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Response of rat rhabdomyosarcoma tumors to split doses of mixed high- and low-let radiation
0360-30 16/X9 53.W + .00 Copyright (~8 1989 Pergamon Press plc
,,,I. J Radratmn Oncolog.~ Em/. Phys.. Vol. 16, pp. 1529-1536 Printed in the U.S.A. All rights reserved.
??Original Contribution
RESPONSE
OF RAT RHABDOMYOSARCOMA TUMORS TO SPLIT DOSES OF MIXED HIGH- AND LOW-LET RADIATION
T. S. TENFORDE, PH.D., V. J. MONTOYA, B. A., S. M. J. AFZAL, S. S. PARR, D.D.S. AND S. B. CURTIS, PH.D. Lawrence
Berkeley Laboratory,
University
of California,
PH.D.,
Berkeley, CA 94720
Radiation-induced growth delay was measured in rat rhabdomyosarcoma tumors exposed to split doses of highLET (linear energy transfer) neon ions in the extended-peak ionization region and low-LET X rays. Top-off doses of 7.5, 15, and 25 Gy of 225-kVp X rays were administered to the tumors at 0.5,4.0, and 24.0 hr following priming doses of either peak neon ions or X rays. The priming doses used were 7 Gy of peak neon ions and 20 Gy of X rays, both of which produced a 10 day delay in tumor regrowth to a volume twice that measured on the day of irradiation. The tumor response to split doses of X rays indicated rapid repair of sublethal damage, with significant recovery occurring at 0.5 hr and complete recovery by 4 hr after the initial 20-Gy X ray dose. The top-off doses of X rays required to produce an additional 10 or 20 days of tumor growth delay were 18 and 7% huger, respectively, when the priming dose was 20 Gy of X rays as compared to 7 Gy of peak neon ions. This result indicates that relatively little interaction of the neon-ion and X ray radiations occurred, even when the time interval between splitdose irradiations was as short as 0.5 hr. Our data indicate that the interaction of high- and low-LET nadiation modalities is small, and approaches a simple additivity of effects when the tumors repair a major portion of the sublethal radiation injury imparted by a priming dose before the second dose is administered. Rat rhabdomyosarcoma tumors, Radiation-induced growth delay, High- and low-LET radiation modalities, Peak neon ions, Radiation damage inieraction.
Interaction of mixed high- and low-LET radiation with tumors and normal tissues is of both practical and theoretical interest. Advantages of a practical and economic nature would be gained in tumor radiotherapy if highLET radiation modalities such as neutrons, pions, and heavy charged-particle beams could be combined efficaciously with low-LET photon radiation in a daily fractionation schedule that produced clinical results comparable to those observed with a fractionated schedule of high-LET radiation alone (5, 29). From a theoretical viewpoint, it is of considerable interest to define the lesions produced by low- and high-LET radiation modalities, and to understand the pathways through which these lesions interact in mixed radiation fields (7, 28, 33). In an effort to define the effects of combined high- and low-LET radiation modalities, a large number of experimental studies have been conducted with cells grown as
monolayers or spheroids in vitro (3, 10, 11, 13, 15, 17, 18,20-23, 3 l), and with various tumors and normal tissues in vivo (2, 12, 14, 19, 24, 30). In general, the results of the in vitro studies have demonstrated that sequential doses of high- and low-LET radiation modalities produce a synergistic effect when administered sequentially within a period of a few hours. Several of the in vivo studies have provided data that are consistent with this observation. However, many of the in vivo experiments have led to results that indicated an independent action of high- and low-LET radiation modalities, especially when the irradiations were performed on a daily fractionation schedule. All of the in vivo studies to date have been performed using neutrons as the high-LET radiation modality. A study was therefore undertaken to determine the response of a rat rhabdomyosarcoma tumor system to a combination of high-LET neon ions and orthovoltage X rays when the fractionation interval between dose installments was varied from 0.5 to 24 hr.
Reprint requests to: T. S. Tenforde, Ph.D., Life Sciences Center (K4- 14), Battelle, Pacific Northwest Laboratories, P.O. Box 999, Richland, WA 99352 USA. Acknowledgments-The skillful assistance of Stephanie O’Rear, Dev Felton, and Michelle Mericka in the preparation of this manuscript is gratefully acknowledged. Research support was
received from Public Health Service Grants CA 174 11 and CA 15 184 awarded by the National Cancer Institute, and from the Office of Health and Environmental Research, U.S. Department of Energy under Contract DE-AC03-76SFOO098 with the University of California. Accepted for publication 14 December 1988.
INTRODUCTION
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Neon ions in the distal extended-peak ionization region were chosen as a source of high-LET radiation in this study for two reasons: (a) peak-to-plateau ratios of the relative biological effectiveness (RBE) for radiation-induced growth delay (9, 27) and for cell killing (8) are significantly greater than unity, indicating the potential advantage of using peak neon ions for the treatment of deep seated tumors with significant sparing of the overlying normal tissue structures located within the plateau ionization region; and (b) RBE values for cell killing and growth delay following exposure of rat rhabdomyosarcoma tumors to 15-MeV neutrons and to extended-peak neon ions are similar (4, 9, 26), which facilitates a comparison of tumor responses when these two types of highLET radiation are used in a mixed high- and low-LET treatment regimen. METHODS
AND MATERIALS
Experimental tumors and volume response measurements Radiation-induced growth delay was determined from the postirradiation volume response characteristics of rhabdomyosarcoma R- 1 tumors transplanted subcutaneously in the thoracic region of syngeneic WAG/Rij rats (9, 27). Although a number of factors such as repair of radiation damage, reoxygenation of hypoxic cells, cellkinetic changes and tumor-host interactions can influence the results of a radiation-induced growth delay assay, this end point was chosen for the present experiments because it corresponds closely with the tumor volume changes that are generally used clinically as an index of response to radiotherapy. A tumor subline designated R2C5 was used in these experiments. The R2C5 tumors have nearly identical cell survival and postirradiation volume response characteristics as the parent cell line R2D2, from which the R2C5 line of rhabdomyosarcoma tumors was derived by cloning (25, 26). Only the R2C5 subline has been used in experiments conducted subsequent to 1982 because of the high plating efficiency of 40-60% that these tumors exhibit when they are excised and plated as single cells in vitro for the assay of postirradiation cell survival based on colony-forming ability. Tumor regression and regrowth following irradiation were monitored by caliper measurements of the tumor volume using methods described previously (9,27). Measurements were made at 2 to 3 day intervals until each tumor reached a volume that was ten times the volume on the day of irradiation. The radiation-induced growth delay was calculated from the difference in time for irradiated and unirradiated tumors to grow to twice the volume measured on the day of irradiation. The average initial volume of 138 tumors used in these experiments
* Model RT200/250,
Philips, Eindhoven,
The Netherlands.
June 1989, Volume 16, Number 6
was 0.508 k 0.020 (S.E.) cm3, and the average doubling time for the volume of unirradiated tumors was 4.5 days. Radiation procedures Tumors were exposed to high-LET radiation in the distal 4-cm extended-peak ionization region of an accelerated neon-ion beam with an initial energy per atomic mass unit (u) of 557 MeV/u, as shown in Figure I. Tumors were irradiated in the distal portion of the extended-peak region to minimize the amount of residual radiation dose delivered to internal normal tissue structures. The variable-thickness absorber (ridge filter) used in these experiments was designed to produce a decreasing physical dose from the proximal to the distal end of the extended-peak region. This physical dose profile offsets the increasing RBE for cell killing across the extended-peak region (8), with a result that the tumor volume receives a nearly uniform biologically effective dose (defined as the product of RBE and physical dose). The residual range of this beam was 22 cm in water, and the dose-averaged LET, values within the tumor treatment zone ranged from 115 to 240 keV/pm. The beam collimation techniques, dosimetry, and tumor irradiation procedures used with accelerated charged-particle beams produced at the Berkeley BEVALAC facility have been described previously (9). The absorbed dose rate in the tumor mass ranged from 5 to 10 Gy/min during irradiations with peak neon ions. Irradiation of tumors was performed with 225-kVp X rays from a therapy unit,* using a total filtration of 0.35 mm Cu (mean photon energy = 75 kV; HVL = 1.08 mm Cu). Collimation of the X ray beam for tumor irradiations followed procedures described previously (9). The absorbed dose rate of X rays in the tumor tissue was 6 Gy/ min, as determined with a 250 R ionization chamber.?
