Int. J. Radiation Oncology Biol. Phys., Vol. 73, No. 5, pp. 1545–1551, 2009
Copyright Ó 2009 Elsevier Inc.
Printed in the USA. All rights reserved
0360-3016/09/$–see front matter
doi:10.1016/j.ijrobp.2008.12.021
BIOLOGY CONTRIBUTION
COMPARISON OF BIOLOGICAL EFFECTIVENESS OF CARBON-ION BEAMS IN
JAPAN AND GERMANY
AKIKO UZAWA, M.SC.,* KOICHI ANDO, D.M.SC.,y SACHIKO KOIKE, M.SC.,* YOSHIYA FURUSAWA, PH.D.,*
YOSHITAKA MATSUMOTO, PH.D.,* NOBUHIKO TAKAI, PH.D.,* RYOICHI HIRAYAMA, PH.D.,*
MASAHIKO WATANABE, M.SC.,* MICHAEL SCHOLZ, PH.D.,z THILO ELSÄSSER, PH.D.,z
x
AND PETER PESCHKE, PH.D.
* Heavy-Ion Radiobiology Research Group and y Particle Therapy Research Group, Research Center of Charged Particle Therapy,
National Institute of Radiological Sciences, Chiba, Japan; z Department of Biophysics, Gesellschaft für Schwerionenforschung,
Darmstadt, Germany; and x Department of Radiation Oncology, Deutsches Krebsforschungszentrum, Heidelberg, Germany
Purpose: To compare the biological effectiveness of 290 MeV/amu carbon-ion beams in Chiba, Japan and in Darmstadt, Germany, given that different methods for beam delivery are used for each.
Methods and Materials: Murine small intestine and human salivary gland tumor (HSG) cells exponentially growing in vitro were irradiated with 6-cm width of spread-out Bragg peaks (SOBPs) adjusted to achieve nearly identical beam depth–dose profiles at the Heavy-Ion Medical Accelerator in Chiba, and the SchwerIonen Synchrotron
in Darmstadt. Cell kill efficiencies of carbon ions were measured by colony formation for HSG cells and jejunum
crypts survival in mice. Cobalt-60 g rays were used as the reference radiation. Isoeffective doses at given survivals
were used for relative biological effectiveness (RBE) calculations and interinstitutional comparisons.
Results: Isoeffective D10 doses (mean ± standard deviation) of HSG cells ranged from 2.37 ± 0.14 Gy to 3.47 ± 0.19
Gy for Chiba and from 2.31 ± 0.11 Gy to 3.66 ± 0.17 Gy for Darmstadt. Isoeffective D10 doses of gut crypts after
single doses ranged from 8.25 ± 0.17 Gy to 10.32 ± 0.14 Gy for Chiba and from 8.27 ± 0.10 Gy to 10.27 ± 0.27 Gy for
Darmstadt, whereas isoeffective D30 doses after three fractionated doses were 9.89 ± 0.17 Gy through 13.70 ± 0.54
Gy and 10.14 ± 0.20 Gy through 13.30 ± 0.41 Gy for Chiba and Darmstadt, respectively. Overall difference of RBE
between the two facilities was 0–5% or 3–7% for gut crypt survival or HSG cell kill, respectively.
Conclusion: The carbon-ion beams at the National Institute of Radiological Sciences in Chiba, Japan and the
Gesellschaft für Schwerionenforschung in Darmstadt, Germany are biologically identical after single and daily
fractionated irradiation. Ó 2009 Elsevier Inc.
Carbon-ion beams, LET, SOBP, Crypt survivals, Fractionated dose.
