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Radiotherapy of the rhabdomyosarcoma R1H of the rat: Kinetics of cellular inactivation by fractionated irradiation
lrr, .I. Rud,arwn On
14. pp. 317-325 Copyright
0360.3016/8X (c: 1988 Pergamon
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??Original Contribution
RADIOTHERAPY OF THE RHABDOMYOSARCOMA RlH OF THE RAT: KINETICS OF CELLULAR INACTIVATION BY FRACTIONATED IRRADIATION HUBERTVOGLER,PH.D.ANDHANS-PETERBECK-BORNHOLDT,PH.D. Institute of Biophysics and Radiobiology,
University of Hamburg, F.R.G.
The kinetics of cellular inactivation by fractionated irradiation in the RlH rhabdomyosarcoma of the rat was studied in the dose range of 1.07 to 12.50 Gy per fraction. Regimens of 1, 3, 5, 7, and 10 fractions per week for several weeks were compared. The number of clonogenic tumor cells per tumor in the course of the different treatment schedules was determined using an in vitro colony assay. The results show that the proliferation of clonogenic tumor cells is decelerated in the course of a fractionated radiotherapy. The deceleration persists for several days after end of treatment, until accelerated repopulation is initiated. The fraction of tumor cells inactivated per week was only dependent on the total dose per week, that is the cellular response was the same whether the weekly dose was applied in 1,3,5,7, or 10 fractions. Thus, the fractionation regimens were considerably more effective than expected from calculations based on single-dose in situ survival curves. Fractionation,
Radiotherapy,
Proliferation,
Clonogenic assay, Tumor.
eral weeks have been survival assay.
INTRODUCTION
examined
using
an excision
cell
The influence of total dose, overall treatment time and number of fractions on normal tissue damage has been studied extensively and a large amount of experimental data provide the basis for several mathematical of repair, reoxymodels.‘3’8338 Though the importance genation, repopulation and redistribution for radiotherapy has been investigated in many studies, the corresponding experiments on tumors were generally performed applying single- or split-dose irradiations. To our knowledge only nine studies on experimental tumors involve fractionation schedules with 10 or more fracti ens. 3,5,7,16,17,21,23,25,28,36 Th’ IS is perhaps surprising because the clinical optimum for fractionated radiotherapy seems to lie in the region of 30 and more fractions. In the last few years, accelerated and hyperfractionation have been discussed as new possibilities to improve radiotherapy by respectively reducing the time for tumor cell repopulation and sparing late reactions in normal tissues. I5 Thus > it seems reasonable to perform systematic studies about different fractionated irradiation schemes, to characterize tumor response to fractionated irradiation, enabling comparison to normal tissues and development of fractionation schemes with optimized therapeutic ratio. In this paper, changes in the number of clonogenic R 1H-tumor cells throughout irradiation regimens of 1, 3, 5, 7 and 10 fractions per week for sev-
The experiments were performed on the rhabdomyosarcoma RlH of the rat (volume doubling time 3 days). This transplantable solid tumor was derived from the rhabdomyosarcoma R- 1 described by Barendsen and Broerse.2 Originally the R- 1 tumor was derived from the Ba 1112 tumor which appeared spontaneously in 1962 in the musculature of a Wistar rat that had been irradiated 8 months before.32 Until 1966, this tumor was alternatingly cloned in vitro and transplanted in vitro. The R-l tumor was kindly provided by Prof. G. W. Barendsen in 1976 and has been maintained since then in our laboratory by serial transplantation on inbred WAG/Rij rats. In 1978 the DNA content of the tumor cells increased spontaneously from 1.4 times*’ to 4.3 times6,i9 that of host cells. The DNA-index has continuously decreased since then equaling about 3.8. Parallel with the increase in DNA-index, the volume of the tumor cells increased. These changes have proved to be highly advantageous because the tumor cells can be distinguished clearly from host cells not only in the flow cytometer, but also on histological slides and when cell suspensions are
Reprint requests to: Hubert Vogler, Institut fur Biophysik, Martin&r. 52, D-2000 Hamburg 20, F.R.G. Acknowledgements-The skillful technical assistance of Mrs.
Maria Omniczynski is gratefully acknowledged. Supported by the Deutsche Forschungsgemeinschaft (Ba 648/2-2). Accepted for publication 28 August 1987.
METHODS
AND MATERIALS
Tumor-host system
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counted in a counting chamber. Tumor take rate, determined after injecting 50 tumor cells per animal, was found to be 100% (20/20). The tumor is non-immunogenic (immunogenicity index 1) (F. Zywietz, oral communication, June, 1985) as was determined according to Embleton.14 Transplantation of the R 1H tumors was performed by implanting a piece of tumor tissue, about 1 mm3 in size, subcutaneously into the back of male WAG/R?) albino rats (200-220 gr) purchased from the TNO-REP institutes, Rijswijk, Netherlands. Three weeks after transplantation the tumors reached a weight of about 0.1 gr. The animals were kept under a 12 hr light, 12 hr dark cycle and provided with food and water ad libitum. The microbiological status of the animals was checked regularly. Irradiation Tumors were locally irradiated with 200 kVp X rays (0.5 mm Cu filtering; dose rate 2 Gy/min). Unanesthetized animals were immobilized in a jig which allowed local tumor irradiation without considerable irradiation of lungs and gut. The jig was built of a Plexiglas tube with variable length to be adjusted to the individual size of the animal. The tube shows an orifice for the tumor. The animals were shielded by a rotable lead cylinder (1 mm) that allowed to adjust the tumor precisely into the radiation beam. The correct positioning of the tumor during irradiation was monitored using a tv-camera. To obtain dose uniformity the tumors were irradiated from alternating sides from day to day. The tumors were irradiated under ambient conditions, except for measurement of the in situ hypoxic dose survival curve, where the animals were sacrificed 5 minutes prior to irradiation. The different fractionation schedules applied are illustrated in Figure 1. The tumors were irradiated with 1 (7.50, 10.00 or 12.50 Gy/fraction), 3 (2.00, 2.50, 2.67. 3.33 or 4.17 Gy/fraction), 5 (1.50, 2.00 or 2.50 Gy/fraction), and 10 (1.50, 2.00 or 2.50 Gy/fraction) fractions per week. The 10 fractions per week were applied from Monday to Friday with a time interval of 12 hr.