0
I
I
I
I
I
5
10
15
20
25
Penetration
Depth
(cm
of
water)
Fig. 1. The depth-dose curve measured in water is shown for the neon-ion beam with an initial energy of 557 MeV/u. The peak ionization region was spread to a width of 4 cm by means of a variable-thickness absorber.
t Victoreen;
Cleveland,
OH.
Mixed high and low LET radiation 0 T. S. TENFORDE
The experimental animals were lightly anesthetized with Metofanet during tumor irradiation with both X rays and peak neon ions. RESULTS Rhabdomyosarcoma tumors were administered a priming dose of 7 Gy of high-LET peak neon ions, followed at intervals of 0.5,4.0, and 24.0 hr by 7.5, 15, and 25 Gy top-off doses of 225-kVp X rays. An identical procedure was used for split doses of X rays, except that the priming X ray dose was 20 Gy. A 7 Gy single dose of peak neon ions and a 20 Gy single dose of X rays produced nearly identical radiation-induced growth delays of 9.95 f 0.85 (S.E.) and 10.83 + 0.88 days, respectively, as shown in Figure 2. The RBE for a single dose of peak neon ions at the lo-day growth delay level was therefore 2.86 in the present experiments, in good agreement with earlier values determined for this tumor line (9, 26). The volume regression and regrowth curves for tumors administered a 7-Gy dose of peak neon ions and 20-, 30-, and 40-Gy doses of 225-kVp X rays are shown in Figure 2. In Figure 3 the volume response curves are plotted for split-dose schedules involving either a priming dose of neon ions followed by graded doses of X rays, or a priming dose of X rays followed by a second dose of X rays. The growth delay curves determined from the volume response data shown in Figures 2 and 3 are presented in Figure 4. These curves demonstrate that the rhabdomyosarcoma tumors exhibited substantial recovery between fractions in a split-dose X ray schedule. The data also indicate that the repair of sublethal damage was nearly complete when the interval between X ray dose fractions
r
100
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,X-rays
(20 Gy)
X-rays(30Gy) X-rays
(40
Gy)
Radiation-Induced Growth
Delay
7 GymNe
0.1
20 30
I 10
SE.
(days)
9.95
k
0.85
GyX-rays(-): Gy X-rays (-):
10.03
t
0.88
40 GY X-rays
0
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(---):
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32.57
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3.07
i 1 30 40 50 Days Postirradiation
20
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60
70
Inc.; Washington
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1531
was as short as 0.5 hr. By 4 hr the recovery from sublethal damage was complete as illustrated by the similar growth delays obtained with interfraction intervals of 4 and 24 hr. Only the data for total X ray doses of 35 and 45 Gy suggest that less recovery occurred with a 0.5 hr split-dose interval than with a 4-hr interval. However, an analysis of variance indicated that this difference is not statistically significant (0.05 < p < 0.10) and therefore only a single line has been drawn through the growth-delay data shown in Figure 4 for split doses of X rays. Overall, the splitdose data are consistent with a rapid recovery from sublethal X ray damage in the rat rhabdomyosarcoma tumors. The data presented in Figure 4 also demonstrate that the growth delay increments produced by administering top-off doses of X rays following a 7-Gy priming dose of peak neon ions were similar to those resulting from split doses of X rays in which the priming X ray dose was 20 Gy. As with split doses of X rays, the total growth delay response was not significantly different when the topoff doses of X rays were administered at 0.5, 4.0, or 24.0 hr following the priming 7-Gy dose of peak neon ions. In Table 1 a comparison is made between the top-off doses of X rays that were required to produce incremental growth delays of 10,20, or 30 days when the priming dose was 7 Gy of peak neon ions or 20 Gy of X rays. A parameter defined as the “incremental dose ratio” is used in Table 1 for this comparison. To form this ratio, the top-off doses of X rays that gave an additional 10,20, or 30 days of radiation-induced growth delay when the priming dose was 20 Gy of X rays were divided by the top-off doses of X rays that gave the same growth delay increments when the priming dose was 7 Gy of peak neon ions. The values of the incremental dose ratio listed in Table 1 range from 1.00 to 1.18, with an average value of 1.08. The fact that the incremental dose ratios have values close to unity indicates that little, if any, interaction occurred between the priming dose of high-LET neon ions and the subsequent top-off dose of low-LET X rays. Table 2 lists RBE values based on the growth-delay end point (RBE,) for mixed neon-ion and X ray irradiation of rhabdomyosarcoma tumors. RBE, values for the mixed-beam treatment were referenced to the split doses of X rays that produced an equivalent growth delay. As shown in Figure 5, the RBE, values are a linear function of the neon-ion dose fraction, f. A linear dependence of RBE, on f would be expected if no interaction occurred between the sequential doses of high- and low-LET radiation (6).
XBL 882 ‘Iii
Fig. 2. Volume regression and regrowth curves are plotted for groups of tumors that received either 7 Gy of peak neon ions or 20, 30, or 40 Gy of X rays. The dotted line is the growth curve for unirradiated tumors. The tumor volumes were normalized to unity on the day of irradiation. Data points represent the mean k 1 S.E. for groups of six tumors.
$ Pittman-Moore,
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DISCUSSION In an early study of the combined effects of X rays and high-LET alpha particles on the proliferative capacity of human T- 1 kidney cells, Barendsen et al. (3) observed no
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June 1989, Volume
16, Number
6
0.5 Hr Split Dose Interval
Growth Growth Pelav 0.1 -
0
, 10
I
20
+ SE.
20 Gy X-rays: 10.03 + 7 5Gy X-rays: 14.22 + 15 Gy X-rays.21 26 + 25 Gy X-rays:36.90 I 1 I 1 30 40 50 60
Ck k + ?
0.88 0.83 216 1.67 1
70
7 Gy + 7.5Gy + 15 Gy + 25 Gy
O.l-
0
I 10
1
20
1
30
Delav
+ S.E. f&y&
“Ne: 9.95 X-rays: 13.54 X-rays: 25.21 X-rays: 29.38
I 40
1 50
k _+ + t
I
60
0.85 2 16 2.12 3.06 1
70
Days Postirradiation
Days Postirradiation
4 Hr Split Dose Interval loo-
100
C
.D 7 GY 20Ne + 7.5 Gy X-rays
20 Gv X-rays .5 Gyi-rays + 25 Gy X-rays
F
Growth
Delay
LLS E !dWL
20 10.03 + 7.5Gy Gy X-rays. X-rays: 14.22 + 15 Gy X-rays 16.66 + 25 Gy X-rays, 28 79
0.1 F
i+ 5 ?
0 1 2 3
88 03 55 70
0
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IO
20
30
40
50
60
70
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Days Postirradiation
24 Hr Split Dose Interval 1OOr
- F 10
Normalized Tumor 1.0 Volume Growth 20 Gy + 7.5Gy + 15 Gy + 25 Gy
0.1
0
10
20
30
40
Delay
-t S.E
X-rays: X-rays. X-rays, X-rays
10 03 14 81 20 70 29.01
50
60
Growth
!&sL -r i f ?
Days Postirradiation
0.88 1.22 2 44 4 92
70
+ S.E. ki&!s~
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0.1 -
0
Delay
I 10
I 20
/ 30
I 40
I 50
I 60
+ 2 + +
0 85 0.49 3.45 3 92
I 70
Days Postirradiation
Fig. 3. Volume response curves are plotted for tumors that received priming doses of 7 Gy of peak neon ions or 20 Gy of X rays, followed by graded X ray doses administered 0.5, 4.0, or 24.0 hr later. Data points represent the mean + 1 S.E. for groups of six tumors.
interaction between the effects of these two radiation modalities. Similar results were later obtained for Chinese hamster cells irradiated with alpha particles and gamma rays (11). The alpha particles used by Barendsen et al.
