This document discusses several key concepts in radiobiology including:
1. The interaction of radiation with cells is probabilistic, with damage occurring through direct and indirect action. Indirect action involves free radicals produced by radiation interacting with water molecules within cells.
2. Different phases of the cell cycle have differing radiosensitivities, with G2/M being most sensitive. Fractionated radiation can exploit this through redistribution effects.
3. The linear quadratic model describes cell survival curves and accounts for both single-hit and double-hit damage from radiation. It is used to calculate biologically equivalent doses.
4. Mechanisms like reoxygenation between fractions can improve the therapeutic ratio by making tumor cells
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Radiobiology
1. IMPROVING
THERAPEUTIC RATIO
RADIOBIOLOGICAL
BACKGROUND
DR ARNAB BOSE
Dept. of Radiotherapy
NRS Medical College, Kolkata
2. Introduction
Radiobiology is a branch of science concerned with the
action of ionizing radiation on biological tissues and living
organisms
Objective of this presentation -
To understand why ionising radiation can be used to
treat malignant cells
To know the type of radiation that does this best
To identify factors of significance to the success of this
process
3. Introduction
The interaction of radiation with a cell is a matter of
chance [probability]. If an interaction occurs, the
damage may not be expressed, in fact damage is more
frequently repaired
The initial deposition of energy occurs very quickly
The radiation is deposited in the cell randomly
Expression of damage occurs after a latent period,
ranging from hours to years or even generations
The DNA is the sensitive target in the cell
4. Cell Cycle
The cell proliferation cycle is defined by two well defined
time periods: Mitosis (M), where division takes place &
the period of DNA synthesis (S).
The S and M portions of the cell cycle are separated by
two periods (gaps) G1 and G2 when, respectively, DNA
has not yet been synthesized or has been synthesized
but other metabolic processes are taking place.
The time between successive divisions (mitoses) is
called the cell cycle time.
5.
6. Cell Cycle
In general, the G2/M phases are the most radiosensitive
and late S phase is most radioresistant.
Transition through the cell cycle is governed by cyclins
and cyclin-dependent kinases (cdk).
List of important checkpoints: G1 →S governed by p53,
Rb, Cyclin D1/Cdk4/6, and Cyclin E/Cdk2 S governed by
Cyclin A/Cdk2 G2 →M governed by Cyclin B/A/Cdk1
For a typical mammalian cell, a single fraction of
radiation (1–2 Gy) results in >1,000 base damage, 1,000
SSB, and 40 DSBs. DSBs are the most relevant in terms
of cell-killing
7. Cell Death
Cell death of non-proliferating (static) cells is defined as
the loss of specific function, while for stem cells and
other cells capable of many divisions it is defined as the
loss of reproductive integrity (reproductive death).
A surviving cell that maintains its reproductive integrity
and proliferates almost indefinitely is said to be
clonogenic.
When cells are exposed to ionizing radiation the
standard physical effects between radiation and the
atoms or molecules of the cells occur first and the
possible biological damage to cell functions follows later.
8. Classification of Radiations in
Radiobiology
For use in radiobiology and radiation protection the
physical quantity that is useful for defining the quality of
an ionizing radiation beam is the linear energy transfer
(LET).
The ICRU defines the LET as follows:
“LET of charged particles in a medium is the quotient 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.”
Unit usually used for the LET is keV/µm.
9. Typical LET values for commonly used radiations are:
250 kVp X rays: 2 keV/µm. Cobalt-60 gamma rays: 0.3
keV/µm. 3 MeV X rays: 0.3 keV/µm. 1 MeV electrons:
0.25 keV/µm.
LET values for other, less commonly used radiations are:
14 MeV neutrons: 12 keV/µm. Heavy charged particles:
100–200 keV/µm. 1 keV electrons: 12.3 keV/µm. 10 keV
electrons: 2.3 keV/µm.
X rays and gamma rays are considered low LET
(sparsely ionizing) radiations, while energetic neutrons,
protons and heavy charged particles are high LET
(densely ionizing) radiations. The demarcation value
between low and high LET is at about 10 keV/µm.
