UMEÅ UNIVERSITY MEDICAL DISSERTATIONS
New series No 315 - ISSN 0346-6612
From the Department of Oncology, University of Umeå, Umeå, Sweden
BIOLOGICAL EFFECTS OF HIGH ENERGY RADIATION
AND
ULTRA HIGH DOSE RATES
Björn Zackrisson
University of Umeå
Umeå 1991
UMEÅ UNIVERSITY MEDICAL DISSERTATIONS
New series No 315 - ISSN 0346-6612
From the Department of Oncology, University of U m eå, Umeå, Sweden
BIOLOGICAL EFFECTS OF HIGH ENERGY RADIATION
AND
ULTRA HIGH DOSE RATES
AKADEMISK AVHANDLING
som med vederbörligt tillstånd av Rektorsämbetet vid
Umeå universitet för avläggande av medicine doktorsexamen
kommer att offentligt försvaras i Onkologiska klinikens föreläsnings­
sal, 244, 2 tr, torsdagen den 21 november 1991, kl 09.00
av
Björn Zackrisson
Umeå 1991
ABSTRACT
BIOLOGICAL EFFECTS OF HIGH ENERGY RADIATION AND ULTRA HIGH
DOSE RATES.
Björn Zackrisson, Department of Oncology, University of Umeå, S-901 85 Umeå,
Sweden.
Recently a powerful electron accelerator, 50 MeV race-track microtron, has been taken
into clinical use. This gives the opportunity to treat patients with higher x-ray and
electron energies than before. Furthermore, treatments can be performed were the
entire fractional dose can be delivered in parts of a second.
The relative biological effectiveness (RBE) of high energy photons (up to 50 MV) was
studied in vitro and in vivo. Oxygen enhancement ratio (OER) of 50 MV photons and
RBE of 50 MeV electrons were investigated in vitro. Single-fraction experiments, in
vitro, using V-79 Chinese hamster fibroblasts showed an RBE for 50 MV x-rays of
approximately 1.1 at surviving fraction 0.01, with reference to the response to 4 MV xrays. No significant difference in OER could be demonstrated. Fractionation
experiments were carried out to establish the RBE at the clinically relevant dose level, 2
Gy. The RBE calculated for the 2 Gy/fraction experiments was 1.17. The RBEs for 20
MV x-rays and 50 MeV electrons were equal to one. In order to investigate the validity
of these results, the jejunal crypt microcolony assay in mice was used to determine the
RBE of 50 MV x-rays. The RBE for 50 MV x-rays in this case was estimated to be 1.06
at crypt surviving fraction 0.1. Photonuclear processes are proposed as one possible
explanation to the higher RBE for 50 MV x-rays.
Several studies of biological response to ionizing radiation of high absorbed dose rates
have been performed, often with conflicting results. With the aim of investigating
whether a difference in effect between irradiation at high dose rates and at conventional
dose rates could be verified, pulsed 50 MeV electrons from a clinical accelerator were
used for experiments with ultra high dose rates (mean dose rate: 3.8 x 10^ Gy/s) in
comparison to conventional (mean dose rate: 9.6 x 10"^ Gy/s). V-79 cells were
irradiated in vitro under both oxic and anoxic conditions. No significant difference in
relative biological effectiveness (RBE) or oxygen enhancement ratio (OER) was
observed for ultra high dose rates compared to conventional dose rates.
A central issue in clinical radiobiological research is the prediction of responses to
different radiation qualities. The choice of cell survival and dose response model greatly
influences the results. In this context the relationship between theory and model is
emphasized. Generally, the interpretations of experimental data are dependent on the
model. Cell survival models are systematized with respect to their relations to
radiobiological theories of cell kill. The growing knowledge of biological, physical, and
chemical mechanisms is reflected in the formulation of new models. This study shows
that recent modelling has been more oriented towards the stochastic fluctuations
connected to radiation energy deposition. This implies that the traditional cell survival
models ought to be complemented by models of stochastic energy deposition processes
at the intracellular level.
Key words: Radiobiology, 50 MV x-rays, 50 MeV electrons, in vitro, in vivo, RBE, OER,
ultra high dose-rate, radiobiological models, model selection, models of stochastic
processes.
UMEÅ UNIVERSITY MEDICAL DISSERTATIONS
New series No 315 - ISSN 0346-6612
From the Department of Oncology, University of Umeå, Umeå, Sweden
BIOLOGICAL EFFECTS OF HIGH ENERGY RADIATION
AND
ULTRA HIGH DOSE RATES
Björn Zackrisson
University of Umeå
Umeå 1991
Copyright 1991 (C) Björn Zackrisson
ISBN 91-7174-614-5
Printed in Sweden by
the Printing Office of Umeå University
Umeå 1991
"The great tragedy of Science the slaying of a beautiful
hypothesis by an ugly fact".
T.H. Huxley 1883
ABSTRACT
BIOLOGICAL EFFECTS OF HIGH ENERGY RADIATION AND ULTRA HIGH
DOSE RATES.
Björn Zackrisson, Department of Oncology, University of Umeå, S-901 85 Umeå,
Sweden.
Recently a powerful electron accelerator, 50 MeV race-track microtron, has been taken
into clinical use. This gives the opportunity to treat patients with higher x-ray and
electron energies than before. Furthermore, treatments can be performed were the
entire fractional dose can be delivered in parts of a second.
The relative biological effectiveness (RBE) of high energy photons (up to 50 MV) was
studied in vitro and in vivo. Oxygen enhancement ratio (OER) of 50 MV photons and
RBE of 50 MeV electrons were investigated in vitro. Single-fraction experiments, in
vitro, using V-79 Chinese hamster fibroblasts showed an RBE for 50 MV x-rays of
approximately 1.1 at surviving fraction 0.01, with reference to the response to 4 MV xrays. No significant difference in OER could be demonstrated. Fractionation
experiments were carried out to establish the RBE at the clinically relevant dose level, 2
Gy. The RBE calculated for the 2 Gy/fraction experiments was 1.17. The RBEs for 20
MV x-rays and 50 MeV electrons were equal to one. In order to investigate the validity
of these results, the jejunal crypt microcolony assay in mice was used to determine the
RBE of 50 MV x-rays. The RBE for 50 MV x-rays in this case was estimated to be 1.06
at crypt surviving fraction 0.1. Photonuclear processes are proposed as one possible
explanation to the higher RBE for 50 MV x-rays.
Several studies of biological response to ionizing radiation of high absorbed dose rates
have been performed, often with conflicting results. With the aim of investigating
whether a difference in effect between irradiation at high dose rates and at conventional
dose rates could be verified, pulsed 50 MeV electrons from a clinical accelerator were
used for experiments with ultra high dose rates (mean dose rate: 3.8 x 10^ Gy/s) in
comparison to conventional (mean dose rate: 9.6 x 10‘2 Gy/s). V-79 cells were
irradiated in vitro under both oxic and anoxic conditions. No significant difference in
relative biological effectiveness (RBE) or oxygen enhancement ratio (OER) was
observed for ultra high dose rates compared to conventional dose rates.