(3), which had an average LET of 170 keV/pm, produced a linear dose-response curve for cell killing. On this basis, Railton et al. (22) have proposed that the lack of interaction of alpha particles with X rays occurred because
Mixed high and low LET radiation 0 T. S. TENFORDE
v-“Ne o-
4-m
225.kVp
single dose
extended-peak X-my
single
dose Rat
‘ONe + x ray
Interval
-5o% 0 540
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0.5 hr 4 h,
. .
A 0
24
.
0
ilr
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I
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8 f
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: E 2
20-
.L 5 f
IO-
.+3 no
I
I
IO
OO
I
I
20
30
Tot01
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IGy)
Fig. 4. Radiation-induced growth delay is plotted as a function of total absorbed dose for rhabdomyosarcoma tumors administered as single doses of extended-peak neon ions (7 Gy) or 225kVp X rays followed at 0.5,4.0, and 24.0 hr by second doses of X rays at dose levels of 7.5, 15, and 25 Gy. For comparison, the
growth delay induced by 20, 30, and 40 Gy single doses of X rays is plotted as a solid line. Data points represent the mean f 1 S.E. for groups of six tumors.
the alpha-particle killing mechanism involved only singlehit events. However, later studies by Ngo et al. (20) demonstrated that Bragg peak neon ions with an average LET of 183 keV/pm could interact synergistically with X rays in the inactivation of Chinese hamster V79 cells, even though the dose-response curve for killing by the neon ions was linear. Ngo et al. (20) demonstrated in split-dose experiments that the neon-ion beam was capable of producing sublethal damage, and that this damage could interact with a subsequent dose of X rays. These results therefore diverge from the findings in earlier experiments with alpha particles and X rays. One explanation of the lack of interaction observed in the early experiments with combined alpha-particle and X ray radiations, as pointed out by McNally et al. ( 18), is that the alpha-particle doses used were too small to induce a significant amount of sublethal damage with which the X rays could subsequently interact. Another interpretation of the combined alpha particle and X ray results is that the alpha particles had such a high LET that no “interactable” damage was
1533
d al.
produced. In the combined neon-ion and X ray experiments, the neon-ion beam had a broad range of LET, the lower-LET portion of which is expected to produce “interactable” damage. In contrast to the earlier studies of Barendsen et al. (3), the results of more recent experiments by Raju and Jett (23) on the killing of human kidney T1 cells exposed to a combination of plutonium alpha particles and X rays are consistent with a small degree of interaction between these two radiation modalities. The existence of synergistic interactions between highand low-LET radiation is supported also by other recent studies with charged-particle beams. Yuhas et al. (3 1) reported that doses of peak pions greater than 0.5 Gy eliminate the shoulder of the subsequent X ray survival curve in cultured CHO-Kl cells. Even at 4 hr following a 3-Gy dose of peak pions the X ray survival curve lacked its normal shoulder, indicating that the repair of pion-induced damage with which the X rays could interact was not complete. In growth delay measurements on spheroids, Yuhas et al. (31) also obtained evidence for a significant interaction between peak pions and X rays. Several in vitro studies have been performed to analyze the interactions between neutrons and low-LET photon radiation. An interaction between ‘j°Co gamma rays and 14-MeV neutrons was observed for cultured Chinese hamster cells by Railton et al. (22), who ascribed this effect to the low-LET portion of the neutron beam interacting with the gamma-ray dose. Using both V79 monolayer cultures and spheroids, Durand and Olive (10) also obtained evidence for the interaction of mixed beams of X rays and neutrons administered as split doses with an interval of 4 hr. They reported that no repair of sublethal damage occurred when either an X ray dose was followed by neutron irradiation, or a neutron dose was followed by X rays. Ngo et al. (2 1) subsequently obtained evidence for repair in V79 cells of neutron-induced sublethal damage that can interact with X ray-induced sublethal damage. This observation was made with both fission neutrons, for which the low-LET component is only 3-4%, and with high-energy neutrons. In a recent study with V79 Chinese hamster cells, McNally et al. (18) reported that neutrons and X rays
Table 1. Mixed neon-ion and X ray irradiation of rat rhabdomyosarcoma
tumors
X ray top-off dose (Gy) required to produce growth delay increment following priming dose of
Growth delay end point (days)
Growth delay increment relative to single doses of X rays or ‘“Ne ions (days)
20-Gy X ray
7-Gy “Ne
Dose increment ratio: [X ray + X ray]/[20Ne + X ray]*
20 30 40t
10 20 30
16.25 24.25 30.50
13.75 22.75 30.50
1.18 1.07 1.00
* The dose increment ratio is formed by dividing the [X ray + X ray] top-off dose by the [20Ne + X ray] top-off dose required to produce a specified increment in growth delay (either 10, 20 or 30 days of additional growth delay). t Data at the 40-day growth delay end point are extrapolated values taken from the growth-delay curves shown in Figure 4.
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Table 2. RBE values for radiation-induced
growth delay
Growth delay
*“Ne
X ray
Percent of total
end point (days)
dose
dose*
dose from *“Ne
(GY)
(GY)
ions
RBL.t
10 20 30 40
7 7 7 7
0 13.75 22.75 30.50
100 34 24 19
2.86 1.75 1.49 1.35
* The top-off X ray dose required to produce a given level of growth delay was determined from the radiation-induced growth delay curves presented in Figure 4. t The RBE values for the mixed neon-ion X ray irradiation were referenced to the equivalent split doses of X rays that pro-
duced the same growth delay: RBE, = [X ray + X ray dose&J [‘“Ne + X ray dose], where g is the growth delay level in days. exerted a synergistic effect, provided that the neutron contribution to the total dose was less than 40%. When the neutron contribution to the dose exceeded this level, the two radiation modalities acted independently in reducing cell survival. The combined effects of neutrons and X rays were found by McNally et al. (18) to be the same whether the two radiation modalities were administered simultaneously, or sequentially in either order. This contrasted with an earlier report by Higgins et al. (13) that sequential irradiation of V79 cells by “Co gamma rays and 14.8-MeV neutrons gave a higher survival level than that resulting from simultaneous irradiation with the two modalities. However, the exposure dose rates used by Higgins et al. (13) were less than 0.1 Gy/min, and Zaider and Brenner (32) have concluded that this lowdose-rate exposure condition would make it difficult to observe damage interactions from sequential high- and low-LET radiations. Another recent in vitro study with V79 cells was reported by Iliakis et al. (15), who exposed plateau-phase
Fig. 5. RBE, values for radiation-induced growth delay are plotted as a function of the fraction, f, of the total dose administered to the tumors as peak neon ions. The straight line fitted to the data was obtained by linear regression analysis. The coefficient of determination, 3, is close to unity, thereby indicating that a linear relationship provides an excellent fit of the experimental data.