10. Cell Damage by Radiation
The biological effects of radiation result mainly from
damage to the DNA, which is the most critical target
within the cell
When directly ionizing radiation is absorbed in biological
material, the damage to the cell may occur in one of
two ways:
4. Direct
5. Indirect.
11. Direct Action
In direct action the radiation interacts directly with the
critical target in the cell. The atoms of the target itself
may be ionized or excited through Coulomb interactions,
leading to the chain of physical and chemical events that
eventually produce the biological damage.
Direct action is the dominant process in the interaction
of high LET particles with biological material.
12. Indirect Action
In indirect action the radiation interacts with other
molecules and atoms (mainly water, since about 80% of
a cell is composed of water) within the cell to produce
free radicals, which can, through diffusion in the cell,
damage the critical target within the cell.
In interactions of radiation with water, short lived yet
extremely reactive free radicals such as H2O+ (water ion)
and OH• (hydroxyl radical) are produced.
The free radicals in turn can cause damage to the target
within the cell.
13. A free radical is a molecule or atom, which is not
combined to anything (free) and carries an unpaired
electron in its outer shell. It is in a state associated with a
high degree of chemical reactivity.
If the water molecule is ionised
H2O = H2O+ + e-
(H2O is the water molecule ; H2O+ is an ion radical )
Ion meaning it is electrically charged, because it has lost
an electron and a radical because it has an unpaired
electron in the outer shell, making it very reactive.
Ion radicals have a short life, usually no more than
10-10 s, before they decay to form free radicals
14. Free radicals are not charged, but do have an unpaired
electron in the outer shell.
The water ion radical can, for example, do the following:
H2O+ + H2O = H3O+ + OH*
(H2O+, H3O+ are the ion radicals H2O is a water molecule)
OH* is a highly reactive hydroxyl radical, with 9
electrons, therefore one is unpaired.
Hydroxyl radicals (OH*), are highly reactive and can go
on to react with DNA. It is estimated that 2/3 of the x-ray
damage to mammalian DNA is by hydroxyl radicals
15.
16. Types of DNA Damage
DNA damage to the cell can come in several forms:
1. Base damage/single-strand breaks (SSBs) – repaired
via base excision repair, not a major contributor to
radiosensitivity.
2. Double-strand breaks (DSBs) – repaired via
homologous recombination repair (in late S/G2, a DNA
template is available) which is accurate, or non
homologous end-joining which is error-prone. DSBs are
a major contributor to radiosensitivity; ~40 DSBs are
required to kill cell.
3.Chromosome aberrations – result from unrepaired or
misrepaired DSBs. Symmetric chromosome damage
(e.g., translocations) tends to be nonlethal, whereas
asymmetric damage (e.g., rings) tends to be lethal due
to the loss of large amounts of DNA.
21. Cell Survival Curve
A cell survival curve describes the relationship between
the surviving fraction of cells (i.e. the fraction of
irradiated cells that maintain their reproductive integrity
(clonogenic cells)) and the absorbed dose.
Cell survival as a function of radiation dose is graphically
represented by plotting the surviving fraction on a
logarithmic scale on the ordinate against dose on a
linear scale on the abscissa.
22. The type of radiation influences the shape of the cell
survival curve.
Densely ionizing radiations exhibit a cell survival curve
that is almost an exponential function of dose, shown by
an almost straight line on the log–linear plot.
For sparsely ionizing radiation, however, the curves
show an initial slope followed by a shoulder region and
then become nearly straight at higher doses.
23. Linear Quadratic Model
During the 1980s the linear-quadratic model has gained
wide acceptance as a mathematical description of
biological response to irradiation. The dose range where
the LQ model is well supported by data is roughly 1–5Gy
per fraction. Extrapolations made outside this range
should be done with extreme caution
It is mainly used for the calculation of treatment
parameters of schedules supposed to be isoeffective.
The simplest adequate mathematical description of
these data is provided by a linear-quadratic function:
24. There is a hypothesis considering two types of radiation
damage
The first type of damage, responsible for the linear
component, is assumed to result from a single event.
This damage is lethal for the cell if it is not or
insufficiently repaired. The probability to produce such a
damage is proportional to dose, while its
probability to be repaired insufficiently is assumed to be
dose independent within the range of clinically relevant
doses.