and have attracted worldwide attention. The therapeutic effectiveness of carbon-ion beams depends on factors including physics and biology. Different from photon beams,
carbon-ion beams are biologically heterogeneous along the
beam path, owing to the change of radiation quality or linear
energy transfer (LET). This heterogeneity is most prominent
at spread-out Bragg peak (SOBP) that targets deep-seated
tumors in the patient body. Because the SOBP of Chiba/HIMAC therapy is provided by use of a scatterer (3), projectile
particles should be more fragmented within the SOBP than in
that of the GSI/SIS, which is made by changing beam energy
and does not use any scattering foils (4). This means that the
INTRODUCTION
A Phase I/II clinical study on carbon-ion radiotherapy started
at the National Institute of Radiological Sciences (NIRS),
Chiba, Japan in 1994. A total of nearly 3000 patients have
been treated with the HIMAC (Heavy-Ion Medical Accelerator in Chiba) synchrotron by 2006 and have been analyzed
for toxicity and local tumor response (1). Clinical studies of
carbon-ion radiotherapy using the SchwerIonen Synchrotron
(SIS) also started at Gesellschaft für Schwerionenforschung
(GSI), Darmstadt, Germany in 1997, and great effort has
been given to treating chordoma of the skull base (2). Clinical
outcomes of these two institutions have been well recognized
Reprint requests to: Koichi Ando, D.D.S., Ph.D., D.M.Sc., Particle Therapy Research Group, National Institute of Radiological Sciences, Anagawa 4-9-1, Inage-ku, Chiba-shi, Chiba 263-8555,
Japan. Tel: (+81) (0) 43-206-3231; Fax: (+81) (0) 43-206-4149;
E-mail: [email protected]
Supported in part by the Special Coordination Funds for Research
Project with Heavy Ions at the National Institute of Radiological
Sciences–Heavy-Ion Medical Accelerator in Chiba.
Conflict of interest: none.
Acknowledgments—The authors thank Prof. Gerhard Kraft,
Dr. Sylvia Ritter, Dr. Suo Sakata, and Dr. Ryonfa Lee for their
help in conducting experiments; and Dr. Naruhiro Matsufuji for statistical analysis.
Received April 10, 2008, and in revised form Dec 9, 2008.
Accepted for publication Dec 9, 2008.
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Dose rate (relative to the peak)
1.4
P
M
Volume 73, Number 5, 2009
D
DE
1.2
1.0
0.8
0.6
0.4
p
GSI/SIS
0.2
0.0
-100
m
d
NIRS/HIMAC
-80
-60
-40
-20
0
20
40
Depth in water (mm : from the center of SOBP)
Fig. 1. Depth–dose distribution of carbon-ion beams with a 6-cm
spread-out Bragg peak (SOBP). Arrows indicate three positions of
gut irradiated with carbon ions. Human salivary gland tumor cells
were irradiated at position P (2.5 cm upstream), D (2.5 cm downstream), or DE (2.8 cm downstream) of middle position (M). Mouse
gut was irradiated at position p (2.0 cm upstream) or d (2.0 cm
downstream) of middle position m. GSI/SIS = Gesellschaft für
Schwerionenforschung/SchwerIonen Synchrotron; NIRS/HIMAC
= National Institute of Radiological Sciences/Heavy-Ion Medical
Accelerator in Chiba.
GSI beams may be more effective than the NIRS beams. It is
therefore important to compare biological effectiveness of
carbon-ion beams between NIRS/HIMAC and GSI/SIS.
We have compared between the two beams using human tumor cell kill in vitro and mouse gut crypt survivals in vivo,
and we report that the two beams are biologically identical
after single as well as daily fractionated irradiation.
METHODS AND MATERIALS
Tumor cells and animals
The animals involved in these studies were procured, maintained,
and used in accordance with the Recommendations for Handling of
Laboratory Animals for Biomedical Research, compiled by the
Committee on the Safety and Handling Regulations for Laboratory
Animal Experiments, National Institute of Radiological Sciences,
Japan. Animal experiments performed at Gesellschaft für Schwerionenforschung, Darmstadt have been reviewed and approved by
a governmental animal protection committee (reference number:
V54-19c20/15-Da17/04). Keeping, care, and handling of experimental animals were carried out in accordance with the guidelines
for laboratory animals committed by the German government.
Human salivary gland tumor (HSG) cells of human salivary gland
origin (5) were cultured in Ham’s F12 medium (Sigma-Aldrich Japan, Tokyo, Japan) supplemented with 10% fetal bovine serum
(JRH Biosciences, Lenexa, KS), 100 U/mL penicillin, and 100 mg/
mL streptomycin, under humidified conditions with 5% CO2 in
a cell culture incubator at 37 C. Cells in logarithmic phase were replated into T25 cell culture flasks (catalog no. 353012; BD Falcon,
Tokyo, Japan) and exponentially grown 2 days before irradiation.