June 1989, Volume 16, Number 6
cultures to sequential doses of fast neutrons and ‘“Co gamma rays with intervals ranging from 0 to 6 hr between the doses. They observed a dose-dependent reduction in the gamma-ray survival level after pre-exposure of the cells to neutrons, and a similar effect occurred in the neutron survival curve when the order of irradiation was reversed. The interaction between these two radiation modalities also was found to decay with time between doses, with kinetics similar to the loss of sensitivity to the PLD repair inhibitor ,&arabinofuranosyladenine (/3-araA). Iliakis et al. (15) suggested on this basis that the interaction between neutrons and gamma rays may involve the set of potentially lethal lesions that cannot be repaired in the presence of /3-araA. The concept that high- and low-LET radiations may interact via a common set of lesions has been described in theoretical terms by Lam (16). In this mathematical model, it is assumed that a common intermediate stage exists in the pathways for lethal lesions produced by different types of radiation. If the lesion precursors of two types of radiation reach the same stage at the same time, then the irradiation effects will not be independent. It is implicit in this model that the time interval between sequential doses of high- and low-LET radiation must be short enough for lesion interactions to occur. Although strong evidence has been obtained for the interaction of mixed high- and low-LET radiation in cell cultures, conflicting observations have been made in previous studies on in vivo normal tissues and tumors exposed to mixed radiation modalities. In an investigation on the effects of a mixed daily fractionation schedule of 14 MeV neutrons and X rays administered to the mouse intestine, Hendry et al. ( 12) found that the effects of the two types of radiation were additive. A contrary finding was later reported by Hornsey et al. (14) who observed interaction of neutron and X ray damage to the mouse intestine when the radiation doses were separated by short intervals ranging from 15 min to 3-4 hr. They attributed the negative findings of Hendry et al. (12) to the fact that recovery of sublethal damage was probably complete within the l-day interval between neutron and X ray dose fractions. Two studies in which mixed regimens of low-LET radiation and neutron radiation were administered on a daily schedule to mouse tumors gave no evidence for an interaction of these two radiation modalities (2, 30). In both cases the tumor response to a daily fractionation schedule using mixed radiation modalities was less than that resulting from fractionated neutron radiation alone, and the overall response to an admixture of high- and low-LET radiation was consistent with that expected if the two radiation modalities acted independently. Two other studies with mouse tumors have led to the finding of a small therapeutic gain resulting from exposure to an admixture of neutrons and X rays given in a daily fractionation schedule ( 19,24). Based on tumor growth delay and 50% tumor cure end points, the combination of two
Mixed high and low LET radiation 0 T. S. TENFORDE
neutron fractions and three X ray fractions was found to reduce the required neutron dose per fraction in comparison with the dose per fraction required if fractionated neutron radiation was given alone. The fact that a small amount of interaction between neutrons and X rays was observed with a daily fractionation schedule in the experiments of Nelson et al. (19) and Rasey et al. (24) suggests that some long-lived, unrepaired lesions may have remained at 1 day following tumor irradiation. The results of experiments described here clearly indicate the absence of a significant amount of damage interaction in rat rhabdomyosarcoma tumors exposed to sequential doses of high-LET radiation from 225-kVp X rays. The lack of significant interaction between these radiation modalities was observed both for a daily interval between dose installments, comparable to the results of previous studies on normal tissues and tumors (2, 12,30), and for relatively short split-dose intervals of 0.5 and 4.0 hr. The fact that no significant interaction between peak neon ions and X rays was observed even with a 0.5-hr interfraction interval probably relates to the very rapid repair of sublethal and potentially lethal radiation lesions exhibited by the rat rhabdomyosarcoma R- 1 tumor line. Recent studies in our laboratory have demonstrated that potentially lethal damage repair by this tumor line is
ef a/.
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complete within an 3-hr interval following irradiation (l), and the results of experiments reported here indicate that sublethal damage repair is nearly complete by 0.5 hr and reaches completion by 4 hr following a priming dose of X rays. Taken together, these results are consistent with the concept that sublethal and potentially lethal repair are manifestations of the repair of the same lesions, as implied in the “lethal-potentially lethal lesion” model of Curtis (7). The observed absence of significant interaction between high- and low-LET radiation modalities in experimental animal tumors has direct implications for clinical radiotherapy procedures that involve daily treatments with mixed beams. The results of studies reported here, as well as previously published observations, suggest that the time interval between sequential treatments with high- and lowLET radiation should be made as short as possible in order to gain a therapeutic advantage in tumor treatment with mixed radiation modalities. In a mixed-beam tumor treatment regimen involving daily dose installments, the experimental information presently available indicates that appropriately RBE-matched doses of high- and lowLET radiation should be administered without assuming that an interaction occurs between the different radiation modalities.
REFERENCES 1. Afzal, S. M. J.; Tenforde, T. S.; Parr, S. S.; Curtis, S. B.
PLD repair in rat rhabdomyosarcoma tumor cells irradiated in vivo and in vitro with high-LET and low-LET radiation. Radiat. Res. 107:354-366; 1986. 2. Ando, K.; Soike, S.; Fukuda, N.; Kanehira, C. Independent
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effect of a mixed-beam regimen of fast neutrons and gamma rays on a murine fibrosarcoma. Radiat. Res. 98:96-106; 1984. Barendsen, G. W.; Beusker, I. L. J.; Vergroesen, A. J.; Budke, L. Effects of different ionizing radiations on human cells in tissue culture. II. Biological experiments. Radiat. Res. 13: 841-849; 1960. Barendsen, G. W.; Broerse, J. J. Experimental radiotherapy of a rat rhabdomyosarcoma with 15 MeV neutrons and 300 kV X rays. I. Effects of single exposures. Eur. J. Cancer 5: 373-391; 1969. Castro, J. R.; Quivey, J. M.; Lyman, J. T.; Chen, G. T. Y.; Phillips, T. L., Tobias, C. A.; Alpen, E. L. Current status of clinical particle radiotherapy at the Lawrence Berkeley Laboratory. Cancer 46:633-64 1; 1980. Curtis, S. B. The OER of mixed high- and low-LET radiation. Radiat. Res. 65:566-572; 1976. Curtis, S. B. Lethal and potentially lethal lesions induced by radiation-a unified repair model. Radiat. Res. 106:252270; 1986. Curtis, S. B.; Schilling, W. A.; Tenforde, T. S.; Crabtree, K. E.; Tenforde, S. D.; Howard, J.; Lyman, J. T. Survival of oxygenated and hypoxic tumor cells in the extendedpeak regions of heavy charged-particle beams. Radiat. Res. 90:292-309; 1982. Curtis, S. B.; Tenforde, T. S.; Parks, D.; Schilling, W. A.; Lyman, J. T. Response of a rat rhabdomyosarcoma to neonand helium-ion irradiation. Radiat. Res. 74:274-288; 1978. Durand, R. E.; Olive, P. L. Irradiation of multi-cell spheroids with fast neutrons versus X rays: A qualitative difference in
1 I.
12.
13.
14.
15.
16. 17.
18.
19.
20.
sublethal damage repair capacity or kinetics. Int. J. Radiat. Biol. 30:588-592; 1976. Elkind, M. M. Damage and repair processes relative to neutron (and charged particle) irradiation. Curr. Top. Radiat. Res. 7: l-44: 1970. Hendry, J. H.; Rosenberg, I.; Greene, D. Addition of neutron and gamma-ray fractions for intestinal damage. Radiol. 12 1: 483-486; 1976. Higgins, P. D.; Deluca, P. M.; Pearson, D. W. V79 survival following simultaneous or sequential irradiation by 15-MeV neutrons and 6”Co photons. Radiat. Res. 95:45-56; 1983. Hornsey, S.; Andreozzi, U.; Warren, P. R. Sublethal damage in cells of the mouse gut after mixed treatment with X rays and fast neutrons. Br. J. Radiol. 50:513-S 17; 1977. Iliakis, G.; Ngo, F. Q. H.; Roberts, N. K.; Youngman, K. Evidence for similarities between radiation expressed by j3amA and damage involved in the interaction effect observed after exposure of V79 cells to mixed neutrons and gamma radiation. Radiat. Res. 104:303-316; 1985. Lam, G. K. Y. The survival response ofa biological system to mixed radiations. Radiat. Res. 110:232-243; 1987. McNally, N. J.; deRonde, J.; Hinchliffe, M. The effect of sequential irradiation with X rays and fast neutrons on the survival of V79 hamster cells. Int. J. Radiat. Biol. 45:301310; 1984. McNally, N. J.; deRonde, J.; Hinchliffe, M. Survival of V79 cells following simultaneous irradiation with X rays and neutrons in air or hypoxia. Int. J. Radiat. Biol. 48:847-855; 1985. Nelson, J. S. R.; Carpenter, R. E.; Nelson, N. J.; Parker, R. G. Response of mouse skin and the C3HBA mammary carcinoma of the C3H mouse to X rays and cyclotron neutrons: Effect of mixed neutron-photon fractionation schemes. Eur. J. Cancer 11:89 I-901; 1975. Ngo, F. Q. H.; Blakely, E. A.; Tobias, C. A. Sequential ex-
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22.
23.
24.
25.
26.