A central issue in clinical radiobiological research is the prediction of responses to
different radiation qualities. The choice of cell survival and dose response model greatly
influences the results. In this context the relationship between theory and model is
emphasized. Generally, the interpretations of experimental data are dependent on the
model. Cell survival models are systematized with respect to their relations to
radiobiological theories of cell kill. The growing knowledge of biological, physical, and
chemical mechanisms is reflected in the formulation of new models. This study shows
that recent modelling has been more oriented towards the stochastic fluctuations
connected to radiation energy deposition. This implies that the traditional cell survival
models ought to be complemented by models of stochastic energy deposition processes
at the intracellular level.
Key words: Radiobiology, 50 MV x-rays, 50 MeV electrons, in vitro, in vivo, RBE, OER,
ultra high dose-rate, radiobiological models, model selection, models of stochastic
processes.
6
7
CONTENTS
ORIGINAL PAPERS
INTRODUCTION
Aspects on radiation energy
Ultra high dose rates
Radiobiological cell survival models
AIMS OF THE STUDY
MATERIALS AND METHODS
Cell cultures
Cell cloning assay
Animals
Jejunal crypt assay
Radiation qualities
Irradiation techniques
Dosimetry
Experimental design and statistical methods
Single-fraction experiments
Three-fractions experiments
Jejunal crypt assay
RESULTS AND COMMENTS
RBE of high energy x-rays and electrons
RBE and OER of pulsed high dose rate electrons
Radiobiological cell survival models
CONCLUSIONS
ACKNOWLEDGEMENTS
REFERENCES
9
11
12
14
15
16
17
17
17
17
17
18
18
19
22
22
22
24
25
25
29
31
33
34
35
8
9
ORIGINAL PAPERS
This thesis is based on the following papers, which will be referred to by their Roman
numerals.
I
Relative biological effectiveness and oxygen enhancement ratio of 50 MV x-rays.
Zackrisson B, Johansson B, Östbergh P. Acta Oncol 1989; 28: 529-35.
II
Relative biological effectiveness of high energy photons (up to 50 MV) and
electrons (50 MeV). Zackrisson B, Johansson B, Östbergh P. Radiat Res. In press
1991.
III
Relative biological effectiveness of 50-MV X rays on jejunal crypt survival, in
vivo. Zackrisson B, Karlsson M. Submitted.
IV
Biological response, in vitro, to pulsed high dose rate electrons from a clinical
accelerator. Zackrisson B, Nyström H, Östbergh P. Acta Oncol. In press 1991.
V
Radiobiological cell survival models - a methodological study. Zackrisson B.
Submitted.
10
11
INTRODUCTION
In radiation therapy, the development of new equipment and techniques has resulted in
an improvement of clinical results. Since megavoltage x-rays became available in clinical
radiotherapy, it has been possible to treat tumours with a high radiation dose without
exceeding the limits for normal tissue tolerance. Today, more tumours can be controlled
by radiotherapy than ever before because of an increased knowledge in radiotherapy,
radiobiology, radiation physics and related research areas. Technical achievements have
made it possible to use some of the new knowledge in clinical practice. Still, there are a
substantial number of tumours that cannot be controlled by radiation therapy or other
treatment modalities. Different approaches are needed to improve treatment results.
In radiotherapy, a major obstacle is that the delivery of higher doses, which can indeed
eradicate the tumour, cause damage to normal tissues in the target volume and result in
side effects that are not acceptable. A small increase in dose significantly increases the
probability of tumour control but the observed side effects increase proportionally. The
dose-effect relationships are often steep at these levels so that a relatively small
difference in dose can have large effects in terms of tumour control and side effects
(Brahme 1984, Nias 1990). The type and extent of side effects also depends on which
tissues are included in the target volume.
Consequently, one objective is to find a way to deliver a higher radiation dose to
tumours but not to normal tissues. Multiple field techniques or arc therapy provides a
means for "focusing" the radiation on the tumour in a way that reduces the dose to
tissues outside the target volume below the tolerance level. To achieve as low a dose as
possible to tissues outside deep sited target volumes, the highest available x-ray energies
are used, usually with accelerating potentials of 10-30 MV. In other tumour locations
multiple or single fields of x-rays of lower energies or high energy electrons (or
combinations) give dose distributions that are suitable. Other types of radiation such as
protons, ir'-mesons, a-particles, and heavy ions give dose distributions that may be of
great value in radiotherapy but are not available for routine clinical use. Presently,
electron accelerators with energies up to 50 MeV are being produced for radiation
therapy to provide electrons with energies up to 50 MeV and the corresponding x-ray
qualities.
Another important issue is to change the effectiveness of the radiation dose by
increasing the sensitivity of tumour cells relative to that of normal cells. This can, at
least theoretically, be done by means of, for example, chemical substances that either act
12
as sensitizers of tumour cells or protectors of normal cells, or by fractionation of the
radiation dose in a way that increases the therapeutic ratio between normal tissues and
tumour. It is also well established that hypoxic cells are less sensitive to radiation than
well oxygenized cells (Holthusen 1921, Read 1952). The dose to anoxic cells has to be
approximately three times as large as to oxic cells in order to give equal proportions of
surviving cells. This relationship is known as the oxygen enhancement ratio (OER)
(ICRU 1979). In normal tissues virtually all cells are well oxygenized, whereas in
tumours, hypoxic cells regularly have been demonstrated (Thomlinson and Gray 1955).
This implies that in a radiation therapy course, these cells are relatively resistant to
radiation, and may survive, causing a treatment failure. In fact, there are clinical data
that support this theory (Henk 1986). Different approaches to overcome the problem
with hypoxic cells have been used. One approach is to specifically sensitize hypoxic cells
by; different chemical compounds acting as a substitute for oxygen (Urtasun et al. 1975),
oxygen treatment of the patient (Churchill-Davidson et al. 1955), increasing the blood
flow to the tumour or combinations of these methods (Rojas 1991, Rojas et al. 1991).
Also drugs that have a toxic effect exclusively on hypoxic cells will belong to this
category (Adams and Stratford 1986). An alternative way to deal with hypoxia is to use
different ways of delivering the radiation dose. It is known that the OER decreases at
smaller doses per fraction (Littbrand 1970). This is the rationale for fractionation of the
radiation into a larger number of fractions with smaller doses per fraction during the
same overall treatment time used in conventional treatment (hyperfractionated
radiotherapy). Also, clinical data have shown improved treatment results with
hyperfractionation (Edsmyr et al. 1985). Delivering the radiation dose continuously over
a prolonged period of time as, for example, in many instances in interstitial or
intracavitary treatments with radioactive isotopes, may also decrease the OER (Hall and
Cavanagh 1967, Ling et al. 1985). The use of densely ionizing radiation e.g. neutron
radiation is yet another way to decrease the OER (Barendsen et al. 1966).
Aspects on radiation energy
The quality of x-ray beams is often described by the accelerating potential in units of
megavolt (MV). In a x-ray beam of 10 MV, there is a spectrum of energies where the
maximum photon energy is 10 MeV. The mean energy of the photons is often about 1/3
of the maximum energy. Regarding electron radiation, the spectrum of energies is often
very narrow and the maximum and mean energies are generally close. However, the
energy given by the manufacturer, often refers to the maximum energy. As for all
particle radiations the electron energy is given in its proper unit, a multiple of eV. Thus,
13
the mean energy of electrons generated by a 10 MV x-ray beam is considerably lower
than that of a 10 MeV electron beam.