C3H/He female mice 10–12 weeks old were used. Mice were produced in specific pathogen-free facilities at NIRS and moved 2 to 3
days before start of irradiation to a conventional environment at either NIRS or GSI. A mixture of anesthetics (ketamine at 100 mg/mL
Fig. 2. Mouse setting for irradiation. Mice under anesthesia were
placed in a jig made of Lucite. The jig had two plates, each 4 mm
thick, separated by 20 mm, and mice were fixed in between the
plates. Whole mice were exposed to beams in an abdominal-to-dorsal direction.
4 mL and xyladine at 20 mg/mL 1 mL) in 24 mL of 0.95% NaCl
solution was used at 0.1 mL/10 g body weight and was intraperitoneally injected 20 min before start of irradiation. The use of anesthesia met German regulations for animal experiments. Mice were kept
in a Lucite jig especially designed for gut irradiation, and they received horizontal beams. Mice were killed 3.5 days after either single-dose irradiation or the second dose of a 3-day fractionated
scheme. Either 3 or 4 mice were used for each dose, and experiments
were at least replicated.
Biological comparison of carbon-ion beams d A. UZAWA et al.
1547
Fig. 3. Method used to determine depth of jejunum in mouse abdomen. Carbon-ion beams (290 MeV/u) with 6-cm spreadout Bragg peak were used. (A) Physical dose fall-off. (B) Crypt survivals after irradiation with the fall-off. Symbol and bar
are mean and standard deviation, respectively.
Irradiation
Carbon ions were accelerated to 290 MeV/u by HIMAC and SIS
and spread out to 6-cm width. The SOBP profile for the SIS used
here was adjusted to be the same as the SOBP profile being used
for therapy at Chiba (3) and different from that being used for therapy at Darmstadt (6). Figure 1 shows the physical depth–dose distribution of carbon-ion beams used in the present study. Irradiation
was made at either three or four positions within the 6-cm SOBP
for mice or cells, respectively. For cell irradiation, a position labeled
P (proximal; approximately 40 keV/mm) was 25 mm upstream of the
center M (middle; approximately 50 keV/mm) of 6-cm SOBP,
whereas positions D (distal; approximately 90 keV/mm) and DE
(distal end; approximately 120 keV/mm) were 25 mm and 28 mm
downstream of the center M, respectively. Mouse jejunum was
irradiated at three positions: the center (m [middle]; approximately
50 keV/mm), 20 mm upstream (p [proximal]; approximately 42
keV/mm) of the center, or 20 mm downstream (d [distal]; approximately 74 keV/mm) of the center. Positions M and m are identical.
The LET values cited above are calculated by the one-dimensional
heavy-ion transport code HIBRAC (7) that has been used for the
current treatment planning of HIMAC beams.
Dose rates for HIMAC and SIS beams depended on irradiation
position and were approximately 3 Gy/min and 1 Gy/min, respec-
tively. Mice received either single-dose irradiation or three fractionated doses with a fixed interval of 24 h for 3 days. Reference beams
used were cobalt-60 g rays.
Endpoint
Cell monolayers (80% confluent) were irradiated, rinsed twice
with phosphate-buffered saline, once with 0.2% trypsin, and incubated for 14 days when colonies were fixed and stained for counting.
We measured cell survival with the colony formation assay. Colonies consisting of more than 50 cells were scored as survivors.
Data were fitted to a linear-quadratic equation. A dose required to
produce 10% survivors was calculated and defined as D10. We performed at least three independent experiments for each survival
curve. Survival curves for g rays were also obtained after experiments in triplicate at Chiba.
Crypt survivals were histologically measured. Jejunum was
removed and fixed in formalin 3.5 days after irradiation. Histology
preparations were made, and hematoxylin and eosin staining was
used to count microscopically the number of crypts surviving and
regenerating. For nonirradiated control, the number of crypts per
circumference section was between 120 and 145. Experiments
were repeated two or three times for each synchrotron facility.