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posures of mammalian cells to low- and high-LET radiations. Radiat. Res. 87:59-78; 1981. Ngo, F. Q. H.; Han, A.; Elkind, M. M. On the repair of sublethal damage in V79 Chinese hamster cells resulting from irradiation with fast neutrons combined with X rays. Int. J. Radiat. Biol. 32:507-511; 1977. Railton, R.; Lawson, R. C.; Porter, C. Interaction of gammaray and neutron effects on the proliferation capacity of Chinese hamster cells. Int. J. Radiat. Biol. 27:75-82; 1975. Raju, M. R.; Jett, J. H. RBE and OER variations for mixtures of plutonium alpha particles and X rays for damage to human kidney cells. Radiat. Res. 60:473-48 1; 1974. Rasey, J. S.; Carpenter, R. E.; Nelson, N. J.; Parker, R. G. Cure of EMT-6 tumors by X rays or neutrons: Effect of mixed-fractionation schemes. Radiol. 123:207-2 12; 1977. Tenforde, T. S.; Afzal, S. M. J.; Parr, S. S.; Howard, J.; Lyman, J. T.; Curtis, S. B. Cell survival in rat rhabdomyosarcoma tumors irradiated in vivo with extended-peak silicon ions. Radiat. Res. 92:208-216; 1982. Tenforde, T. S.; Curtis, S. B.; Crabtree, K. E.; Tenforde, S. D.; Schilling, W. A.; Howard, J.; Lyman, J. T. In vivo cell survival and volume response characteristics of rat rhabdomyosarcoma tumors irradiated in the extended peak region of carbon- and neon-ion beams. Radiat. Res. 83:4256; 1980.
June 1989, Volume 16, Number 6 27. Tenforde, T. S.; Tenforde, S. D.; Crabtree, K. E.; Parks, D. L.; Schilling, W. A.; Parr, S. S.; Flynn, M. J.; Howard, J.; Lyman, J. T.; Curtis, S. B. RBE values for radiationinduced growth delay in rat rhabdomyosarcoma tumors exposed to plateau and peak carbon, neon and argon ions. Int. J. Radiat. Oncol. Biol. Phys. 7:2 17-221; 1981. 2%. Tobias, C. A.; Blakely, E. A.; Ngo, F. Q. H.; Yang, T. C. H. The repair-misrepair model of cell survival. In: Meyn, R. E., Withers, H. R., eds. Radiation biology in cancer research. New York, NY: Raven Press; 1980: 195-230. 29. Tsunemoto, H.; Morita, S.; Arai, T.; Kutsutani, Y.; Kurisu, A.; Umegaki, Y. Results of clinical trial with 30 MeV d-Be neutrons at NIRS. In: Abe, M., Sakamoto, K., Phillips, T. L., eds. Treatment of radioresistant cancer. Amsterdam: Elsevier/North-Holland; 1977: 1 l5- 126. 30. Urano, M.; Koike, S. Comparison of the effects of neutron and/or photon irradiation on spontaneous squamous-cell carcinoma in mice. Radiol. 134:219-225; 1980. 31. Yuhas, J. M.; Li, A. P.; Khgerman, M. M. Present status of the proposed use of negative pi mesons in radiotherapy. Adv. Radiat. Biol. 851-83; 1979. 32. Zaider, M.; Brenner, D. J. Comments on “V79 survival following simultaneous or sequential irradiation by l5-MeV neutrons and M’Cophotons.” Radiat. Res. 99:438-441; 1984. 33. Zaider, M.; Rossi, H. H. The synergistic effects of different radiations. Radiat. Res. 95:732-739; 1980.
,,,I. J Radratmn Oncolog.~ Em/. Phys.. Vol. 16, pp. 1529-1536 Printed in the U.S.A. All rights reserved.
??Original Contribution
RESPONSE
OF RAT RHABDOMYOSARCOMA TUMORS TO SPLIT DOSES OF MIXED HIGH- AND LOW-LET RADIATION
T. S. TENFORDE, PH.D., V. J. MONTOYA, B. A., S. M. J. AFZAL, S. S. PARR, D.D.S. AND S. B. CURTIS, PH.D. Lawrence
Berkeley Laboratory,
University
of California,
PH.D.,
Berkeley, CA 94720
Radiation-induced growth delay was measured in rat rhabdomyosarcoma tumors exposed to split doses of highLET (linear energy transfer) neon ions in the extended-peak ionization region and low-LET X rays. Top-off doses of 7.5, 15, and 25 Gy of 225-kVp X rays were administered to the tumors at 0.5,4.0, and 24.0 hr following priming doses of either peak neon ions or X rays. The priming doses used were 7 Gy of peak neon ions and 20 Gy of X rays, both of which produced a 10 day delay in tumor regrowth to a volume twice that measured on the day of irradiation. The tumor response to split doses of X rays indicated rapid repair of sublethal damage, with significant recovery occurring at 0.5 hr and complete recovery by 4 hr after the initial 20-Gy X ray dose. The top-off doses of X rays required to produce an additional 10 or 20 days of tumor growth delay were 18 and 7% huger, respectively, when the priming dose was 20 Gy of X rays as compared to 7 Gy of peak neon ions. This result indicates that relatively little interaction of the neon-ion and X ray radiations occurred, even when the time interval between splitdose irradiations was as short as 0.5 hr. Our data indicate that the interaction of high- and low-LET nadiation modalities is small, and approaches a simple additivity of effects when the tumors repair a major portion of the sublethal radiation injury imparted by a priming dose before the second dose is administered. Rat rhabdomyosarcoma tumors, Radiation-induced growth delay, High- and low-LET radiation modalities, Peak neon ions, Radiation damage inieraction.
Interaction of mixed high- and low-LET radiation with tumors and normal tissues is of both practical and theoretical interest. Advantages of a practical and economic nature would be gained in tumor radiotherapy if highLET radiation modalities such as neutrons, pions, and heavy charged-particle beams could be combined efficaciously with low-LET photon radiation in a daily fractionation schedule that produced clinical results comparable to those observed with a fractionated schedule of high-LET radiation alone (5, 29). From a theoretical viewpoint, it is of considerable interest to define the lesions produced by low- and high-LET radiation modalities, and to understand the pathways through which these lesions interact in mixed radiation fields (7, 28, 33). In an effort to define the effects of combined high- and low-LET radiation modalities, a large number of experimental studies have been conducted with cells grown as
monolayers or spheroids in vitro (3, 10, 11, 13, 15, 17, 18,20-23, 3 l), and with various tumors and normal tissues in vivo (2, 12, 14, 19, 24, 30). In general, the results of the in vitro studies have demonstrated that sequential doses of high- and low-LET radiation modalities produce a synergistic effect when administered sequentially within a period of a few hours. Several of the in vivo studies have provided data that are consistent with this observation. However, many of the in vivo experiments have led to results that indicated an independent action of high- and low-LET radiation modalities, especially when the irradiations were performed on a daily fractionation schedule. All of the in vivo studies to date have been performed using neutrons as the high-LET radiation modality. A study was therefore undertaken to determine the response of a rat rhabdomyosarcoma tumor system to a combination of high-LET neon ions and orthovoltage X rays when the fractionation interval between dose installments was varied from 0.5 to 24 hr.
Reprint requests to: T. S. Tenforde, Ph.D., Life Sciences Center (K4- 14), Battelle, Pacific Northwest Laboratories, P.O. Box 999, Richland, WA 99352 USA. Acknowledgments-The skillful assistance of Stephanie O’Rear, Dev Felton, and Michelle Mericka in the preparation of this manuscript is gratefully acknowledged. Research support was
received from Public Health Service Grants CA 174 11 and CA 15 184 awarded by the National Cancer Institute, and from the Office of Health and Environmental Research, U.S. Department of Energy under Contract DE-AC03-76SFOO098 with the University of California. Accepted for publication 14 December 1988.