The relative biological effectiveness (RBE) describes the effectiveness of a radiation
quality, compared to a reference radiation, in producing the same response (ICRU
1979). The RBE of sparsely ionizing radiation has been the objective of several studies
(Sinclair 1962, Hettinger et al. 1965, Wambersie et al. 1966, Kim et al. 1968, Dutreix et
al. 1969, Robinson and Ervin 1969, Huang et al. 1974, Williams and Hendry 1978,
Amols et al. 1986, Bistrovic et al. 1986). It is well known that x-rays with accelerating
potentials up to about 300 keV have a higher RBE than megavoltage x-rays and GOO)
gamma-rays. This is explained by the fact that kilovoltage x-rays, when interacting with
matter, have a larger proportion of more densely ionizing radiation of low energy.
Densely ionizing radiation is known to be more effective in cell killing per unit dose
than sparsely ionizing. The term linear energy transfer (LET) describes ionization
density. Photon and electron radiations are usually referred to as low-LET radiation
while e.g. neutron radiation is a high-LET quality.
The photoelectric absorption dominates the interaction processes for x-rays up to ~0.1
MV. Compton scattering dominates for x-rays from about 0.1 MV. Above 15-20 MV,
production of electron pairs becomes a significant ( - 10%) photon interaction process
(Hubbel 1969). In addition to this, photonuclear processes occur to a small extent for
high photon energies. The probability for photonuclear processes is largest for photon
energies between 20 and 25 MeV (NCRP 1984). The given examples are approximately
valid for water, however, the elemental composition of the irradiated material will
determine at which energy, and in which proportion, each interaction will take place.
Biological experiments have been carried out to study the RBE of megavoltage x-rays of
different accelerating potentials (Sinclair 1962, Robinson and Ervin 1969, Amols et al.
1986). Most commonly, x-ray qualities up to 30 MV have been studied but results from
up to 42 MV are available (Bistrovic et al. 1986). No significant differences in RBE are
reported from these studies.
The interactions of electrons of different energies with matter roughly consist of
ionization, excitation and loss of energy by setting delta rays in motion (ICRU 1984).
The probability for an electron to interact directly with a nucleus is about two orders
lower than for corresponding x-ray qualities (SCRAD 1966, Laughlin et al. 1979).
Several studies of RBE for different electron energies compared to megavoltage x-rays
have shown an RBE of unity (Sinclair 1962, Hettinger et al. 1965, Wambersie et al.
14
1966, Kim et al. 1968, Dutreix et al. 1969, Robinson and Ervin 1969, Huang et al. 1974,
Williams and Hendry 1978).
Ultra high dose rates
The mean absorbed dose rates used in clinical external beam therapy are often in the
range of 1.7 x 10"2 to 5.0 x 10"2 Gy/s (1-3 Gy/min). With the introduction of scanning
electron beams, it has become possible to treat patients with ultra high dose rates (i.e.
the entire radiation dose is delivered in parts of a second, which usually will mean > > 2
Gy/s (Hall 1972)).
The interest in ultra high dose rates increased dramatically when Dewey and Boag
(1959, 1960) showed that bacteria irradiated with ultra high dose rates under hypoxic
conditions, at high doses, exhibited the characteristics of being anoxic. This phenomenon
was due to oxygen consumption in the cells by the radiation. Investigations of this
phenomenon in mammalian cells failed to reveal a similar behaviour of the mammalian
cell survival curves (Todd et al. 1968, Nias et al. 1969 & 1970, Berry and Stedeford 1972,
Epp et al. 1972, Michaels et al. 1978).
The attention of the modified OER was due to the hypothetical possibility of making all
cells in a radiation target volume act anoxically, thus eliminating the difference in
response of hypoxic tumour cells compared to normal cells.
An inability of cells to repair sublethal damage after irradiation with ultra high dose
rates have been reported (Nias et al. 1973). In opposition to this, Schultz et al. (1978)
and Gerweck et al. (1979) found evidence for retained repair capacity.
Purdie et al. (1978, 1980) found that human kidney cells showed an increased sensitivity
to high dose rates under both oxic and anoxic conditions. In addition, there was a
reduction of OER. This was the case for both single pulses of electrons with dose rates
of 2.5 x 107 . 2.5 x 10^ Gy/s and gamma irradiation with 0.7 Gy/s. Furthermore, Ling et
al. (1984) found a decreased OER when comparing pulsed x-rays at mean dose rate 1.7 x
10'1 to 1.7 x 10'2 Gy/s. The dose rates in each 2 microsecond pulse were 300 and 150
Gy/s, respectively. In summary, despite many investigations in this research area,
seemingly diverging results have been reported.
Recently a powerful electron accelerator, 50 MeV race-track microtron, has been taken
into clinical use. This provides the opportunity to treat patients with higher x-ray and
15
electron energies than before. Furthermore, treatments can be performed where the
entire fractional radiation dose can be delivered in parts of a second (i.e. an extremely
high dose rate compared to conventional radiotherapy).
Radiobiological cell survival models
Radiobiological research usually precedes the start of clinical trials or the use of new
equipment. This is important since new hypotheses relevant for clinical research may be
formulated. In order to predict the effects, advantages, or hazards of a new treatment,
an important role is also played by experimental radiobiology. Different theories in
radiobiology, as in many other research fields, have resulted in different mathematical
expressions to predict dose-response relationships. These models are often used to
analyze experimental results. The ability to predict certain effects from these models
depends on the validity of the theories related to the models.
At low doses of radiation, the dose response relationships are difficult to establish
experimentally since the response is relatively small in comparison to the experimental
variations. Subsequently, the studied phenomenon or the experimental system used
make the inference from low doses (i.e. < 2 Gy) complicated. Therefore, the use of
models for cell survival are used in radiobiology to interpret experimental results. This
means that the results from levels of response, where the response to a small change in
dose is relatively large, can be used for inference from the low dose levels. However, the
conclusions drawn under such circumstances will be critically dependent on the validity
of the theory behind the model. A number of cell survival models and related theories
have been proposed. This prediction aspect has been paid special attention by some
investigators (Littbrand and Révész 1969, Goodhead 1983, Palcic et al. 1983), and
important deviations from the predicted- results have been shown. The dominating cell
survival models have been the single-hit multitarget model (Lea 1955), and the linearquadratic (LQ) model (Kellerer and Rossi 1972, Chadwick and Leenhouts 1973, 1981).
The growing knowledge of biological, physical and chemical mechanisms of cell killing
by radiation is reflected in the formulation of new models (Kappos and Pohlit 1972,
Tobias et al. 1980, Goodhead 1985, Curtis 1986). Recently, the modelling process has
been more aware of stochastic fluctuations of the radiation effects (Albright and Tobias
1985, Curtis 1988, Albright 1989, Sachs et al. 1990). An alternative approach for studying
the stochastic processes in radiation effects is to empirically study the cell kill at fixed
doses.