Fig. 4. Histology of jejunum transverse sections obtained after irradiation with spread-out Bragg peak (SOBP) fall-off. (A)
Unirradiated jejunum; (B, C) jejunum irradiated with the SOBP at binary filter thickness of 118–120 mm. B shows only two
to three crypts surviving, whereas crypts in C are abundant at the upper hemisphere but null at the lower hemisphere. This
indicates the sharp drop-off of the beam within the jejunum.
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Fig. 5. Human salivary gland tumor cell survivals after irradiation with carbon ions. (A–D) Data are obtained by irradiating
cells at positions shown in Fig. 1 as proximal (P), middle (M), distal (D), or distal-end (DE), respectively. Symbol and bar
are mean and standard deviation, respectively. GSI = Gesellschaft für Schwerionenforschung; NIRS = National Institute of
Radiological Sciences.
The isoeffective doses at D10 for single doses and D30 for fractionated doses were obtained from the crypt survival curves, representing doses required to reduce survivals to 10 and 30 crypts,
respectively. Isoeffective doses were used to compare biological
effectiveness between the two facilities.
Depth of jejunum from abdominal skin surface was measured by
a range absorber method. Specifically, mice were placed in a Lucite
jig and received horizontal beams. Carbon ions pass through a 4mm-thick Lucite plate and abdominal wall before reaching jejunum
(Fig. 2). First, physical doses of the carbon 6-cm SOBP at various
positions were measured using HIMAC beams (Fig. 3A). Changing
thickness of binary filters, doses sharply drop at the distal fall-off of
the SOBP. Within 2 mm from the initial drop, doses decreased to
nearly 1/10. The middle position of this drop was made by use of
a 129.5-mm-thick binary filter. Second, jejunum crypt survivals
were measured at various positions within the same 6-cm SOBP. After a fixed dose of 10 Gy at a position made by a 109.0-mm-thick
binary filter, the number of crypts surviving this dose was 1.0
(Fig. 3B). The number of crypts gradually increased when the thickness of the binary filter increased from 113.5 mm and reached 130,
a nearly unirradiated level, at 123.0-mm thickness. This increase is
due to the decrease of physical doses at the distal fall-off. What was
different from physical fall-off was that the range of crypts increase
was 9.5 mm (123.0–113.5 mm) and wider than the range of dose decrease. This indicates that jejunum was not flat to the horizontal
beam and partly bending back and forth in the body. The middle position of the crypt drop was made by a use of 118.0-mm-thick binary
filter. Subtracting 118.0 mm from 129.5 mm, the resulting 11.5 mm
includes the thickness of both the Lucite (4 mm) and the abdominal
wall, the thickness of which could easily be calculated as 7 mm (the
water equivalent thickness of 4 mm Lucite is 4.64 mm). Therefore,
the average depth of jejunum was 7 mm in the abdomen. It is also
noted that variation of crypt counts was large at the depth of binary
filter 118–120 mm (Fig. 3B). When we observed histologic sections
of this depth, some crypts presented such a unique feature that crypts
at a hemisphere almost disappeared, whereas crypts at another
sphere were intact and showed no damage at all (Fig. 4). This means
that the jejunum at the depth of 118–120 mm was irradiated with the
distal fall-off of SOBP, where physical dose sharply dropped within
a range of 2 mm, the same thickness as mouse jejunum (Fig. 3A).
RBE calculation
The obtained data in log scale were plotted against doses in normal scale. For each experiment, a linear-quadratic model was used
to fit data of HSG cells, whereas exponential curve fitting was
used for gut crypts. Isoeffective doses obtained from fitted curves
were compared between carbon ions and reference g rays to calculate the mean and standard deviation of RBE.