INTRODUCTION
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Neon ions in the distal extended-peak ionization region were chosen as a source of high-LET radiation in this study for two reasons: (a) peak-to-plateau ratios of the relative biological effectiveness (RBE) for radiation-induced growth delay (9, 27) and for cell killing (8) are significantly greater than unity, indicating the potential advantage of using peak neon ions for the treatment of deep seated tumors with significant sparing of the overlying normal tissue structures located within the plateau ionization region; and (b) RBE values for cell killing and growth delay following exposure of rat rhabdomyosarcoma tumors to 15-MeV neutrons and to extended-peak neon ions are similar (4, 9, 26), which facilitates a comparison of tumor responses when these two types of highLET radiation are used in a mixed high- and low-LET treatment regimen. METHODS
AND MATERIALS
Experimental tumors and volume response measurements Radiation-induced growth delay was determined from the postirradiation volume response characteristics of rhabdomyosarcoma R- 1 tumors transplanted subcutaneously in the thoracic region of syngeneic WAG/Rij rats (9, 27). Although a number of factors such as repair of radiation damage, reoxygenation of hypoxic cells, cellkinetic changes and tumor-host interactions can influence the results of a radiation-induced growth delay assay, this end point was chosen for the present experiments because it corresponds closely with the tumor volume changes that are generally used clinically as an index of response to radiotherapy. A tumor subline designated R2C5 was used in these experiments. The R2C5 tumors have nearly identical cell survival and postirradiation volume response characteristics as the parent cell line R2D2, from which the R2C5 line of rhabdomyosarcoma tumors was derived by cloning (25, 26). Only the R2C5 subline has been used in experiments conducted subsequent to 1982 because of the high plating efficiency of 40-60% that these tumors exhibit when they are excised and plated as single cells in vitro for the assay of postirradiation cell survival based on colony-forming ability. Tumor regression and regrowth following irradiation were monitored by caliper measurements of the tumor volume using methods described previously (9,27). Measurements were made at 2 to 3 day intervals until each tumor reached a volume that was ten times the volume on the day of irradiation. The radiation-induced growth delay was calculated from the difference in time for irradiated and unirradiated tumors to grow to twice the volume measured on the day of irradiation. The average initial volume of 138 tumors used in these experiments
* Model RT200/250,
Philips, Eindhoven,
The Netherlands.
June 1989, Volume 16, Number 6
was 0.508 k 0.020 (S.E.) cm3, and the average doubling time for the volume of unirradiated tumors was 4.5 days. Radiation procedures Tumors were exposed to high-LET radiation in the distal 4-cm extended-peak ionization region of an accelerated neon-ion beam with an initial energy per atomic mass unit (u) of 557 MeV/u, as shown in Figure I. Tumors were irradiated in the distal portion of the extended-peak region to minimize the amount of residual radiation dose delivered to internal normal tissue structures. The variable-thickness absorber (ridge filter) used in these experiments was designed to produce a decreasing physical dose from the proximal to the distal end of the extended-peak region. This physical dose profile offsets the increasing RBE for cell killing across the extended-peak region (8), with a result that the tumor volume receives a nearly uniform biologically effective dose (defined as the product of RBE and physical dose). The residual range of this beam was 22 cm in water, and the dose-averaged LET, values within the tumor treatment zone ranged from 115 to 240 keV/pm. The beam collimation techniques, dosimetry, and tumor irradiation procedures used with accelerated charged-particle beams produced at the Berkeley BEVALAC facility have been described previously (9). The absorbed dose rate in the tumor mass ranged from 5 to 10 Gy/min during irradiations with peak neon ions. Irradiation of tumors was performed with 225-kVp X rays from a therapy unit,* using a total filtration of 0.35 mm Cu (mean photon energy = 75 kV; HVL = 1.08 mm Cu). Collimation of the X ray beam for tumor irradiations followed procedures described previously (9). The absorbed dose rate of X rays in the tumor tissue was 6 Gy/ min, as determined with a 250 R ionization chamber.?
0
I
I
I
I
I
5
10
15
20
25
Penetration
Depth
(cm
of
water)
Fig. 1. The depth-dose curve measured in water is shown for the neon-ion beam with an initial energy of 557 MeV/u. The peak ionization region was spread to a width of 4 cm by means of a variable-thickness absorber.
t Victoreen;
Cleveland,
OH.
Mixed high and low LET radiation 0 T. S. TENFORDE
The experimental animals were lightly anesthetized with Metofanet during tumor irradiation with both X rays and peak neon ions. RESULTS Rhabdomyosarcoma tumors were administered a priming dose of 7 Gy of high-LET peak neon ions, followed at intervals of 0.5,4.0, and 24.0 hr by 7.5, 15, and 25 Gy top-off doses of 225-kVp X rays. An identical procedure was used for split doses of X rays, except that the priming X ray dose was 20 Gy. A 7 Gy single dose of peak neon ions and a 20 Gy single dose of X rays produced nearly identical radiation-induced growth delays of 9.95 f 0.85 (S.E.) and 10.83 + 0.88 days, respectively, as shown in Figure 2. The RBE for a single dose of peak neon ions at the lo-day growth delay level was therefore 2.86 in the present experiments, in good agreement with earlier values determined for this tumor line (9, 26). The volume regression and regrowth curves for tumors administered a 7-Gy dose of peak neon ions and 20-, 30-, and 40-Gy doses of 225-kVp X rays are shown in Figure 2. In Figure 3 the volume response curves are plotted for split-dose schedules involving either a priming dose of neon ions followed by graded doses of X rays, or a priming dose of X rays followed by a second dose of X rays. The growth delay curves determined from the volume response data shown in Figures 2 and 3 are presented in Figure 4. These curves demonstrate that the rhabdomyosarcoma tumors exhibited substantial recovery between fractions in a split-dose X ray schedule. The data also indicate that the repair of sublethal damage was nearly complete when the interval between X ray dose fractions
r
100
c
,X-rays
(20 Gy)
X-rays(30Gy) X-rays
(40
Gy)
Radiation-Induced Growth
Delay
7 GymNe
0.1
20 30
I 10
SE.
(days)
9.95
k
0.85
GyX-rays(-): Gy X-rays (-):
10.03
t
0.88
40 GY X-rays
0
+
(---):
(j:
23.10
+
2.26
32.57
+
3.07
i 1 30 40 50 Days Postirradiation
20
I
I
60
70
Inc.; Washington
Crossing,
a/.
1531
was as short as 0.5 hr. By 4 hr the recovery from sublethal damage was complete as illustrated by the similar growth delays obtained with interfraction intervals of 4 and 24 hr. Only the data for total X ray doses of 35 and 45 Gy suggest that less recovery occurred with a 0.5 hr split-dose interval than with a 4-hr interval. However, an analysis of variance indicated that this difference is not statistically significant (0.05 < p < 0.10) and therefore only a single line has been drawn through the growth-delay data shown in Figure 4 for split doses of X rays. Overall, the splitdose data are consistent with a rapid recovery from sublethal X ray damage in the rat rhabdomyosarcoma tumors. The data presented in Figure 4 also demonstrate that the growth delay increments produced by administering top-off doses of X rays following a 7-Gy priming dose of peak neon ions were similar to those resulting from split doses of X rays in which the priming X ray dose was 20 Gy. As with split doses of X rays, the total growth delay response was not significantly different when the topoff doses of X rays were administered at 0.5, 4.0, or 24.0 hr following the priming 7-Gy dose of peak neon ions. In Table 1 a comparison is made between the top-off doses of X rays that were required to produce incremental growth delays of 10,20, or 30 days when the priming dose was 7 Gy of peak neon ions or 20 Gy of X rays. A parameter defined as the “incremental dose ratio” is used in Table 1 for this comparison. To form this ratio, the top-off doses of X rays that gave an additional 10,20, or 30 days of radiation-induced growth delay when the priming dose was 20 Gy of X rays were divided by the top-off doses of X rays that gave the same growth delay increments when the priming dose was 7 Gy of peak neon ions. The values of the incremental dose ratio listed in Table 1 range from 1.00 to 1.18, with an average value of 1.08. The fact that the incremental dose ratios have values close to unity indicates that little, if any, interaction occurred between the priming dose of high-LET neon ions and the subsequent top-off dose of low-LET X rays. Table 2 lists RBE values based on the growth-delay end point (RBE,) for mixed neon-ion and X ray irradiation of rhabdomyosarcoma tumors. RBE, values for the mixed-beam treatment were referenced to the split doses of X rays that produced an equivalent growth delay. As shown in Figure 5, the RBE, values are a linear function of the neon-ion dose fraction, f. A linear dependence of RBE, on f would be expected if no interaction occurred between the sequential doses of high- and low-LET radiation (6).
XBL 882 ‘Iii
Fig. 2. Volume regression and regrowth curves are plotted for groups of tumors that received either 7 Gy of peak neon ions or 20, 30, or 40 Gy of X rays. The dotted line is the growth curve for unirradiated tumors. The tumor volumes were normalized to unity on the day of irradiation. Data points represent the mean k 1 S.E. for groups of six tumors.
$ Pittman-Moore,
ef
NJ.