16
AIMS OF THE STUDY
The development, in recent years, of new technical equipment for radiation therapy,
includes the use of powerful electron accelerators. Before these accelerators are taken
into clinical practice the biological effects of the radiation beams produced by this type
of equipment need to be investigated. In the present thesis, the effects of high energy
and ultra high dose rates were investigated according to the following specific aims:
- To determine the RBE and OER of high energy x-rays (50 MV) in relation to well
known radiation qualities (I).
- To find plausible explanations to a difference in RBE (II).
- To investigate whether the difference in RBE can be detected at clinically relevant
dose levels (i.e. ~2 Gy/fraction) (II).
- To evaluate the validity of the results in vitro, in an in vivo experimental system (III).
- To investigate potential clinical benefits from using ultra high dose rates (IV).
- To study radiobiological survival models from a methodological point of view (V).
- To find a useful experimental method of determining RBE with limited model
dependence (II).
17
MATERIALS AND METHODS
Cell cultures (I,II,IV)
Fibroblasts from lung tissue of Chinese hamster (V-79-379-A) were used for the
experiments. Monolayers of exponentially growing cells were cultured on glass plates
(diameter 16 mm, thickness 0.1 mm) (I,IV) or plastic tissue culture discs (Lux Scientific,
diameter 11 mm, thickness 0.1 mm) (H) on the bottom of Petri dishes in Eagle’s MEM
supplemented with NaHCC^, L-glutamine, penicillin, streptomycin, amphotericin B and
fetal calf serum. The cells were incubated in an CC^-incubator at 37°C and a relative
humidity of 90% in an atmosphere containing 5% CC>2 in air. The plates were
transferred to the irradiation chamber, which is described in more detail below.
Cell cloning assay (I,II,IV)
The cells were trypsinized not more than 15 min after the irradiation, and then plated
for cloning assay. Seven days after irradiation, clones were stained in situ and those
comprising more than 50 cells were counted as survivors.
Animals (HI)
Male, pathogen free, CBA/J mice aged 8 weeks (range 7-9), were used. All mice were
obtained in the same shipping in order to minimize inter-individual variation.
Jejunal crypt assay Hill
After total body irradiation the mice were kept under standard conditions for 72 hours,
at which time they were sacrificed. The duodenum was identified and the following 10
cm of the jejunum was used for the study of microcolonies.
Neutral formalin was used for fixation of jejunum. Following fixation, each specimen
was divided into 6-10 pieces, which were then bound together in a bundle with surgical
tape (Potten and Hendry 1985). In this way the pieces from one individual mouse could
be handled easily during the embedding procedure, and the specimen could also be
oriented in such a way as to ensure true cross-sections were obtained. The specimens
were embedded in paraffin.
18
Histological sections (thickness approximately 5 mm) were prepared from the embedded
specimens. When several sections were made from each specimen, they were taken at
least 200 /xm apart. The sections were stained with haematoxylin and eosin. Surviving
microcolonies were counted in a light microscope. In order to be counted as a survivor,
the crypt should contain 10 or more cells with prominent nuclei and basophilic
cytoplasm. Cells of Paneth type were excluded (Withers and Elkind 1970, Potten and
Hendry 1985). Since the assessment of crypt viability is partly subjective, the slides were
coded in order to avoid bias.
Radiation qualities (I,II,m,IV)
20 and 50 MV x-rays and 50 MeV electrons were produced by a race-track microtron
(MM50, Scanditronix, Uppsala, Sweden). The 4 MV x-rays used for reference (I,II) were
produced by a linear accelerator (Clinac 4-800, Varian, USA). No flattening filters are
used in the beams of the race-track microtron since they are generated by scanning 20 or
50 MeV electrons on a target. Therefore, the dose distribution and energy spectrum are
constant over the radiation field. The race-track microtron was set to give a
homogeneous dose distribution to the cells during the scanning cycle, and to give a mean
dose rate, pulse dose rate, pulse repetition frequency, and pulse duration similar to the 4
MeV linear accelerator (I,II,HI). The radiation qualities were specified according to the
NACP-protocol (NACP 1980) and the IAEA-protocol (IAEA 1987) (I-IV).
In order to get a ultra high dose rate (IV), the 50 MeV electron beam was extracted
from the race-track microtron by switching off the first gantry bending magnet. Thus, the
beam did not pass the built-in system of scanning magnets, foils, collimation and dosemonitoring devices. Also, the pulse duration was set at its maximum (6 ms). T o increase
the homogeneity of the dose distribution at the position of the cells, a thin gold
scattering foil was positioned at the exit window.
Irradiation techniques (I,II,III,IV)
A specially designed irradiation chamber was used in the in vitro studies (I,II,IV). The
glass (I,IV) or plastic (H) plate on which the cells had been grown was attached to a
holder in the chamber. The chamber was then sealed and filled with cell growth medium
which thus surrounded the cell culture plate. The chamber was attached to a gas flow
system that allowed the medium to equilibrate with a desired mixture of gases. For the
experiments performed under anoxic conditions (I,IV), a mixture of 95% purified
nitrogen and 5% purified carbon dioxide was used (in total < 2 ppm oxygen as measured
19
with a Hagan oxygen meter). In the oxic experiments, a mixture of 95% air and 5%
carbon dioxide was used. When the chamber was attached to the gas-flow system the gas
went through the cell growth medium in order to increase the gas exchange. Therefore,
only serum-free medium was used since the serum protein would produce foam when
gas passed through the medium. The irradiation chamber and gas flow system were
manufactured from metal and glass since all plastic materials contain oxygen and may
compromise the anoxic environment. This is also the reason why cells were grown on
glass plates in some investigations (I,TV). The chamber was then put into a water
phantom. The water was heated to a temperature that kept the cell growth medium in
the chamber at a temperature of 37°C. The cells were irradiated after gas and
temperature equilibration (15 min). After irradiation, cells were removed and plated for
the cloning assay described above.
The mice were given total-body irradiation (HI). They were irradiated four at a time in a
perforated cylindrical Lucite cage. The cage was divided into six compartments by
Lucite walls. During the irradiations one mouse was kept in each of four compartments.
The mice were unanaesthetised during the experiments. All irradiations were performed
on the same day. The different doses and energies were delivered in random order.
In all cases (I,II,HI,TV) Lucite of appropriate thickness was positioned between the
source and the cells (I,n,IV) or the abdominal cavity (HI) in order to ensure that they
were positioned at the maximum dose for the radiation quality tested.
Dosimetry (I,II,IH,IV)
The absorbed doses were calculated using the NACP-protocol (NACP 1980) (I) or the
LAEA-protocol (IAEA 1987) (n,HI,TV). In the conventional dose rate experiments, the
mean absorbed dose in the cells was calculated from the measured mean dose to a
ferrous sulphate solution (Fricke and Hart 1966). The ferrous sulphate solution was
irradiated in cylindrical Lucite capsules in the same position as the culture plates.
Corrections were made for any dose gradient over the dosimeter. Measurements for
calibration of the dose monitor were made in the same way. Additional measurements
were performed with air ionization chambers to confirm the results of the FeSC^dosimetry.