25. The second type of damage, responsible for the
quadratic component, is by itself not lethal for the cell. It
is a so-called sublethal damage.
Only the combination of two such lesions can yield a
lethal event for the cell. The probability to produce a
single sublethal damage is again proportional to dose.
The probability to produce two of such lesions is
proportional to the square of dose, i.e. Again
the probability of insufficient repair is assumed to be
dose independent within the range of clinically relevant
doses.
26. Typically, survival curves are continuously bending, with
a slope that steepens as the dose increases. The ratio α/
β gives the relative importance of the linear dose term
and the quadratic dose term for those cells, and controls
the shape of the survival curve. When α/β is large, the
linear term predominates, so a plot of log (SF) against d
is relatively straight, while if α/β is small, the quadratic
term is more important, giving a plot with greater
curvature. For cells whose survival curves have a lower
α/β ratio, doubling the dose leads to more than doubling
of the effect on log (SF). Such cells will be particularly
sensitive to changes in fraction size when radiation is
given as fractionated schedule.
27. The earlier multitarget single
hit model described the slope
of the survival curve by D0
(the dose to reduce survival to
37% of its value at any point
on the final near exponential
portion of the curve) and the
extrapolation number n (the
point of intersection of the
slope on the log survival axis).
Dq was the quasi-threshold
dose. However, this model
does not have any current
biological basis.
28. The linear quadratic model
assuming that there are two
components to cell kill by
radiation
where
S(D) is the fraction of cells
surviving a dose D; alpha is a
constant describing the initial
slope of the cell survival curve;
beta is a smaller constant
describing the quadratic
component of cell killing.
The ratio gives the dose
at which the linear and
quadratic components of cell
killing are equal (8 Gy in the
example shown)
29. High α/β [straighter curve], characteristic of cell with little
repair capability e.g. tumour cells [from 5 - 20 Gy]
Low α/β [more curved], characteristic of high repair
potential e.g. late responding normal tissue [1-4 Gy]
This difference in cell survival curves provides rationale
for fractionated radiation therapy treatment and explains
therapy treatment and explains radiobiological
advantage
The biological equivalent dose (BED) refers to the
effective total absorbed dose (in Gy) for a given
fractionation scheme if it were given by standard
fractionation (1.8–2.0 Gy/day).
BED = nd[1+d/(a(alpha)b(beta))], where n = number of
fractions and d = the dose per fraction.
30.
31. In the past few decades great efforts have been made
to apply radiobiological concepts to design safer and
more effective therapeutic strategies
Withers (1975) suggested four basic mechanisms that
contribute to the diverse reactions of different tissues to
irradiation:
Re distribution of cells in the cell cycle
Re oxygenation of hypoxic cells in the tumor
Repair of cellular radiation damage
Re population of surviving cells during radiotherapy
treatment
32. Re distribution
The radio sensitivity of cells varies considerably when
they transit through the cell cycle
Radiation-induced partial synchrony is a consequence
from selective killing of cells in a sensitive phase of the
cell cycle as well as by progression delay in late G2-
phase
Cells surviving irradiation are preferentially those which
were in relatively resistant phases during fractionated
radiotherapy
33.
34. Re distribution
Redistribution of surviving cells within the mitotic cycle
results in self-sensitization of proliferating cell
populations
This process, however, only affects cells that divide
frequently during the 4 to 8 weeks commonly taken to
administer a course of curative radiotherapy, but there is
little or no such an effect in slowly or non-proliferating
tissues
Assuming a proliferating tumor surrounded by non-
proliferating normal tissue, small doses per fraction and
time intervals sufficient for redistribution, should result in
an improved therapeutic differential
35. Re oxygenation
Hypoxic cells are about 2.5 to 3.0 times more resistant to
X-irradiation than euoxic cells
In tumors, hypoxic cells arise because of imbalances
between the rate of production of new cells and the
vascularization of the tumor
Cells are well oxygenated to a distance of about 100 mm
from a capillary. At greater distances partial oxygen
pressure is so low that cells die and later become
necrotic. At intermediate distances, the oxygen
concentration is high enough to keep cells viable but at
the same time low enough to increase their resistance to
X-rays
These chronically hypoxic cells might limit radio curability
of the tumor
36. Oxygen “fixes” the free radical damage to DNA caused
by X-rays. For this effect to be observed, oxygen must
be present in the target at the time of irradiation or
microseconds afterwards. Generally, at least 2% oxygen
concentration results in maximum radiosensitization.