DISCUSSION In an early study of the combined effects of X rays and high-LET alpha particles on the proliferative capacity of human T- 1 kidney cells, Barendsen et al. (3) observed no
1532
I. J. Radiation Oncology 0 Biology 0 Physics
June 1989, Volume
16, Number
6
0.5 Hr Split Dose Interval
Growth Growth Pelav 0.1 -
0
, 10
I
20
+ SE.
20 Gy X-rays: 10.03 + 7 5Gy X-rays: 14.22 + 15 Gy X-rays.21 26 + 25 Gy X-rays:36.90 I 1 I 1 30 40 50 60
Ck k + ?
0.88 0.83 216 1.67 1
70
7 Gy + 7.5Gy + 15 Gy + 25 Gy
O.l-
0
I 10
1
20
1
30
Delav
+ S.E. f&y&
“Ne: 9.95 X-rays: 13.54 X-rays: 25.21 X-rays: 29.38
I 40
1 50
k _+ + t
I
60
0.85 2 16 2.12 3.06 1
70
Days Postirradiation
Days Postirradiation
4 Hr Split Dose Interval loo-
100
C
.D 7 GY 20Ne + 7.5 Gy X-rays
20 Gv X-rays .5 Gyi-rays + 25 Gy X-rays
F
Growth
Delay
LLS E !dWL
20 10.03 + 7.5Gy Gy X-rays. X-rays: 14.22 + 15 Gy X-rays 16.66 + 25 Gy X-rays, 28 79
0.1 F
i+ 5 ?
0 1 2 3
88 03 55 70
0
0%%-Cdd+O
IO
20
30
40
50
60
70
Days Postirradiation
Days Postirradiation
24 Hr Split Dose Interval 1OOr
- F 10
Normalized Tumor 1.0 Volume Growth 20 Gy + 7.5Gy + 15 Gy + 25 Gy
0.1
0
10
20
30
40
Delay
-t S.E
X-rays: X-rays. X-rays, X-rays
10 03 14 81 20 70 29.01
50
60
Growth
!&sL -r i f ?
Days Postirradiation
0.88 1.22 2 44 4 92
70
+ S.E. ki&!s~
9.95 + 7.5Gy X-rays: 13.24 + 15 Gy X-rays: 20.84 + 25 Gy X-rays: 38.18
0.1 -
0
Delay
I 10
I 20
/ 30
I 40
I 50
I 60
+ 2 + +
0 85 0.49 3.45 3 92
I 70
Days Postirradiation
Fig. 3. Volume response curves are plotted for tumors that received priming doses of 7 Gy of peak neon ions or 20 Gy of X rays, followed by graded X ray doses administered 0.5, 4.0, or 24.0 hr later. Data points represent the mean + 1 S.E. for groups of six tumors.
interaction between the effects of these two radiation modalities. Similar results were later obtained for Chinese hamster cells irradiated with alpha particles and gamma rays (11). The alpha particles used by Barendsen et al.
(3), which had an average LET of 170 keV/pm, produced a linear dose-response curve for cell killing. On this basis, Railton et al. (22) have proposed that the lack of interaction of alpha particles with X rays occurred because
Mixed high and low LET radiation 0 T. S. TENFORDE
v-“Ne o-
4-m
225.kVp
single dose
extended-peak X-my
single
dose Rat
‘ONe + x ray
Interval
-5o% 0 540
X my+ Xroy
0.5 hr 4 h,
. .
A 0
24
.
0
ilr
rhabdomyos~rcomo R-l tumorr in
I
WAG/RI1
rofs
T
8 f
z
30-
: E 2
20-
.L 5 f
IO-
.+3 no
I
I
IO
OO
I
I
20
30
Tot01
dose
IGy)
Fig. 4. Radiation-induced growth delay is plotted as a function of total absorbed dose for rhabdomyosarcoma tumors administered as single doses of extended-peak neon ions (7 Gy) or 225kVp X rays followed at 0.5,4.0, and 24.0 hr by second doses of X rays at dose levels of 7.5, 15, and 25 Gy. For comparison, the
growth delay induced by 20, 30, and 40 Gy single doses of X rays is plotted as a solid line. Data points represent the mean f 1 S.E. for groups of six tumors.
the alpha-particle killing mechanism involved only singlehit events. However, later studies by Ngo et al. (20) demonstrated that Bragg peak neon ions with an average LET of 183 keV/pm could interact synergistically with X rays in the inactivation of Chinese hamster V79 cells, even though the dose-response curve for killing by the neon ions was linear. Ngo et al. (20) demonstrated in split-dose experiments that the neon-ion beam was capable of producing sublethal damage, and that this damage could interact with a subsequent dose of X rays. These results therefore diverge from the findings in earlier experiments with alpha particles and X rays. One explanation of the lack of interaction observed in the early experiments with combined alpha-particle and X ray radiations, as pointed out by McNally et al. ( 18), is that the alpha-particle doses used were too small to induce a significant amount of sublethal damage with which the X rays could subsequently interact. Another interpretation of the combined alpha particle and X ray results is that the alpha particles had such a high LET that no “interactable” damage was
1533
d al.
produced. In the combined neon-ion and X ray experiments, the neon-ion beam had a broad range of LET, the lower-LET portion of which is expected to produce “interactable” damage. In contrast to the earlier studies of Barendsen et al. (3), the results of more recent experiments by Raju and Jett (23) on the killing of human kidney T1 cells exposed to a combination of plutonium alpha particles and X rays are consistent with a small degree of interaction between these two radiation modalities. The existence of synergistic interactions between highand low-LET radiation is supported also by other recent studies with charged-particle beams. Yuhas et al. (3 1) reported that doses of peak pions greater than 0.5 Gy eliminate the shoulder of the subsequent X ray survival curve in cultured CHO-Kl cells. Even at 4 hr following a 3-Gy dose of peak pions the X ray survival curve lacked its normal shoulder, indicating that the repair of pion-induced damage with which the X rays could interact was not complete. In growth delay measurements on spheroids, Yuhas et al. (31) also obtained evidence for a significant interaction between peak pions and X rays. Several in vitro studies have been performed to analyze the interactions between neutrons and low-LET photon radiation. An interaction between ‘j°Co gamma rays and 14-MeV neutrons was observed for cultured Chinese hamster cells by Railton et al. (22), who ascribed this effect to the low-LET portion of the neutron beam interacting with the gamma-ray dose. Using both V79 monolayer cultures and spheroids, Durand and Olive (10) also obtained evidence for the interaction of mixed beams of X rays and neutrons administered as split doses with an interval of 4 hr. They reported that no repair of sublethal damage occurred when either an X ray dose was followed by neutron irradiation, or a neutron dose was followed by X rays. Ngo et al. (2 1) subsequently obtained evidence for repair in V79 cells of neutron-induced sublethal damage that can interact with X ray-induced sublethal damage. This observation was made with both fission neutrons, for which the low-LET component is only 3-4%, and with high-energy neutrons. In a recent study with V79 Chinese hamster cells, McNally et al. (18) reported that neutrons and X rays
Table 1. Mixed neon-ion and X ray irradiation of rat rhabdomyosarcoma
tumors
X ray top-off dose (Gy) required to produce growth delay increment following priming dose of
Growth delay end point (days)
Growth delay increment relative to single doses of X rays or ‘“Ne ions (days)
20-Gy X ray
7-Gy “Ne
Dose increment ratio: [X ray + X ray]/[20Ne + X ray]*
20 30 40t
10 20 30
16.25 24.25 30.50
13.75 22.75 30.50
1.18 1.07 1.00
* The dose increment ratio is formed by dividing the [X ray + X ray] top-off dose by the [20Ne + X ray] top-off dose required to produce a specified increment in growth delay (either 10, 20 or 30 days of additional growth delay). t Data at the 40-day growth delay end point are extrapolated values taken from the growth-delay curves shown in Figure 4.