RESULTS
Figure 5 shows HSG cell survivals at four positions within
the SOBPs. Survival curves of the proximal position were
distinctively different from and less curvy than that of
reference g rays (Fig. 5A). The differences between the
Table 1. Isoeffective doses and RBEs of 6-cm SOBP for HSG cell kill
Isoeffective dose (Gy)*
RBE
Position within
the SOBP
GSI
NIRS
GSI
NIRS
RBE ratio
(GSI/NIRS)
Proximal
Middle
Distal
Distal end
Cobalt-60 g rays
3.66 0.17
3.22 0.12
2.43 0.11
2.31 0.11
5.59 0.42
3.47 0.19
3.44 0.44
2.55 0.12
2.37 0.14
1.53 0.13
1.74 0.14
2.30 0.20
2.42 0.21
1.61 0.15
1.63 0.24
2.19 0.19
2.36 0.22
0.95 0.12
1.07 0.18
1.05 0.13
1.03 0.13
Abbreviations: RBE = relative biological effectiveness; SOBP = spread-out Bragg peak; HSG = human salivary gland tumor; GSI = Gesellschaft für Schwerionenforschung; NIRS = National Institute of Radiological Sciences.
Values are mean standard deviation.
* D10 (dose required to reduce surviving fraction down to 10%).
Biological comparison of carbon-ion beams d A. UZAWA et al.
1549
Fig. 6. Crypt survivals after single and fractionated irradiation with carbon ions. Mice received single doses (A–C) or 3
fractions/3 days (D–F) with carbon ions. Corresponding positions within 6-cm spread-out Bragg peak shown in Fig. 1
are proximal (p) for A and D, middle (m) for B and E, and distal (d) for C and F. Symbol and bar are mean and standard
deviation, respectively. GSI = Gesellschaft für Schwerionenforschung; NIRS = National Institute of Radiological Sciences.
NIRS and GSI carbon beam results in vitro were small. When
irradiation positions move downstream, survival curves of
carbon-ion beams became steep, so that the distal-end position (Fig. 5D) produced the steepest survival curves. The
D10 and RBE values are listed in Table 1.
Figure 6 shows crypt survivals after a single dose of carbon-ion irradiation. Proximal and distal positions produced
survival curves identical between GSI and NIRS, whereas
a slight but not significant difference was noticed for the middle position. Fractionated irradiation also produced survival
curves identical between the two facilities. With depth, survival curves became steeper after both single and fractionated
irradiations.
Isoeffective doses and RBE values for single and fractionated irradiation are listed in Tables 2 and 3, respectively.
DISCUSSION
We here presented and compared between GSI and NIRS
for inactivation of human tumor cells and gut crypt survivals
after irradiation with 6-cm carbon-ion SOBP profiles.
Because carbon ions are more effective than protons, RBE
values are very critical to the design of SOBP. In NIRS, clinical RBE of the carbon-ion SOBP is determined by incorporating fast neutron’s RBE before the start of therapy (3),
whereas GSI therapy uses the local effect model and uses
an a/b value obtained by photon therapy (8). Because carbon-ion therapy has been conducted almost exclusively in
NIRS and GSI, clinical results obtained by them are important for the radiation oncology society to evaluate this therapy. Clinical doses used in both institutions are expressed
as gray equivalent. Physical comparison between the two facilities has been done for dose but not for LET distribution
yet (9). If biological effectiveness of carbon-ion beams
were different between the two facilities, we would have difficulty interpreting clinical results based on clinical dose.
Thus, biological comparison is essential.
Several articles have reported biological effectiveness of
carbon ions using the same endpoint of gut crypt survivals.
In the 1980s, RBE values of carbon ions obtained by Bevalac
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Table 2. Isoeffective doses and RBEs of 6-cm SOBP for gut crypt survivals after single doses
Isoeffective dose (Gy)*
RBE
Position within
the SOBP
GSI
NIRS
GSI
NIRS
RBE ratio
(GSI/NIRS)
Proximal
Middle
Distal
Cobalt-60 g rays
10.27 0.20
9.10 0.23
8.27 0.10
14.86 0.08
10.32 0.14
9.45 0.08
8.25 0.17
1.47 0.03
1.63 0.04
1.80 0.02
1.44 0.02
1.57 0.02
1.80 0.03
1.00 0.02
1.04 0.03
1.00 0.02
Abbreviations as in Table 1.
Values are mean standard deviation.