An air ionization chamber was also used for control measurements during the cell
irradiation experiments with conventional dose rates (H, TV). This ionization chamber
was positioned behind the cell irradiation chamber in a fixed geometry.
20
In paper (I), the additional dose from backscatter from the glass, the cells were attached
to was considered. Since the amount of backscatter could be assumed to be dependent
on the radiation quality, paired experiments were performed with cells on glass plates
being irradiated in parallel to cells on plastic plates. The ratios of survival from each
experiment were used as response (Fig. 1). The dose modifying factor obtained in the
analysis of these experiments was then used for correcting the absorbed dose calculated
in the conventional way. The analysis showed that the dose modifying factor was
significantly different from unity for 4 MV, but not for 50 MV x-rays.
3.0-
2.0-
.5-
8
9
10
11
12
Absorbed dose / Gy
Figure 1. The ratio of surviving fraction as a function of dose for cells cultured on glass
plates and plastic plates irradiated with 4 MV x-rays. The steepness of the curve
represents the dose modifying factor (1.085 with 95% confidence interval ± 0.011) due
to backscatter from the glass. This includes an assumption of approximate log-linearity
of the survival curve in the dose range investigated. Each symbol represents the mean
value of 9 individual experiments. The error bars represent the 95% confidence
intervals.
In the high dose rate experiments, a coil (pulse transformer) was used for monitoring
the beam current (IV). The beam passes through the coil and a current is therefore
induced which is proportional to the dose rate. The total dose could be determined, by
integrating the pulse transformer signals from the radiation pulses delivered. The
calibration of the transformer was done by irradiation of ferrous sulphate solution. The
21
calibration of the transformer was done by irradiation of ferrous sulphate solution. The
results of ferrous sulphate dosimetry is linear beyond the doses per pulse («1.6 Gy) used
in this study (ICRU 1982).
The target volume in the animal experiment (ID) is the abdominal cavity of the mouse.
A cylindrical (Sinclair 1969) perspex phantom with dimensions according to those
measured on the mice was manufactured for the dosimetry. A calibrated air ionization
chamber was inserted in the phantom at the position of the abdominal cavity. The
dosimetry was done according to the LAEA-protocol (IAEA 1987) and verified at the
time of the animal experiments. A comparison between this dosimetry and Fricke
dosimetry was made for the two beam qualities. The radiation dose was delivered as a
single field from below. In order to achieve a homogeneous dose in the abdominal
cavity, specially designed build up and back scattering material was used. The thickness
was individually optimized to the different beam qualities so that the dose variation
within the target volume should vary less than 1 percent from the estimated average
dose value (Fig. 2).
100
50 MV
0
1
100
2
3
4
5
wh
Buildup material..Lucite
I
Abdominal wall
I I Abdominal cavity
20 MV
0
1
2 3 4 5
Depth / cm
Figure 2. Schematic description of the irradiation geometry. Depth dose curve, build-up
material, mouse anatomy and back scattering material. The air space surrounding the
mice in the experimental situation (not shown in the figure) does not significantly affect
the dose distribution.
22
Experimental design and statistical methods (I-IV)
Single-fraction experiments (I,II,IV)
The survival data were analyzed using the LQ-model (Chadwick and Leenhouts 1973).
In spite of its weak theory connection, it is widely used in radiobiological research for
evaluating data. This model is of the form
InS = - aD - ßE>2
(1)
where S is the surviving fraction, a and ß are constants and D is the absorbed dose. The
doses in the experiments were chosen partly because of the relatively small experimental
errors in this dose domain. The model was given an intercept by the unirradiated
controls. The parameters were estimated by the ordinary least squares method. The
model was then adjusted to pass through S = 1 when D = 0. The model parameters were
tested simultaneously by application of a dummy variable. The statistical F-test was
applied for tests of significance. To calculate the confidence intervals a normal
distribution of the experimental errors was assumed. The confidence intervals of RBE
were transformed from the confidence interval of the model parameters. For each
experiment a number of different dose levels and an unirradiated control were used.
Each experiment was repeated at least 7 times. For practical reasons only one
experiment could be performed each day.
Three-fractions experiments (II)
By using multiple fractions of radiation the possibility of detecting small differences in
response is increased. In this case, 3 fractions of 2.0 Gy were delivered to the cells. The
interval between each fraction was 4 h to allow for repair of sublethal damage. In each
experiment, one tissue culture plate was irradiated with 50 MV one with 20 MV, and
one plate remained unirradiated as a control. All three plates were drawn from the
same tissue culture vessel. The cells were treated in parallel throughout the experiment.
Due to the experimental design, a situation with paired observations in the statistical
analysis is required. The experimental series consists of 14 pairs.
In this experiment, a partial synchronization of cells in the cell cycle might occur.
However, it is shown at small doses per fraction (1.5 - 3 Gy) the effect of
synchronization is very small (McLarty 1976). Furthermore, since these experiments
compare two radiation qualities which are only moderately different, the possible
23
influence of redistribution is therefore assumed to affect the cells similarly in both arms
and thus be reduced due to the experimental design.
In fractionated-dose experiments, in vitro, where there is complete repair between each
fraction, the dose-response relationship will be approximately log-linear, provided the
number of fractions is small (< 5), and the fraction sizes are equal (McNally and de
Ronde 1976). In this case, the assumption of equal effect per fraction means that making
observations after one or two fractions will give information of limited practical value.
The surviving fractions are described by the equations
' lnS20MV = kl x D 1
(2)
-lnS50MV = k2 x D2
(3)
and
where S20MV an<^ S50MV rePresents surviving fraction at 20 and 50 MV x-rays
respectively,
and k2 are the regression coefficients.
and D2 are the total absorbed
doses. The intercepts are 0 since S = 1 when D = 0. By definition RBE = 'D^/T>2 wken
-lnS20MV = "^n^50MV an<^ consequently
RBE = 0 ^ 2 = k2/k 1
(4)
The statistical t-test for paired observations was applied for test of significance.
Experiments were also carried out with the purpose of verifying that complete repair of
sublethal damage was completed after 4 hours. V-79 cells were exposed to two fractions
of 60(2o radiation and incubated at 37°C for various time intervals between the two
doses. The cells were treated in exactly thè same way as described for the three fraction
experiments, except for being irradiated only twice. For practical reasons the experiment
could not be designed to allow analysis as paired observations. The results are shown in
Fig. 3. In the case where 2 Gy x 2 were delivered, a small increase in surviving fraction is
indicated after 0.5 h. However, no statistically significant differences between the
surviving fractions are present with intervals between fractions up to 8 h. In order to
investigate whether any inhibition of repair was present, a second series of experiments
was performed where 4 Gy x 2 were delivered to the cells. The results are shown in Fig.
3. In this case a statistically significant increase of the surviving fraction is present, as an
expression of repair of sublethal damage. The repair process is virtually complete after 2
h. Obviously, four hours seems to allow for complete repair of sublethal damage in this
system. Furthermore, the experiments with 2 Gy x 2 indicate that any effects from
24
redistribution of cells within the cell cycle will be of minor importance, in agreement
with the findings of McLarty (1976). The inability to demonstrate evidence of repair
after a fraction of 2 Gy is most likely due to the increase in surviving fraction being small
compared to the experimental errors.