2
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TOTAL TREATMENT TIME (days) Fig. 1. Schematic presentation of the different fractionation regimens. Every single dash represents a dose fraction.
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February 1988. Volume 14. Number 2
Tumor volume Tumor volume was determined by measuring the tumors in two perpendicular dimensions using vernier calipers, assuming the shape to be a rotational ellipsoid and correcting for skin thickness. A one to one correlation was found between tumor volume and tumor weight determined after tumor excision. Volume was measured twice a week. For reasons of convenience tumors were included into the experiment only once a week. The minimal volume for inclusion was 0.2 cm3. Due to tumor growth from week to week the maximal volume included amounted to about 3.7 cm3 (median 1.1 cm3; 68% of the tumors had starting volumes between 0.7 and 1.8 cm3). Thus, the range of variation of the tumor volumes at start of treatment was about 1 decade. For the largest tumors absorption led to a dose difference of 20% between the proximal and the distal side of the tumor. This problem of dose inhomogeneity was partly overcome by alternating irradiation ofthe tumors from opposite sides, leaving a dose difference between tumor centre and periphery of less than 10%. However the dose difference between tumor center and periphery for most of the tumors was less than 5%, which with regard to other sources of error seems to be defendable. Colony assay The number of clonogenic tumor cells per tumor was determined at different intervals during fractionation treatment by means of an in vitro colony assay. Animals and were asphixiated in CO*, their tumors removed weighed, and single cell suspensions prepared by trypsinization. 34 The total number of tumor cells per tumor was determined using a hemocytometer, carefully discriminating between host and neoplastic cells. The discrimination between tumor and host cells in the hemocytometer was checked by flow cytometry and a satisfactory agreement was found. A yield of 2.8. lo6 tumor cells per gram tissue was obtained for untreated controls. The cells were subsequently plated in plastic petri dishes and the clonogenic fraction was determined. The mean plating efficiency PE of control tumors was 63%. The effects of cytotoxic treatments are often expressed as surviving fraction (SF = PE(treated)/PE(control)). However, during extended treatments the surviving fraction may be misleading because of preferential loss of sterilized cells or proliferation of survivors. To overcome this problem, the number of clonogenic tumor cells per tumor N has been used, since this takes account of treatment-induced cell yield changes7,‘9.34 and allows for proliferation. To compensate the relatively large range of tumor volumes at the start of treatment, the cell numbers were normalized to this volume Vo : N = Total tumor
cell number
per tumor*
PE/Vo. ( 1)
The cell yield per gram of tumor tissue was found to be independent of the tumor volume. A significant de-
Fractionated irradiation in the R 1H rhabdomyosarcoma
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Fig. 2. Dose survival curves of (a) Rl asphixiation in CO2 (0) and R 1H cells by fitting the linear-quadratic equation = 0.010 Gy-‘, /3 = 0.008 1 GY-~). The mined by the vertical distance between
10
. 10
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Flow cytometry Samples of the single cell suspensions prepared for the colony assay were fixed in ethanol and used for flow cytometric analysis. The cells were stained in the dark for 10 minutes with a fluorescent dye solution (10 pg/ml Hoechst 33258 dissolved in phosphate-buffered saline containing 25% ethanol). The flow cytometric measurements were performed using an ICP-22 detector* equipped with a high pressure mercury lamp, excitation filters BG38 and BG3t and a barrier filter &X10$. The histograms were evaluated with a computer program correcting for background.4
RESULTS Figure 2 shows the single-dose survival curves for Rl H rhabdomyosarcomas obtained under in vitro, in situ (airbreathing) and in situ (hypoxic) conditions. The solid lines indicate the fit obtained with the linear-quadratic
20
25
equation. The fraction of hypoxic cells under in situ airbreathing conditions can be determined by the vertical distance between the ambient data (broken line) and the curve for the completely hypoxic population.40 The line for the ambient points was calculated as follows: the fitted line of the hypoxic points was shifted to the ambient points to give the best least square fit considering the error bars. A hypoxic fraction of 50% is obtained with this method. However, due to statistical errors and intrinsic methodological problems29 the accuracy of this result is very poor. The cellular composition ofthe tumor cell suspensions prepared for colony assay was analyzed by flow cytometry using DNA stains (Fig. 3). This analysis revealed that untreated R 1H rhabdomyosarcomas contained a substantial subpopulation of diploid host cells that can be clearly distinguished from the nearly octoploid tumor cells.6,‘9 Figure 4 shows the number of all tumor cells (clonogenie and non-clonogenic) and the number of nucleated host cells released per gram as a function of total dose. An exponential decrease with a rate of 0.071 + 0.003 Gy-’ and 0.026 + 0.003 Gyp’, respectively, is observed. No significant difference between the different fractionation schedules was found. As mentioned previously, the surviving fraction can give a misleading impression of the overall effect of ex-
$ Leitz, Wetzlar, F.R.G.