In addition to rendering cells more radioresistant, both
chronic and acute hypoxia also contribute to malignant
and metastatic progression.
37. Re oxygenation
Irradiation preferentially sterilizes cells that are
adequately oxygenated. If a mixed population is
irradiated, a biphasic dose response curve results which
is steep at low doses but shallower at higher doses due
to preferential survival of the more resistant hypoxic cells
Between fractions hypoxic cells may be re oxygenated
which increases radio curability of the tumor
There had been many attempts to overcome hypoxia by
specific radio sensitizers, by improving oxygenation
pharmacologically or by irradiation under hyperbaric
oxygen pressure or by breathing carbogen
38.
39. Oxygen Enhancement Ratio
The ratio of doses without and with oxygen (hypoxic versus well
oxygenated cells) to produce the same biological effect is called
the oxygen enhancement ratio (OER).
OER = Dose to produce a given effect without oxygen
Dose to produce the same effect with oxygen
The OER for X rays and electrons is about three at high doses
and falls to about two for doses of 1–2 Gy.
The OER decreases as the LET increases and approaches
OER = 1 at about LET = 150 keV/mm,
41. Relative Biological Effectiveness
The relative biological effectiveness (RBE) compares the
dose of test radiation to the dose of standard radiation to
produce the same biological effect. The standard
radiation has been taken as 250 kVp X rays for historical
reasons, but is now recommended to be 60Co g rays.
RBE = Dose from standard radiation to produce a given biological effect
Dose from test radiation to produce the same biological effect
The RBE varies not only with the type of radiation but
also with the type of cell or tissue, biologic effect under
investigation, dose, dose rate and fractionation.
In general, the RBE increases with the LET to reach a
maximum RBE of 3–8 (depending on the level of cell kill)
at LET ª 200 keV/m and then decreases because of
energy overkill
43. Repair
The influence of repair of molecular injury on cell survival
and the response of tissue to irradiation can be inferred
from in vitro survival curves and from changes in the
total dose required to produce a certain level of injury as
a function of changes in dose per fraction, i.e. from
isoeffect curves
Fractionation responses can be modeled in terms of two
types of radiation-induced cellular injury,
one resulting in a logarithmic decline in target cell
survival that is linear with dose , and
another in which the decline increases proportionally to
the square of the dose
44. Repair
The linear component is assumed to reflect cell kill from
a single molecular event, while the quadratic component
might be due to two independent so-called sub lethal
events that have to interact to become lethal for the cell
Sub lethal events may be repaired with half-times in the
order of 20 minutes to some hours
If a dose is split into two fractions with a time interval of
several hours then a substantial portion of sub lethal
damage induced by the first fraction is already repaired
when the second fraction is given
45. Repair
Thus the likelihood for interaction of two sub lethal
damages is diminished, resulting in less cell kill due to
the quadratic component , as compared to the same
dose given in a single session
Thus not only total dose but also the number of
fractions or the dose per fraction, respectively,
determine the magnitude of the radiation effect.
46. If radiation dose is
delivered in a series of
equal fractions (F),
separated by a time
interval that allows
complete SLD repair, the
effective dose survival
curve becomes an
exponential function of
dose Shoulder of the
survival curve is repeated
many times; the effective
survival curve is a straight
line from the origin through
point on the single-dose
survival curve
corresponding to the daily
dose (F)
D0 (the reciprocal of the
slope), has a value close
to 3 Gy for human cells
47. In mammalian cells 3 types of radiation damage
described :
Lethal damage
Sub lethal damage
Potentially lethal damage
Lethal Damage - Irreversible and irreparable
Leads to cell death
Potentially Lethal Damage - Component of radiation
damage that can be modified by post
irradiation environmental conditions
48. Sub lethal Damage -
Under normal circumstances can be repaired in hours
usually considered to be complete within 24 h
If additional sub lethal damage added within this time
then can interact to form lethal damage
Sub lethal damage repair observed as an increase in
survival if a dose of radiation is split into 2 equal
fractions separated by a time interval fractions
49. If dose is split into 2
fractions separated by a
time interval more cells
survive than for the
same total dose given
in a single fraction,
because the shoulder of
the curve must be
repeated each time.