1. J. Radiation Oncology 0 Biology 0 Physics
1534
Table 2. RBE values for radiation-induced
growth delay
Growth delay
*“Ne
X ray
Percent of total
end point (days)
dose
dose*
dose from *“Ne
(GY)
(GY)
ions
RBL.t
10 20 30 40
7 7 7 7
0 13.75 22.75 30.50
100 34 24 19
2.86 1.75 1.49 1.35
* The top-off X ray dose required to produce a given level of growth delay was determined from the radiation-induced growth delay curves presented in Figure 4. t The RBE values for the mixed neon-ion X ray irradiation were referenced to the equivalent split doses of X rays that pro-
duced the same growth delay: RBE, = [X ray + X ray dose&J [‘“Ne + X ray dose], where g is the growth delay level in days. exerted a synergistic effect, provided that the neutron contribution to the total dose was less than 40%. When the neutron contribution to the dose exceeded this level, the two radiation modalities acted independently in reducing cell survival. The combined effects of neutrons and X rays were found by McNally et al. (18) to be the same whether the two radiation modalities were administered simultaneously, or sequentially in either order. This contrasted with an earlier report by Higgins et al. (13) that sequential irradiation of V79 cells by “Co gamma rays and 14.8-MeV neutrons gave a higher survival level than that resulting from simultaneous irradiation with the two modalities. However, the exposure dose rates used by Higgins et al. (13) were less than 0.1 Gy/min, and Zaider and Brenner (32) have concluded that this lowdose-rate exposure condition would make it difficult to observe damage interactions from sequential high- and low-LET radiations. Another recent in vitro study with V79 cells was reported by Iliakis et al. (15), who exposed plateau-phase
Fig. 5. RBE, values for radiation-induced growth delay are plotted as a function of the fraction, f, of the total dose administered to the tumors as peak neon ions. The straight line fitted to the data was obtained by linear regression analysis. The coefficient of determination, 3, is close to unity, thereby indicating that a linear relationship provides an excellent fit of the experimental data.
June 1989, Volume 16, Number 6
cultures to sequential doses of fast neutrons and ‘“Co gamma rays with intervals ranging from 0 to 6 hr between the doses. They observed a dose-dependent reduction in the gamma-ray survival level after pre-exposure of the cells to neutrons, and a similar effect occurred in the neutron survival curve when the order of irradiation was reversed. The interaction between these two radiation modalities also was found to decay with time between doses, with kinetics similar to the loss of sensitivity to the PLD repair inhibitor ,&arabinofuranosyladenine (/3-araA). Iliakis et al. (15) suggested on this basis that the interaction between neutrons and gamma rays may involve the set of potentially lethal lesions that cannot be repaired in the presence of /3-araA. The concept that high- and low-LET radiations may interact via a common set of lesions has been described in theoretical terms by Lam (16). In this mathematical model, it is assumed that a common intermediate stage exists in the pathways for lethal lesions produced by different types of radiation. If the lesion precursors of two types of radiation reach the same stage at the same time, then the irradiation effects will not be independent. It is implicit in this model that the time interval between sequential doses of high- and low-LET radiation must be short enough for lesion interactions to occur. Although strong evidence has been obtained for the interaction of mixed high- and low-LET radiation in cell cultures, conflicting observations have been made in previous studies on in vivo normal tissues and tumors exposed to mixed radiation modalities. In an investigation on the effects of a mixed daily fractionation schedule of 14 MeV neutrons and X rays administered to the mouse intestine, Hendry et al. ( 12) found that the effects of the two types of radiation were additive. A contrary finding was later reported by Hornsey et al. (14) who observed interaction of neutron and X ray damage to the mouse intestine when the radiation doses were separated by short intervals ranging from 15 min to 3-4 hr. They attributed the negative findings of Hendry et al. (12) to the fact that recovery of sublethal damage was probably complete within the l-day interval between neutron and X ray dose fractions. Two studies in which mixed regimens of low-LET radiation and neutron radiation were administered on a daily schedule to mouse tumors gave no evidence for an interaction of these two radiation modalities (2, 30). In both cases the tumor response to a daily fractionation schedule using mixed radiation modalities was less than that resulting from fractionated neutron radiation alone, and the overall response to an admixture of high- and low-LET radiation was consistent with that expected if the two radiation modalities acted independently. Two other studies with mouse tumors have led to the finding of a small therapeutic gain resulting from exposure to an admixture of neutrons and X rays given in a daily fractionation schedule ( 19,24). Based on tumor growth delay and 50% tumor cure end points, the combination of two
Mixed high and low LET radiation 0 T. S. TENFORDE
neutron fractions and three X ray fractions was found to reduce the required neutron dose per fraction in comparison with the dose per fraction required if fractionated neutron radiation was given alone. The fact that a small amount of interaction between neutrons and X rays was observed with a daily fractionation schedule in the experiments of Nelson et al. (19) and Rasey et al. (24) suggests that some long-lived, unrepaired lesions may have remained at 1 day following tumor irradiation. The results of experiments described here clearly indicate the absence of a significant amount of damage interaction in rat rhabdomyosarcoma tumors exposed to sequential doses of high-LET radiation from 225-kVp X rays. The lack of significant interaction between these radiation modalities was observed both for a daily interval between dose installments, comparable to the results of previous studies on normal tissues and tumors (2, 12,30), and for relatively short split-dose intervals of 0.5 and 4.0 hr. The fact that no significant interaction between peak neon ions and X rays was observed even with a 0.5-hr interfraction interval probably relates to the very rapid repair of sublethal and potentially lethal radiation lesions exhibited by the rat rhabdomyosarcoma R- 1 tumor line. Recent studies in our laboratory have demonstrated that potentially lethal damage repair by this tumor line is
ef a/.
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complete within an 3-hr interval following irradiation (l), and the results of experiments reported here indicate that sublethal damage repair is nearly complete by 0.5 hr and reaches completion by 4 hr following a priming dose of X rays. Taken together, these results are consistent with the concept that sublethal and potentially lethal repair are manifestations of the repair of the same lesions, as implied in the “lethal-potentially lethal lesion” model of Curtis (7). The observed absence of significant interaction between high- and low-LET radiation modalities in experimental animal tumors has direct implications for clinical radiotherapy procedures that involve daily treatments with mixed beams. The results of studies reported here, as well as previously published observations, suggest that the time interval between sequential treatments with high- and lowLET radiation should be made as short as possible in order to gain a therapeutic advantage in tumor treatment with mixed radiation modalities. In a mixed-beam tumor treatment regimen involving daily dose installments, the experimental information presently available indicates that appropriately RBE-matched doses of high- and lowLET radiation should be administered without assuming that an interaction occurs between the different radiation modalities.
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June 1989, Volume 16, Number 6 27. Tenforde, T. S.; Tenforde, S. D.; Crabtree, K. E.; Parks, D. L.; Schilling, W. A.; Parr, S. S.; Flynn, M. J.; Howard, J.; Lyman, J. T.; Curtis, S. B. RBE values for radiationinduced growth delay in rat rhabdomyosarcoma tumors exposed to plateau and peak carbon, neon and argon ions. Int. J. Radiat. Oncol. Biol. Phys. 7:2 17-221; 1981. 2%. Tobias, C. A.; Blakely, E. A.; Ngo, F. Q. H.; Yang, T. C. H. The repair-misrepair model of cell survival. In: Meyn, R. E., Withers, H. R., eds. Radiation biology in cancer research. New York, NY: Raven Press; 1980: 195-230. 29. Tsunemoto, H.; Morita, S.; Arai, T.; Kutsutani, Y.; Kurisu, A.; Umegaki, Y. Results of clinical trial with 30 MeV d-Be neutrons at NIRS. In: Abe, M., Sakamoto, K., Phillips, T. L., eds. Treatment of radioresistant cancer. Amsterdam: Elsevier/North-Holland; 1977: 1 l5- 126. 30. Urano, M.; Koike, S. Comparison of the effects of neutron and/or photon irradiation on spontaneous squamous-cell carcinoma in mice. Radiol. 134:219-225; 1980. 31. Yuhas, J. M.; Li, A. P.; Khgerman, M. M. Present status of the proposed use of negative pi mesons in radiotherapy. Adv. Radiat. Biol. 851-83; 1979. 32. Zaider, M.; Brenner, D. J. Comments on “V79 survival following simultaneous or sequential irradiation by l5-MeV neutrons and M’Cophotons.” Radiat. Res. 99:438-441; 1984. 33. Zaider, M.; Rossi, H. H. The synergistic effects of different radiations. Radiat. Res. 95:732-739; 1980.