,y
H cells irradiated in vitro; (b) RlH cells irradiated in situ 5 minutes after irradiated in situ in air breathing animals (A). The solid lines were obtained to the data (in vitro: cy = 0.20 Gy-‘, p = 0.041 Gym2, in vivo hypoxic: a fraction of hypoxic cells under in situ air-breathing conditions was deterthe ambient (broken line) and the completely hypoxic population.
crease in cell yield, that is number of tumor cells (clonogenie and non-clonogenic) released per gram of tumor tissue, was found with increasing dose. However, the dissociation efficiency, that is the proportion of clonogenic tumor cells released per clonogenic tumor cell in the tissue, was shown to be approximately constant in the course of a 6 week fractionated irradiation treatment8
* Phywe, Giittingen, F.R.G. t Jenaer Glaswerke Schott, Mainz,
b
F.R.G.
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1-a
Host cells
.I
-2_
-3
Tumour cells Pl 0
Gl/o
90
60
30
Relative
DNA content
120
per cell
Fig. 3. DNA histogram of the RI H tumor showing host and tumor cell population with their corresponding G l/GO and G2 peaks.
Total dose
Fig. 5. Surviving fraction of R 1H tumor cells in the course of two different fractionation schemes. (0) 3 fractions of 2.00, 2.50, and 2.67 Gy per week. (0) 10 fractions of 1.50, 2.00, and 2.50 Gy per week. Note that about the same dose of(O) is given in the threefold time in (0).
(Gy)
Fig. 4. Number of (a) tumor cells and (b) host cells suspended per gram of tumor tissue as a function of total dose. The number of tumor and of host cells decreases exponentially with a rate of 0.071 f 0.003 Gy-’ and 0.026 f 0.003 Gym’, respectively.
tended treatments due either to lysis of radiation sterilized cells or proliferation by survivors.’ The surviving fraction data (Fig. 5) may enable some assessment of the contributions of cell loss and proliferation during the different fractionation schedules. After an initial steep decrease. the surviving fraction plateaued at average doses of about 1 Gy per day, which suggests that an equilibrium between inactivation and removal ofdead tumor cells and proliferation of surviving cells may have been reached after about 30 days of treatment. For an average daily dose of about 3 Gy equilibrium was not reached, and the surviving fraction continued to fall with increasing total dose. To overcome the ambiguity of surviving fraction as an endpoint in fractionation experiments, we used the number of clonogenic tumor cells per tumor to assess the effect ofthe different fractionation treatments (Eq. 1). Figure 6 shows an example of the results obtained for a particular fractionation scheme (3 fractions per week and 2.00 Gy per fraction). The number of clonogenic tumor cells per tumor is plotted as a function of time after start of treatment. The solid line indicates the expected slope of the kinetics of clonogenic tumor cells in the course of a fractionated treatment: every fraction causes a reduction of clonogenic tumor cells (vertical segments) while in the time interval between the fractions, the surviving tumor cells proliferate (rising segments). As will be shown later on, the data of all fractionation schemes (Fig. 7) can be described by a common inactivation rate (l/Do) and a common proliferation rate (In 2/T,) in the course of treatment. Thus, the lines shown in Figures 6 and 7 are the result of a simultaneous fit of all data obtained for all fractionation schemes of this study. The simplest assumptions possible for the description of the inactivation and repopulation kinetics of clono-
Fractionated
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scribes the number of clonogenic tumor cells per tumor during fractionated irradiation as a function of time t and total dose D is obtained: N(t, D) = N(0, 0) exp(t In 2/Td - D/Do). Equation 4 can be transformed into a linear that can be used to determine Do and Td: In2 y=---x Td
(4)
expression
1 Do
with y=ln(s)/tandx=D/t.
n = 3ffw d = 2.0 Gy D/t
= 6.0
10
Days
after
Gy
20
start
30
LO
of treatment
Fig. 6. Illustration of the evaluation method for the colony assay data. The number of clonogenic tumor cells per tumor is plotted as a function of time during a treatment of 3 fractions of 2.00 Gy per week. The kinetics of cell inactivation in the course of a fractionated irradiation is assumed to depend on two main processes: inactivation due to irradiation (vertical segments) and proliferation of clonogenic tumor cells (rising segments).
genie tumor cells in the course of a fractionated irradiation treatment are a constant repopulation rate with time and a constant i’nactivation rate with dose. Then repopulation is given by:
t , N(t) = N(0) exp y ( d 1
(2)
where N(0) is the number of clonogenic tumor cells at start of treatment and N(t) the number of clonogenic tumor cells at time t. Td represents the doubling time for the number of clonogenic tumor cells N during treatment. If all fractions are assumed to be isoeffective, cell inactivation is described by: N(D) = N(0) exp(-D/Do),
Figure 8 shows all results of Figure 7 plotted as ln(N(t, D)/N(O, O))/t as a function of D/t. N(0, 0) was determined as the log mean value of all control tumors. The solid line shows the linear regression obtained for the data. The intercept with the y-axis yields In 2/Td and the slope yields 1/Do. All fractionation regimens can be fitted with one single set of parameters Td = 9 + 2 d and Do = 4.3 t 0.2 Gy. The solid lines in Figure 7 indicate the slopes calculated for each fractionation scheme using Eq. 5; the vertical lines represent the inactivation per fraction and the rising horizontal lines represent proliferation. There is a good agreement between fit and data, perhaps excepting the experiment with one fraction per week, where the inactivation rate seems to be higher as compared to the other regimens. The constant proliferation rate during treatment that was obtained from evaluation of the data shown in figure 7 seems to contradict the surviving fraction data of figure 5. As shown in figure 4, cell yield per gram tumor depends on total dose alone. Thus, the difference in surviving fraction indicates more repopulation in the 3 X 2.5 Gy per week schedule at later times in treatment (>30 days). Nevertheless, the general conclusion concerning a decelerated proliferation as compared to unirradiated tumors seems to be defendable. Additional experiments are necessary to elucidate the problem of proliferation during treatment. The cellular proliferation kinetics after end of treatment was investigated using a protocol where 2.50 Gy were delivered as one fraction per day for 10 days. From Figure 9, it is clear, that the number of clonogenic tumor cells remained fairly constant for the first 6 days after irradiation was finished. The data, however, are also consistent with a doubling time of 9 days as was determined for the clonogenic tumor cells in the course of the different fractionation schedules. By 10 days accelerated repopulation was apparent (doubling time 1.5 days).