0
0.8
I— 0.7
^
0.6
Î
o,
g
0.4
>
>
H
0.3
DC
z>
U)
0.2
0.1
-
0
0.5
1
3
4
5 .........
6...
TIME B E T W E E N F R A C T I O N S
7
£
(hours)
Figure 3. Surviving fraction as a function of time intervals between fractions for cells
irradiated with 2 Gy x 2 (□) and 4 Gy x 2 (■). Symbols represent the mean values of 9
individual experiments. Error bars represent the 95% confidence intervals.
Jejunal crypt assay (ID)
Five different dose levels were used in order to acquire a survival curve for each of the
two x-ray energies. Since small differences in survival between the two qualities were
expected, special attention was given to reducing inter-individual variations. All animals
were irradiated within 3 hours, in order to avoid the influence of diurnal variations in
radiosensitivity. At all dose levels, 8 mice were irradiated at each quality. 8 unirradiated
mice were used as a control in order to assess the surviving fraction.
25
In the dose domain investigated (9.5 - 15.5 Gy) the data were analyzed by a double loglinear model which is an approximation often advocated (Gilbert 1974, Moore et al.
1983, Potten and Hendry 1985, Thames and Hendry 1987). The model has the form
ln(-lnP) = ln N - D /D 0
(5)
P denotes the probability of the crypt being sterilized (i.e. 1 - crypt surviving fraction).
The expression -In P is considered to be proportional to the number of surviving
clonogenic cells per crypt. D is the absorbed dose, D q is the absorbed dose that is
required to decrease the number of clonogens per crypt to 0.37 of its previous value (i.e.
it describes the steepness of the curve), and N is the intercept, proportional to the initial
number of clonogens per crypt. The parameters were estimated by the ordinary least
squares method. The statistical t-test was applied for the tests of significance. To
calculate the confidence intervals of the parameters a normal distribution of the
experimental errors was assumed. The confidence intervals of the RBE were
transformed from the confidence intervals of the model parameters.
RESULTS AND COMMENTS
RBE of high energy x-rays and electrons (1-ffi)
The RBE for 50 MV x-rays was found to be significantly greater than unity, when using 4
MV x-rays as reference (I). The RBE at surviving fraction (S) 0.1 was estimated to 1.12
with 95% confidence interval (Cl) ±0.08 and 1.10 with 95% Cl ± 0.07 at S 0.01, for oxic
cells (I). The corresponding values for the anoxic cases were 1.11 ±0.09 at S 0.1 and 1.10
±0.06 at S 0.01. There is a tendency toward an increase in RBE at lower doses. This
increase is not statistically significant. The results suggest that a phenomenon related to
the radiation energy could be responsible for the higher RBE of 50 MV x-rays compared
to 4 MV x-rays. The difference between the LET-distributions for 4 and 50 MV are
moderate, if just the electron fluences in the irradiated phantom are considered.
However, the photonuclear reactions ( y,p; y ,n; y ,a; y,2n; y,np) fluences make a small
contribution to the absorbed dose. In tissue such reactions are mainly due to photon
interactions with oxygen, carbon and nitrogen. The threshold energies for the (y ,n)
reaction with these elements are between 10.6-18.7 MeV. The giant resonance peaks for
the interactions occur at photon energies between 20-25 MeV (Bülow and Forkman
1974, NCRP 1984). It should therefore be expected that such reactions are of minor
importance for x-ray qualities below 20 MV (i.e. having a mean photon energy much
26
lower than 20 MeV). For electrons, the dose contribution due to nuclear reactions is
very small (SCRAD 1966, Laughlin et al. 1979). In order to investigate whether
photonuclear reactions offer a plausible explanation to the higher RBE of 50 MV x-rays,
a new set of experiments were set up in which 50 and 20 MV x-rays and 50 MeV
electrons were used (H). 4 MV x-rays were chosen as reference quality. The results
confirm those of (I) for 50 MV x-rays. However, the results from 20 MV x-rays and 50
MeV electrons showed that the RBE for those qualities did not differ significantly from
unity (II). The estimates of RBE are given in Table 1.
Table I
Estimates of RBE for single fraction experiments, in vitro, with 4 MV x-rays as reference
(II). (The figures in brackets represent the 95 % Cl).
Quality
RBE at surviving
fraction 0.1
RBE at surviving
fraction 0.01
50 MV x-rays
20 MV x-rays
50 MeV electrons
1.14 (± 0.07)
0.99 (± 0.07)
1.03 (± 0.08)
1.12 (± 0.05)
1.00 (± 0.05)
1.02 (± 0.07)
These results support the hypothesis that a high-LET component is responsible for the
higher RBE of 50 MV x-rays. In this experiment there also is a tendency for an
increasing RBE at lower doses, which is consistent with a high-LET component.
The OER was 2.83 with 95 % Cl ± 0.02 for 4 MV x-rays at surviving fraction 0.01. The
corresponding result for 50 MV x-rays was 2.81 ± 0.03. This difference is not statistically
significant. A decreased OER is expected if high-LET is involved (Hall 1978). The
expected decrease in OER is, however, hard to predict quantitatively. There are
experimental data from some studies on OER for mixed low- and high-LET beams
(Railton et al. 1974). The fitted data of Railton et al. for different compositions of the
mixed gamma-ray and neutron beam can provide a crude estimate of the expected
decrease in OER in this case. Analyzing their data this way, predicts an approximate
decrease in OER from 2.72 to 2.66 for 1 % neutrons and to 2.42 for 5 % neutron
contribution. The predicted decrease in OER for 1 % neutron contribution is small and
of the same order as the estimate in the present study.
27
Several investigators have made both actual measurements, and theoretical calculations
based on available cross sections for photonuclear processes in different atoms, of this
high-LET component, i.e. the total dose arising from photonuclear reactions, of high
energy photon radiation (Horsley et al. 1953, Frost and Michel 1964, Allen and
Chaudhri 1982, 1988). Allen and Chaudhri (1982) calculated the dose contribution from
neutrons, protons and a-particles produced by photonuclear processes in tissue for 24
MV x-rays. The dose contribution of all these particles together was calculated to be
0.1% of the photon dose. In another later study (Allen and Chaudri 1988), the
photoneutron yield from tissue-equivalent material irradiated with 28 MV x-rays from a
betatron was measured. The authors found that the measured results were in good
agreement with the calculated results. The total dose contribution from all photonuclear
processes for 30 MV x-rays was calculated to be 0.22% of the photon dose. A fourfold
increase of neutron dose at 50 MV x-rays compared to 30 MV has been calculated
(Laughlin et al. 1979). This estimate of the size of the high-LET component means that
a dose contribution of «0.9% would be expected for the 50 MV x-rays in this
investigation. However, deviations from this value might follow from different energy
distributions of the x-rays from different accelerators.
Most authors conclude that in radiotherapy, no significant biological effect should be
expected from photonuclear processes. However, for tissues irradiated with 24 MV xrays, protons, neutrons and a-particles contribute 69%, 24% and 7%, respectively of the
additional dose (Biilow and Forkman 1974, Allen and Chaudhri 1982). Recoil nuclei
also contribute to the absorbed dose (Horsley et al. 1953). With present knowledge, it is
not possible to predict a common RBE for this mixture of particles. Consequently,
biological experiments are needed to investigate the effect of photonuclear processes.