50. As time interval between 2 F
increases see rapid increase
in SF, usually complete within
2 h in culture but longer in
vivo, particularly for some
late responding tissues
As time interval increases
may see dip in SF due to
movement of surviving cells
through the cell cycle; only
observed in cycling cells
If time interval exceeds the
cell cycle, see increase in SF
due to proliferation
51. Conventional fractionation is explained as follows:
division of dose into multiple fractions spares normal
tissues through repair of sublethal damage between
dose fractions and repopulation of cells. The former is
greater for late reacting tissues and the latter for early
reacting tissues.
Concurrently, fractionation increases tumour damage
through reoxygenation and redistribution of tumour cells.
A balance is achieved between the response of tumour
and early and late reacting normal tissues, so that small
doses per fraction spare late reactions preferentially, and
a reasonable schedule duration allows regeneration of
early reacting tissues and tumour reoxygenation to likely
occur.
52. The current standard fractionation is based on five daily
treatments per week and a total treatment time of
several weeks. This regimen reflects the practical
aspects of dose delivery to a patient, successful
outcome of patient treatments and convenience to the
staff delivering the treatment.
Conventional fractionation consists of daily fractions of
1.8 to 2.0 Gy, 5 days per week; the total dose is
determined by the tumor being treated and the tolerance
of critical normal tissues in the target volume (usually 60
to 75 Gy).
53. Hyperfractionation uses an increased total dose, with the
size of dose per fraction significantly reduced and the
number of fractions increased; overall time is relatively
unchanged
In accelerated fractionation, overall time is significantly
reduced; the number of fractions, total dose, and size of
dose per fraction are unchanged or somewhat reduced,
depending on the overall time reduction
Accelerated hyperfractionation has features of both
hyperfractionation and accelerated fractionation.
Hypofractionation uses decreased number of fractions
with increased fraction size
54. Concomitant boost is an additional dose delivered 1 or
more times per week to selected target volumes (i.e.,
gross tumor volume) through smaller field(s), along with
the conventional dose to larger irradiated volumes.
To achieve an increase in tolerance of late-responding
tissues through dose fractionation, the time interval
between the dose fractions must be long enough (6
hours) to allow cellular repair to approach completion.
55.
56. Dose-rate effect refers to repair of SLD that occurs
during long radiation exposure. Smaller doses per
fraction lead to a repeat of the shoulder on the survival
curve. Continuous low-date irradiation (such as I-125
seeds) would be considered an infinite number of
infinitely small fractions leading to a survival curve with
no shoulder and far shallower compared to acute
exposures.
The inverse-dose effect occurs when decreasing dose
rate actually increases cell killing. This is because higher
dose rates (HDRs) would cause arrest in radioresistant
phases of the cell cycle
57. Together with the total dose and fractionation
schedule, target volume is a major variable in
radiotherapy. For a given fractionation regimen,
higher doses can usually be given when volumes at
the same site are small rather than large
Volume is also an important determinant of normal
tissue response to a given dose, first because larger
volumes provide less opportunity for tissues to draw
on their ‘functional reserve’ and second because
larger irradiated volumes make it more likely that a
critical volume element will exceed some upper
dose limit
58. Re population
Early reacting tissues like skin and mucosa counteract
cell depletion by repopulation, usually after a delay that
depends on the degree of denudation. Cells in late
reacting tissues proliferate very slowly if at all.
Prolongation of treatment time might spare acute normal
tissue damage but not late reactions
Proliferation of surviving tumor cells during treatment is
one of the main factors that determine the outcome of
fractionated radiotherapy
An increase in the number of viable tumor cells between
fractions or during treatment interruptions is assumed to
result in a failure to control the tumor. Irradiation
treatment should be performed in as short a time as
possible
59.
60. Radiation kills cells randomly, which means that each
tumour cell has the same probability of surviving
irradiation, that probability depending on the given dose.
SF2 is the probability of any cell surviving a single dose
of 2 Gy, the most commonly used fraction size.
Generally, after F fractions, the final survival probability
will be (SF2)F.