(3)
where D is the total dose delivered up to the time t. If these two equations are combined, an equation that de-
(5)
DISCUSSION In the present cell depopulation
study the kinetics of clonogenic tumor during the administration of one to ten
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Fig. 7. The number of clonogenic tumor cells per tumor as a function of time after start of treatment is shown for the different fractionation regimens. The data were simultaneously fitted by a common inactivation rate of 4.3 i 0.2 Gy and a common proliferation rate corresponding to a doubling time of 9 2 2 days. The solid lines show the corresponding fit.
doses per week of fractionated radiotherapy to an experimental rat tumor was examined. It was found that the tumor response does not depend on the number of fractions at a given total dose. This finding was unexpected, since from extrapolation of the results obtained by single-dose and split-dose experiments a decreasing tumor response with increasing number of fractions is predicted. As already mentioned in the introduction, studies on experimental tumors using “clinical” fractionation and hyperfractionation schedules are scarce. We are aware of only one other study’ of the type described here, in which the kinetics of cellular inactivation by fractionated irradiation in Lewis lung carcinoma was determined by means of an excision survival assay. Regimens of one and two fractions per day (dose range: 2.3 to 6.5 Gy per fraction) for 10 days were compared and it was found that the fraction of tumor cells inactivated per day was only dependent on the total dose per day, that is, the cellular response was the same whether the daily dose was given in one or two fractions. The results of the present study confirm this finding for a very different experimental tumor system.
This independence of the tumor response in the number of fractions might be explained either if the acute survival curve had no shoulder or if it had a very large shoulder with a substantial initial slope (flexure dose above 12.5 GY).~~ However, the pronounced shoulders on the single-dose survival curves in Figure 2 suggest that these interpretations are very unlikely. There are several possibilities to explain why the tumor response is independent of the number of fractions: 1. Cell cycle effects: Irradiation could induce partial synchrony. The results would require an increasing number of cells in the sensitive phases with decreasing interfraction intervals. 2. Reoxygenation: Increasing number of fractions could spare endothelial cells and thus, improve reoxygenation.5 3. Diminution of repair capacity during fractionated irradiation: This effect has been reported for cells in ~i/ro.~~,~~,~’On the other hand in a comprehensive survey of fractionated data for tumors irradiated in situ it was shown that there seems to be no diminution of repair up to 10 fractions.42 The results of the present paper also show that the repopulation of clonogenic tumor cells is considerably de-
Fractionated irradiation in the RlH rhabdomyosarcoma
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T&9 +2d -lO-
Do=4.3*0.2
+0
Gy
3
2
1
c
5
L
6
D/t P,/d-‘I Fig. 8. Data of Figure 7 transformed according to Eq. 5.ln(N(t,D)/N(O,O))/t is plotted as a function of D/t according to Eqn. 5. The solid line shows the linear regression obtained for the data. The y-intercept yields In 2/Td and the slope yields l/Do. Error bars (S.D.) are shown only for a few points.
celerated during the course of treatment. This finding seems to be contradictory to other studies that report a compensatory accelerated proliferation following irradiation or other types of cytotoxic treatments,2,22,24,33.34 whereby the speed of proliferation seems to depend on
t
/ -ii
f-i L
I
0
1
”
2
‘1
4
Days after
‘1
end60f
‘1’
8
10
treatment
Fig. 9. Number of clonogenic tumor cells per tumor as a function of time after end of a fractionated course of 10 fractions of 2.50 Gy given in IO days. The number of clonogenic tumor cells was fairly constant between day 0 and 6. After this lag period repopulation is accelerated (Td = 1.5 days). The line until day 6 corresponds to a Td of 9 days which was the result of the analysis from Figure 7.
