38. ◦ OER is a function of dose and dose rate- at low
doses (& low dose rate )
◦ OER tend to be lower at low doses than OER at
high doses( & dose rates)
◦ Reason is Oxygen reacts with broken DNA in β-
type damage – but at low doses α- type damage
dominates , so the effect of O2 sensitization is
reduced .
◦ Reduced effect of O2 means lower OER.
◦ Modification of the L-Q model to account for
oxygen effect and reoxygenation have been
published but are not widely used.

39. Cell Cycle effect
◦ Cell cycle effect relates to Redistribution.
◦ Cells are most sensitive at or close to
mitosis
◦ Survival curves for cells in the M phase are
linear , indicating the absence of any repair
◦ Cells in late G2 are usually sensitive ,
perhaps as sensitive as cells in M phase
◦ Resistance is usually greater in the later part
of S phase .

40. THANK
YOU