It is also known that a synergistic effect' occurs when cells are irradiated sequentially
with a small dose of a high-LET radiation and a dose of x-rays (Durand and Olive 1976,
Hornsey et al. 1977, Ngo et al. 1977, Ngo et al. 1981, Bird et al. 1983, McNally et al.
1984, McNally et al. 1988). This synergism is reported to be even more pronounced
when the two radiation qualities are used simultaneously (Higgins et al. 1983).
For the race-track microtron the neutron contamination of the beam is very small
(Gudowska 1985) as flattening filters are not used. However, the dose contribution from
in-phantom photonuclear reactions might be of biological significance. In this
experimental situation (1,11), photonuclear reactions will be produced mainly in the
Lucite, in water or in the cells. The main elements for production of photonuclear
processes are oxygen and carbon. In soft tissues nitrogen may also contribute. The
28
photonuclear cross-sections for these elements show only small differences (NCRP
1984). Therefore, the situation in this experiment should simulate the situation in soft
tissues reasonably well (II). An aluminium chamber was used partly because of the
similarities between the cross sections for photonuclear reactions and in the relevant
elements (O, C, N). However, slightly more photonuclear reactions are to be expected in
aluminium, and the high-LET contribution might therefore be slightly larger than in
tissue. This is only true for the neutron component and not for the other components
since the range of these particles is too short to reach the cells in this system (II). From
the cross-section data (NCRP 1984) the increase in dose from photonuclear reactions in
aluminium compared to soft tissue will not exceed 20%.
In radiotherapy, the RBE at clinically relevant dose levels (i.e. doses in the order of 2
Gy/fraction) is of importance. For that reason, fractionated experiments were
performed with 2 Gy given three times to the cells with 4 hours intervals (II). The RBE
for 50 MV x-rays was estimated to 1.17 with 95% Cl ±0.11. The results obtained with
this method (II) will be independent from any cell survival model and give an estimate
of the RBE at 2 Gy. The RBE obtained with this method does have a higher value
(although, not statistically significant) than with high single doses, which is consistent
with the hypothesis of a high-LET component.
Since a higher RBE will have consequences for radiation therapy, it is important to
investigate the validity of the in vitro experiments by using a different biological system.
The jejunal crypt survival, in vivo, was studied in mice for this purpose (III). The
parameters of the models were significantly different, both with respect to D q and N
(p< 0.001 in both cases). The estimated RBE shows a higher RBE for 50 MV x-rays
compared to 20 MV. At a crypt surviving fraction of 0.5, the RBE is estimated to be 1.02
with 95% Cl ±0.14. For higher doses the RBE was significantly greater than unity. RBE
was 1.06 (±0.02) at a crypt surviving fraction of 0.1 and the corresponding result was
1.10 (±0.04) at a crypt surviving fraction of 0.01. The estimates of RBE in this
experiment have a tendency to increase as the dose increases, ranging from 1.02-1.10 for
the surviving fraction levels 0.5-0.01. However, when the confidence intervals are taken
into account, they do not differ significantly. In most cases, the RBE show a variation
with absorbed dose, often pronounced only at low doses. In this experiment, the
absorbed doses are considerably higher than in most cell survival studies. For this reason
it might be expected that the variation of RBE as a function of dose would be virtually
zero, and the variation observed in this case might only reflect experimental variation
and possible shortcomings of the survival model used. Since N is proportional to the
initial number of clonogens per crypt, according to the assumptions of the model, there
29
is no obvious reason for N to differ between the two treatments. Consequently, one
possibility of analyzing the data would be to force the two dose-response curves to the
same intercept. However, in this case there would be an obvious risk for introducing an
error in the analysis of the data.
The values of RBE are similar in both the in vitro, and in vivo studies. The point
estimates of the RBE in the in vivo study seem to be somewhat lower compared to in
vitro studies. The differences could be explained by stochastic variations. No conclusions
of the differences can be drawn from this material. However, there are some possible
explanations for the different RBE values that may be worthy of further discussion.
Firstly, a high-LET component could be responsible for the RBE. Different biological
systems may give different RBE values (Kellerer and Rossi 1972). Secondly, the
experimental irradiation system (i.e. the aluminium irradiation chambers) used for the
in vitro studies might have introduced a source of error, as mentioned earlier.
In conclusion, it is known that in a target with a composition comparable to human
tissue, high energy x-rays will produce a small high-LET contribution to the absorbed
dose. Also, low doses of high-LET radiation can have a fairly high RBE. Furthermore,
the synergism between high- and low-LET radiation will give an additional biological
effect. It seems reasonable that these effects will explain the RBE of 50 MV x-rays. In
clinical radiotherapy an RBE higher than unity should be taken into consideration since
even quite small deviations in dose-effect might, together with uncertainties in the
radiotherapy procedure, influence the outcome of the treatment (ICRU 1976). Both in
vitro and in vivo experiments show an increased RBE when the radiation effect of 50
MV x-rays is compared to 4 or 20 MV. The results strongly indicate that the higher RBE
should be considered in the clinical use of this high energy. A reduction of the total dose
of 5-10 % would be reasonable. However, it is not possible to deduce a fixed "correction
factor" from this material since the RBE may be different from one tissue to another.
Furthermore, a higher radiation dose-effect than conventionally used might be
acceptable if a decrease in energy imparted follows from careful shielding and
advantages from the depth dose distribution for the high photon energy.
RBE and P E R of pulsed high dose rate electrons (IV)
The results of Purdie et al. (1978, 1980) and Ling et al. (1984) indicate that ultra high
dose rates might give a different biological response, mainly due to a reduced OER.
Such effects could be of major interest in clinical radiotherapy since hypoxic cells can be
demonstrated in human tumours.
30
However, in the present investigation there was no statistically significant difference
between the response to high dose rates (mean dose rate: 3.8 x 102 Gy/s; pulse dose
rate 2.7 x 10^ Gy/s) and conventional (mean dose rate: 9.6 x 10'2 Gy/s; pulse dose rate
1.2 x 102 Gy/s), in either the oxic or anoxic cases. Subsequently, there was no significant
difference in RBE or OER. The estimates of RBE at survival level 0.01 were 1.03 with
95% Cl ±0.08 in the oxic state, and 1.02 (±0.07) for the anoxic state. OER at the same
level of survival were 2.78 (±0.24) for the high dose rate cases and 2.74 (±0.14) for the
conventional dose rate. The conventional dose rate in this investigation is higher than in
common clinical practice. The purpose was to minimize the influence of recovery from
sublethal damage during irradiation.
Although technical considerations prevented the use of single pulses, each pulse used
contained a dose of approximately 1.6 Gy which is close to the usual dose per fraction
given in clinical radiotherapy. Of course, the negative results of this study do not
preclude the existence of dose rate effects at even higher dose rates and doses per pulse.
If considerably larger doses per radiation pulse are needed in order to obtain a different
biological response, it is unlikely that this effect will be useful in clinical radiotherapy.