the dose that is, the amount of cell killing,22.35 and tumors which normally have a high cell loss seem to respond quicker.‘0,24 However, most of these experiments were single dose experiments, high doses were used and the repopulation response was measured after the end of treatment. In case of fractionated irradiation the response seems to be different. The repeated irradiations in short intervals within the fractionated treatment apparently delay the accelerated repopulation until the end of treatment. This delay seems to last for a considerable time period as is indicated by the results of the fractionation experiment shown in Figure 9, where the accelerated proliferation only starts after a delay of about 1 week. This phenomenon could be explained by radiation induced division delay.‘2~‘3,37~43 This delay roughly amounts to 2 hr/Gy,*’ which does not seem to be enough to explain the whole effect. More probable seems to be an explanation given by Suit et al.36 During fractionated radiotherapy of the C3H murine mammary carcinoma, the influence of the overall treatment time was found to be high under clamp conditions, lower under ambient conditions and nearly no influence was observed under hyperbaric oxygen. This result was interpreted by considering the local distribution of the surviving tumor cells. Under ambient irradiation conditions the cells of the hypoxic region show higher surviving fractions. Since these cells proliferate very slowly because of deprivation of other nutrients as well, the influence of overall treatment time on the tumor control dose is small. The data presented in this paper indicate that the extrapolation of results from single-dose and split-dose ex-
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periments to the situation during fractionated irradiation seems to be questionable. This view is supported for example by experiments showing that the oxygen enhancement ratio depends on the dose per fraction,9,3’ which in fractionated therapy is much smaller then in single dose experiments. Further, it is to be expected that in the course of a fractionated treatment repair, reoxygenation, repopulation, and redistribution interact with each other and are influenced by physiological changes caused by the irradiation. Thus, to find out the effect of a particular fractionation scheme with a high number of fractions it seems to be necessary to perform the complete scheme. Even top-up dose experiments can be misleading, because they are performed in a relatively short time not
February 1988. Volume 14, Number 2
including long-term physiological changes which may be of radiobiological importance. In this study no dose sparing in the tumor during hyperfractionation was found. Since dose sparing in normal tissues is seen during hyperfractionation, irradiation with 7 or 10 fractions per week should yield better results in treatment of the Rl H rhabdomyosarcoma. This does not seem to be a peculiarity of the RIH-tumor, since similar results have been reported for the Lewis lung carcinoma.’ To establish the generality of the superiority of hyperfractionation treatment, more studies with more fractions per day, using different methods. different tumor host systems, and different end points would be desirable.
REFERENCES 1. Barendsen,
G.W.: Dose fractionation, dose rate and isoeffect relationships for normal tissue responses. Int. J. Radiat. Oncol. Biol. Phys. 8: 1981-1997, 1982.
2. Barendsen,
3.
4.
5.
6.
7.
8.
9.
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. Europ. J. Cuncer 5: 373-393, 1969. Barendsen, G.W., Broerse, J.J.: Experimental radiotherapy of a rat rhabdomyosarcoma with 15 MeV neutrons and 300 kV x-rays. II. Effects of fractionated treatments, applied five times a week for several weeks. Europ. J. C’ancer 6: 89-109, 1970. Beck, H.-P.: Evaluation of flow cytometric data of human tumours. Correction procedures for background and cell aggregations. Cell Tissue Kinet. 13: 1’?3- I8 1, 1980. Beck-Bornholdt, H.-P.: Parameters affecting the response of experimental tumors to fractionated irradiation. Struhlentherapie (In press). Beck, H.-P., Omniczynski, M.: Comparison of the applicability of flow cytometry and autoradiography for cell kinetic studies. Radiotoxic effects of incorporated 3H-thymidin and tumour response to irradiation. Acta Puthol. Microbiol. Scund. A 274 (Suppl.): 327-330, 198 1. Beck-Bornholdt, H.-P., Peacock, J.H., Stephens, T.C.: Kinetics of cellular inactivation by fractionated and hyperfractionated irradiation in Lewis lung carcinoma. Znt. J. Radiat. Oncol. Biol. Phys. 11: 1171-I 179, 1985. Beck-Bornholdt, H.-P., Vogler, H.: The colony assay in dose fractionation experiments. In Rodent Tumor Models. In Experimental Cancer Therapy, Kallman, R.F. (Ed.). Oxford, Pergamon. 1987, pp. 106-107. Brosing, J.W., Palcic, B., Skarsgard, L.D.: Cell survival at low doses and the measurement of O.E.R. Radial. Res. 87:
501-507,198l. 10. Denekamp, J.: Cell Kinetics and Cancer Therapy. Springfield, IL, C. C. Thomas, 1982. 11. Denekamp, J., Stewart, F.A.: Evidence for repair capacity in mouse tumors relative to skin. Int. J. Rudiat. Oncol.
Biol. Phys. 5: 2003-20 10, 1979. 12. Dewey, W.C., Highfield, D.P.: G2-block in Chinese Hamster cells induced by x-irradiation, hyperthermia. cycloheximide, or actinomycin D. Radiut. Res. 65: 51 l-528, 1976. 13. Ehmann. U.K., Nagasawa, K., Petersen, D.F., Lett, J.T.: Symptoms of x-ray damage to radiosensitive mouse leukemic cells: asynchronous populations. Rudiut. Res. 60:
453-472,
1974.