* D10 (dose required to reduce the number of crypts per circumference to 10).
accelerator in Lawrence Berkeley Laboratory were reported
for mouse gut crypt survivals. Goldstein et al. (10) reported
that the RBE of 4-cm SOBP (400 MeV/u) for D10 is 1.28,
whereas that of 10-cm SOBP ranges from 1.1 to 1.16
depending on positions within the SOBP (11). Using
a 184-in synchrocyclotron, Alpen et al. (12) reported the
RBE for D10 of 4-cm SOBP (400 MeV/u) as 1.3–1.4 for
dose-averaged LET of 55–160 keV/mm. We also reported
the RBE of 3-cm SOBP for D10 (135 MeV/u) as 2.08–
2.59 for dose-averaged LET of 65–120 keV/mm (13).
Gueulette et al. (14) reported the RBE for D10 of 6-cm
SOBP (290 MeV/u) as 1.6–1.9 for a dose-averaged LET
of 41–71 keV/mm. The RBE values of carbon ions with
single doses in the present studies ranged from 1.44 to
1.80 (Table 2) and agreed with the other reports mentioned
above.
Because mouse crypt survivals are widely used to measure
biological effectiveness of particle beams (14, 15), we used
this endpoint along with colony formation of HSF cells in
the present study. These in vivo and in vitro assays have
been successfully used to measure and compare biological effectiveness of protons and carbon ions between several facilities in Japan (15–17).
We measured the depth of mouse jejunum tissue in the
SOBP (Fig. 3). The variation of crypt counts was large at
the depth of binary filter 118–120 mm. When we observed
histologic sections of this depth, some crypts presented
such a unique feature that crypts at a hemisphere almost disappeared, whereas crypts at another sphere were intact and
showed no damage at all (Fig. 4). This means that the jejunum
at the depth of 118–120 mm was irradiated with the distal
fall-off of SOBP, where physical dose sharply dropped
within a range of 2 mm, the same thickness of mouse jejunum (Fig. 3).
The above-stated results clearly show that the carbon-ion
beams at NIRS and GSI are biologically identical, even
though a small difference of RBE is noticed. This statement
does not, however, mean that the clinical doses being used in
NIRS and GSI are comparable. Preliminary studies show
that the difference of physical dose, not biological dose,
between NIRS and GSI is approximately 15% when treatment planning for the same model tumor to receive a given
biological dose is compared between the two institutions
(Dr. T. Kanai, private communication). Because the two institutions independently use RBE values that are calculated
from clinical data of patients receiving fast neutron therapy
in years past, the reason why the clinical RBE of carbon ions
is different between NIRS and GSI should be clarified in future studies. The present finding that the RBE of carbon-ion
beams is not different between NIRS and GSI would
exclude the possibility that difference, if any, in clinical
outcome of carbon-ion therapy is due to difference in biological effectiveness between NIRS and GSI. In addition,
the present results clearly show that biology is very important for carbon-ion radiotherapy. The role of biology would
become more important in the future, when this type of
charged particle radiotherapy is more commonly practiced
worldwide.
We conclude that the carbon-ion beams at NIRS and GSI
are biologically identical after single and daily fractionated
irradiation, even though a <7% difference of RBE is noticed
between the two facilities.
Table 3. Isoeffective doses and RBEs of 6-cm SOBP for gut crypt survivals after fractionated doses
Isoeffective dose (Gy)*
RBE
Position within
the SOBP
GSI
NIRS
GSI
NIRS
RBE ratio
(GSI/NIRS)
Proximal
Middle
Distal
Cobalt-60 g rays
13.30 0.41
11.68 0.15
10.14 0.20
22.75 0.19
13.70 0.54
12.24 0.37
9.89 0.17
1.71 0.05
1.95 0.03
2.24 0.04
1.66 0.07
1.86 0.06
2.30 0.3
1.03 0.05
1.05 0.04
0.98 0.02
Abbreviations as in Table 1.
Values are mean standard deviation.
* D30 (dose required to reduce the number of crypts per circumference to 30).
Biological comparison of carbon-ion beams d A. UZAWA et al.
1551
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