Some physico-chemical mechanisms for effects of ultra high dose rates:
- Oxygen is consumed by radiation (Dewey and Boag 1959, 1960, Epp et al. 1968). This
is not relevant in radiotherapy since doses in the kGy range would be needed in order to
obtain enough hypoxia to be detected in biological materials (Kiefer and Ebert 1970).
- At extremely high dose rates, a greater number of free radicals will be present
simultaneously in the irradiated volume. These free radicals may recombine in a way
that decreases the radiation effect. This will require pulse doses of a magnitude that is
not relevant in radiation therapy (Tallentire and Barber 1978).
These mechanisms are clearly beyond clinical application. Furthermore, biological
mechanisms are not likely to be differently affected by ultra high dose rates than by
conventional doses since the known repair processes in the cells will act during minutes
and hours, and not during the irradiation lasting for parts of a second. Some
investigators, however, have reported results that might seem to be in conflict with this
(Purdie et al. 1978, Ling et al. 1984). They observed a significant variation in cell
survival depending on dose rate. One explanation of this conflict is that the reference
dose rate used by Purdie et al. and Ling et al (3.5 x IO'2 Gy/s and 1.6 x IO'2 Gy/s
31
respectively) requires exposure times which are too long to eradicate the effect of repair
of sublethal damage during the irradiation. Also, Ling et al. (1985) show a variation of
OER with dose rate in the dose rate range 2 x 10"2 - 7.6 x 10'2 Gy/s which could offer
an explanation of their results.
In conclusion, the results of this study are in agreement with several earlier
investigations (Todd et al. 1968, Nias et al. 1969, Berry and Stedeford 1972, Epp et al.
1972, Michaels 1978). However, small differences in biological response could be
overlooked due to experimental variations. In this investigation, a difference in OER or
RBE in the order of 10% would have been required to find a statistically significant
effect of high dose rates. However, a decrease in OER of about 10% or less would be
too small to be of practical value in fractionated radiotherapy (Denekamp and Joiner
1982). From a clinical point of view, there seems to be few arguments for further
investigations on effects of ultra high dose rates.
Radiobiological cell survival models (V)
The study has established that in radiobiology, a number of models have been utilized,
and empirical studies have verified many effects of them. A given set of experimental
data has also shown a good fit to different models (Palcic et al. 1983). As a consequence,
questions concerning the methodology for model evaluation and clinical implementation
arise. The key question can be formulated; How does one select or develop an effective
quantitative model?
The issue is of great clinical relevance and concerns the prediction aspect from the high
dose domain ( >4 Gy) to the low (< 2 Gy). In the high dose domain, several models
have a good fit to observations, but the predictions of the low dose response may diverge
significantly. Portier and Hoel (1983) tried a low dose extrapolation from carcinogenesis
data and verified the expectation. Their argument to choose a multistage model was that
it was the only one which has any basis in the biological theory of carcinogenesis. The
authors also conclude: "The results of this research emphasizes the need for
incorporation of the biological theory in model selection". The conclusion is easy to
accept, but on the other hand, many theories can be associated with a specific model and
many models can be related to a single theory. Simplicity (e.g. in context of the number
of explaining mechanisms) is another argument for model choice, which further
complicates the issue. Otherwise, it is not possible to provide an unambiguous answer to
the question raised. Clinical experience, evaluation of experimental results, and new
research are natural elements which interact in a process to come closer to the truth. In
32
this view development of new quantitative models can be characterized as a permanent
search process closely related to the development of new theories.
The question can also be answered in connection with the research process. The
interaction between theories (explanations), models, and observations can be regarded
as the driving force in the research process. That view has been demonstrated through
this study. Many models have been tried, some of them have been long-lived, and
dominated (e.g. the LQ approach) the growth of knowledge. Obviously, the propensity
of model changes is not of the same order as the propensity of theory changes. That
implies, in the long run, a risk of conservatism which makes the interpretations of
experimental studies inefficient and perhaps also invalid. One of the major reasons
appears to be the inability to establish an effective methodology for model evaluation to
sort out deficient models and with which users can be guided to the model best suited
for their purposes.
The experimental research in radiobiology is dependent on the measuring capability,
and thus directed to a high dose domain. Palcic et al. (1983) demand experimental data
from all the dose domains. Without measurements in the low dose domain, a model is
not valid. They go very far and conclude that statements of low dose effects are not
meaningful. This describes the ideal situation of model use, but never possible to satisfy
in research. Prediction is always a part of the research process to create new hypotheses
and hence new knowledge, at least from a popperian point of view (Popper 1959). In
this context, models and theories are considered as preliminary. Model approaches will
always be replaced. On the contrary, the research and the clinical experience in
radiobiology have shown that such predictions are not only meaningful, but also
valuable instruments in the development of new knowledge.
In conclusion: What is expected in future is a closer relationship between theories of
cellular mechanisms and the model choice and development. An application of
stochastic models and the theory of stochastic processes seem to be very fruitful and will
extend the ability to improve new theories. This development will provide a new
direction for radiobiological cell survival model formulation in direction to an
infinitesimal approach, even if the phenomenological one will still be a useful
simplification of very complicated systems. The approach, however, demands a strict
problem and theory formulation to be effective.
33
CONCLUSIONS
- The RBE of 50 MV x-rays is significantly greater than unity compared to 4 MV xrays. No statistically significant difference in OER could be detected.
- Photonuclear processes at high photon energies seem to offer a plausible explanation
for the higher RBE of 50 MV x-rays.
- The higher RBE of 50 MV x-rays is present at clinically relevant doses i.e. 2
Gy/fraction.
- Similar results for 50 MV x-rays, are obtained from jejunal crypt assay, in vivo as from
in vitro experiments.
- Ultra high dose rates do not have a different biological effect with respect to RBE or
OER, when compared to a conventional dose rate.
- A closer relationship between description of cellular mechanisms and the model
development is required for more effective analyses of experimental results.
- RBE at doses around 2 Gy can be determined, in vitro, with a reasonable sensitivity
by paired experiments with fractionated radiation.
34
ACKNOWLEDGEMENTS
I wish to express my sincere gratitude to:
Professor Bo Littbrand, Head of the Department of Oncology, my tutor, for introducing
me to the field of radiobiology, for constructive criticism, and valuable advice during the
investigation.
Professor Hans Svensson, Head of the Department of Radiation Physics and Head of
Dosimetry Section IAEA, for taking so much interest in this study, and for generously
sharing his vast knowledge.
Bengt Johansson, Peter Östbergh, Håkan Nyström, Mikael Karlsson, for stimulating
collaboration, and for co-authorship.
Margaretha Bäckström, Kjell Brännström, Cenneth Forsmark, Tor Granqvist, Rolf
Hahlin, Annika Holmberg, Yvonne Jonsson, Stellan Sandström, for expert technical
assistance in the experimental work.
Karin Gladh, Anna Wernblom, for excellent typing, editing and helping in
administrative problems.
Rut Jonsson, Parviz Mothlag, Allan Sandström, Helena Tano, for excellent stand-in
performances of technical assistance.
Christina, Hanna and Emil, for being there.
This study was supported by grants from the Swedish Cancer Society and Lion’s
Research Foundation, University of Umeå.
35
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