14. Embleton. M.J.: How to determine tumourimmunogenicity. In Rodent Tumor Models in Experimental Cancer Therapy>. Kallman, R.F. (Ed.). New York, Pergamon Press. 1987, pp. 19-22. radiotherapy? Br. 15. Fowler, J.F.: What next in fractionated .I. Cancer 49 (Suppl. VI): 285-300. 1984. 16. Fowler, J.F., Sheldon, P.W., Harris, S.R., Hill, S.A., Ayres, S.E.: Relative effectiveness of 12-hourly fractionation and a non-uniform X-ray schedule in the optimum fractionation ofC3H mouse mammary tumours. Br. J. Radiol. 48:
58 1-589. 1975. 17. Gonzalez, D., Haveman, J.: Effects of irradiation by single or multiple fractions per day on a transplantable murine mamma carcinoma. Br. J. Rudiol. 55: 9 16-92 1, 1982. for normal tissue dam18. Hornsey, S.: Isoeffect relationships age: high and low linear energy transfer radiations. In The Biological Busis ofRadiotherapy>. New York, Elsevier Publishers. 1983, pp. 167- 197. 19. Jung, H., Beck, H.-P., Brammer, I., Zywietz, F.: Depopulation and repopulation of the RI H rhabdomyosarcoma of the rat after X-irradiation. Europ. .I Cancer 17: 375-386,
1981. Kal, H.B.: Proliferation behaviour of P and Q cells in a rat rhabdomyosarcoma after irradiation as determined by DNA measurements. Europ. J. Cuncer9: 753-757, 1973. 21. Kob, D.. Kloetzer, K.H., Arndt, J., Kriester, A., Magdon, E.: Ergebnisse tierexperimenteller Untersuchungen bei Anwendung unterschiedlicher Fraktionierungsrhythmen und Strahlenarten niedriger LET. Arch. Geschwulstforsch. 20
46: 529-537, 1976. und Repopulierung des 22. Kruger, H.-J.: Depopulierung transplantablen Rhabdomyosarkoms R 1H der Ratte nach Rontgenbestrahlung. Thesis, University of Hamburg, 1984. J.: Regeneration in tumours. Presented at 23. Kummermehr, the 33rd Annual Meeting of the Radiation Research Society. Los Angeles, 1985, (Abstr). 24. Kummermehr, J., Trott, K.-R.: Rate of repopulation in a slow and a fast growing mouse tumor. In Progress in Radio-Oncology, Karcher, K.H., Kogelnik, H.D., Reinhartz, G. (Eds.). New York, Raven Press. 1982, pp. 299-307. 25. Looney, W.B., Hopkins, H.A.: Solid tumor modells for the assessment of different treatment modalities: XXV. Comparison of the effect of one radiation fraction per day with multiple fractions per day (MFD) given either continuously or intermittently on tumor response and normal tis-
Fractionated irradiation in the R 1H rhabdomyosarcoma sue reaction,
Int. J. Radiat. Oncol. Biol. Ph~x 12: 203-
210, 1986. 26. McNally, N.J., de Ronde, J.: The effect of repeated small doses of radiation on recovery from sub-lethal damage by Chinese hamster cells irradiated in the plateau phase of growth. Int. J. Radiat. Biol. 29: 221-234, 1976. 21. Mitchell, J.B., Bedford, J.S., Bailey, SM.: Dose-rate effects in mammalian cells in culture. III. comparison of cell killing and cell proliferation during irradiation for six different cell lines. Radial. Res. 19: 537-555, 1979. 28. Moulder, J.E., Fischer, J.J., Milardo, R.: Time-dose relationships for the cure of an experimental rat tumor with fractionated radiation. Int. J. Radiat. Oncol. Biol. Phys. 1: 43 l-438, 1976. 29. Moulder, J.E.. Rockwell, S.: Hypoxic fractions of solid tumors: Experimental techniques, methods of analysis and a survey of existing data. Int. J. Rudiat. Oncol. Bioi. Phys.
10: 695-7 12, 1984. 30. Ngo, F.Q.H., Youngman. K., Suzuki, S., Koumoundouros, J.. Iliakis, G.: Evidence for reduced repair capacity for damage accumulation and repair in plateau phase C3H lOT1/2 cells following multiple dose irradiation with Xrays. Radiat. Rex 106: 380-395, 1986. 31. Palcic, B., Brosing, J.W., Skarsgard, L.D.: Survival measurements at low doses: Oxygen enhancement ratio. Br. J. Cancer 46: 980-984, 1982. 32. Reinhold, H.S.: A cell dispersion technique for use in quantitative transplantation studies with solid tumours. Europ. J. Cancer 1: 61-7 1, 1965. 33. Rowley, R., Hopkins, H.A.. Betsill, W.L., Ritenour, E.R., Looney, W.B.: Response and recovery kinetics of a solid tumor after irradiation. Br. J. Cancer 42: 586-595, 1980. 34. Stephens, T.C., Currie, G.A.. Peacock. J.H.: Repopulation of x-irradiated Lewis lung carcinoma by malignant cells
0 H. VOGLER AND H.-P. BECK-B• RNHOLDT and host macrophage
progenitors.
325
Br. J. Cancer 38: 573-
582, 1978. 35. Stephens, cytotoxic
T.C., Steel, G.G.: Regeneration of tumors after treatment. In Radiation Biology in Cancer Reseurch. New York, Raven Press. 1980, pp. 385-395. 36. Suit, H.D., Howes, A.E., Hunter, N.: Dependence of response of a C3H mammary carcinoma to fractionated irradiation on fraction number and intertreatment interval.
Radiat. Res. 72: 440-454,
1977.
prolifera37. Szczepanski, L.V., Trot& K.R.: Post-irradiation tion kinetics of a serially transplanted murine adenocarcinoma. Br. J. Radio/ 48: 200-208, 1975. 38. Thames, H.D., Withers, H.R., Peters, J.P., Fletcher, G.H.: Changes in early and late radiation responses with altered dose fractionation: implications for dose-survival relationships. Znt. J. Radiat. Oncol. Biol. Phys. 8: 219-226, 1982. 39. Tucker, S.L., Thames, H.D.: Flexure dose: the low-dose limit of effective fractionation. Int. J. Radiat. Oncol. Biol. Phys. 9: 1373-l 383, 1983. 40. Van Putten, L.M., Kallman, R.F.: Oxygenation status of a transplantable tumor during fractionated radiotherapy. J. Nut/. Cancer Inst. 40: 44 l-45 1, 1968. 41. Van Rongen, E.: Analysis of cell survival after multiple fractions per day and low-dose irradiation of two in-vitro cultured rat tumor cell lines. Radiat. Rex 104: 28-46,
1985. 42. Williams, M.V., Denekamp, J., Fowler, J.F.: A review ofd/ s ratios for experimental tumors: Implications for clinical Znt. J. Radiat. Oncol. Biol. studies of altered fractionation. Phvsics 11: 87-96, 1985. 43. Zywietz, F., Jung, H.: Partial synchronisation ofthree solid animal tumours by X-rays. Europ. J. Cancer 16: 13811388, 1980.
14. pp. 317-325 Copyright
0360.3016/8X (c: 1988 Pergamon
$3.00 + .oO Journals Ltd.
??Original Contribution
RADIOTHERAPY OF THE RHABDOMYOSARCOMA RlH OF THE RAT: KINETICS OF CELLULAR INACTIVATION BY FRACTIONATED IRRADIATION HUBERTVOGLER,PH.D.ANDHANS-PETERBECK-BORNHOLDT,PH.D. Institute of Biophysics and Radiobiology,
University of Hamburg, F.R.G.
The kinetics of cellular inactivation by fractionated irradiation in the RlH rhabdomyosarcoma of the rat was studied in the dose range of 1.07 to 12.50 Gy per fraction. Regimens of 1, 3, 5, 7, and 10 fractions per week for several weeks were compared. The number of clonogenic tumor cells per tumor in the course of the different treatment schedules was determined using an in vitro colony assay. The results show that the proliferation of clonogenic tumor cells is decelerated in the course of a fractionated radiotherapy. The deceleration persists for several days after end of treatment, until accelerated repopulation is initiated. The fraction of tumor cells inactivated per week was only dependent on the total dose per week, that is the cellular response was the same whether the weekly dose was applied in 1,3,5,7, or 10 fractions. Thus, the fractionation regimens were considerably more effective than expected from calculations based on single-dose in situ survival curves. Fractionation,
Radiotherapy,
Proliferation,
Clonogenic assay, Tumor.
eral weeks have been survival assay.
INTRODUCTION
examined
using
an excision
cell
The influence of total dose, overall treatment time and number of fractions on normal tissue damage has been studied extensively and a large amount of experimental data provide the basis for several mathematical of repair, reoxymodels.‘3’8338 Though the importance genation, repopulation and redistribution for radiotherapy has been investigated in many studies, the corresponding experiments on tumors were generally performed applying single- or split-dose irradiations. To our knowledge only nine studies on experimental tumors involve fractionation schedules with 10 or more fracti ens. 3,5,7,16,17,21,23,25,28,36 Th’ IS is perhaps surprising because the clinical optimum for fractionated radiotherapy seems to lie in the region of 30 and more fractions. In the last few years, accelerated and hyperfractionation have been discussed as new possibilities to improve radiotherapy by respectively reducing the time for tumor cell repopulation and sparing late reactions in normal tissues. I5 Thus > it seems reasonable to perform systematic studies about different fractionated irradiation schemes, to characterize tumor response to fractionated irradiation, enabling comparison to normal tissues and development of fractionation schemes with optimized therapeutic ratio. In this paper, changes in the number of clonogenic R 1H-tumor cells throughout irradiation regimens of 1, 3, 5, 7 and 10 fractions per week for sev-
The experiments were performed on the rhabdomyosarcoma RlH of the rat (volume doubling time 3 days). This transplantable solid tumor was derived from the rhabdomyosarcoma R- 1 described by Barendsen and Broerse.2 Originally the R- 1 tumor was derived from the Ba 1112 tumor which appeared spontaneously in 1962 in the musculature of a Wistar rat that had been irradiated 8 months before.32 Until 1966, this tumor was alternatingly cloned in vitro and transplanted in vitro. The R-l tumor was kindly provided by Prof. G. W. Barendsen in 1976 and has been maintained since then in our laboratory by serial transplantation on inbred WAG/Rij rats. In 1978 the DNA content of the tumor cells increased spontaneously from 1.4 times*’ to 4.3 times6,i9 that of host cells. The DNA-index has continuously decreased since then equaling about 3.8. Parallel with the increase in DNA-index, the volume of the tumor cells increased. These changes have proved to be highly advantageous because the tumor cells can be distinguished clearly from host cells not only in the flow cytometer, but also on histological slides and when cell suspensions are
Reprint requests to: Hubert Vogler, Institut fur Biophysik, Martin&r. 52, D-2000 Hamburg 20, F.R.G. Acknowledgements-The skillful technical assistance of Mrs.
Maria Omniczynski is gratefully acknowledged. Supported by the Deutsche Forschungsgemeinschaft (Ba 648/2-2). Accepted for publication 28 August 1987.
METHODS
AND MATERIALS
Tumor-host system
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counted in a counting chamber. Tumor take rate, determined after injecting 50 tumor cells per animal, was found to be 100% (20/20). The tumor is non-immunogenic (immunogenicity index 1) (F. Zywietz, oral communication, June, 1985) as was determined according to Embleton.14 Transplantation of the R 1H tumors was performed by implanting a piece of tumor tissue, about 1 mm3 in size, subcutaneously into the back of male WAG/R?) albino rats (200-220 gr) purchased from the TNO-REP institutes, Rijswijk, Netherlands. Three weeks after transplantation the tumors reached a weight of about 0.1 gr. The animals were kept under a 12 hr light, 12 hr dark cycle and provided with food and water ad libitum. The microbiological status of the animals was checked regularly. Irradiation Tumors were locally irradiated with 200 kVp X rays (0.5 mm Cu filtering; dose rate 2 Gy/min). Unanesthetized animals were immobilized in a jig which allowed local tumor irradiation without considerable irradiation of lungs and gut. The jig was built of a Plexiglas tube with variable length to be adjusted to the individual size of the animal. The tube shows an orifice for the tumor. The animals were shielded by a rotable lead cylinder (1 mm) that allowed to adjust the tumor precisely into the radiation beam. The correct positioning of the tumor during irradiation was monitored using a tv-camera. To obtain dose uniformity the tumors were irradiated from alternating sides from day to day. The tumors were irradiated under ambient conditions, except for measurement of the in situ hypoxic dose survival curve, where the animals were sacrificed 5 minutes prior to irradiation. The different fractionation schedules applied are illustrated in Figure 1. The tumors were irradiated with 1 (7.50, 10.00 or 12.50 Gy/fraction), 3 (2.00, 2.50, 2.67. 3.33 or 4.17 Gy/fraction), 5 (1.50, 2.00 or 2.50 Gy/fraction), and 10 (1.50, 2.00 or 2.50 Gy/fraction) fractions per week. The 10 fractions per week were applied from Monday to Friday with a time interval of 12 hr.
2
+I 2 ; ; 2 z
‘8 30 30 0
I I I I I III III III III III III 1111111111 1111111111 llllllllll lllll Illll lllll lllll Illll IIIII IlIIIIIIllIIIlIIIIlllIlIIIIlIIIIllIIIIIIll 1
0
10
20
30
LO
TOTAL TREATMENT TIME (days) Fig. 1. Schematic presentation of the different fractionation regimens. Every single dash represents a dose fraction.
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February 1988. Volume 14. Number 2
Tumor volume Tumor volume was determined by measuring the tumors in two perpendicular dimensions using vernier calipers, assuming the shape to be a rotational ellipsoid and correcting for skin thickness. A one to one correlation was found between tumor volume and tumor weight determined after tumor excision. Volume was measured twice a week. For reasons of convenience tumors were included into the experiment only once a week. The minimal volume for inclusion was 0.2 cm3. Due to tumor growth from week to week the maximal volume included amounted to about 3.7 cm3 (median 1.1 cm3; 68% of the tumors had starting volumes between 0.7 and 1.8 cm3). Thus, the range of variation of the tumor volumes at start of treatment was about 1 decade. For the largest tumors absorption led to a dose difference of 20% between the proximal and the distal side of the tumor. This problem of dose inhomogeneity was partly overcome by alternating irradiation ofthe tumors from opposite sides, leaving a dose difference between tumor centre and periphery of less than 10%. However the dose difference between tumor center and periphery for most of the tumors was less than 5%, which with regard to other sources of error seems to be defendable. Colony assay The number of clonogenic tumor cells per tumor was determined at different intervals during fractionation treatment by means of an in vitro colony assay. Animals and were asphixiated in CO*, their tumors removed weighed, and single cell suspensions prepared by trypsinization. 34 The total number of tumor cells per tumor was determined using a hemocytometer, carefully discriminating between host and neoplastic cells. The discrimination between tumor and host cells in the hemocytometer was checked by flow cytometry and a satisfactory agreement was found. A yield of 2.8. lo6 tumor cells per gram tissue was obtained for untreated controls. The cells were subsequently plated in plastic petri dishes and the clonogenic fraction was determined. The mean plating efficiency PE of control tumors was 63%. The effects of cytotoxic treatments are often expressed as surviving fraction (SF = PE(treated)/PE(control)). However, during extended treatments the surviving fraction may be misleading because of preferential loss of sterilized cells or proliferation of survivors. To overcome this problem, the number of clonogenic tumor cells per tumor N has been used, since this takes account of treatment-induced cell yield changes7,‘9.34 and allows for proliferation. To compensate the relatively large range of tumor volumes at the start of treatment, the cell numbers were normalized to this volume Vo : N = Total tumor
cell number
per tumor*
PE/Vo. ( 1)
The cell yield per gram of tumor tissue was found to be independent of the tumor volume. A significant de-
Fractionated irradiation in the R 1H rhabdomyosarcoma
\
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319
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+a %f 4\ 4’ \
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Fig. 2. Dose survival curves of (a) Rl asphixiation in CO2 (0) and R 1H cells by fitting the linear-quadratic equation = 0.010 Gy-‘, /3 = 0.008 1 GY-~). The mined by the vertical distance between
10
. 10
15
Flow cytometry Samples of the single cell suspensions prepared for the colony assay were fixed in ethanol and used for flow cytometric analysis. The cells were stained in the dark for 10 minutes with a fluorescent dye solution (10 pg/ml Hoechst 33258 dissolved in phosphate-buffered saline containing 25% ethanol). The flow cytometric measurements were performed using an ICP-22 detector* equipped with a high pressure mercury lamp, excitation filters BG38 and BG3t and a barrier filter &X10$. The histograms were evaluated with a computer program correcting for background.4
RESULTS Figure 2 shows the single-dose survival curves for Rl H rhabdomyosarcomas obtained under in vitro, in situ (airbreathing) and in situ (hypoxic) conditions. The solid lines indicate the fit obtained with the linear-quadratic
20
25
equation. The fraction of hypoxic cells under in situ airbreathing conditions can be determined by the vertical distance between the ambient data (broken line) and the curve for the completely hypoxic population.40 The line for the ambient points was calculated as follows: the fitted line of the hypoxic points was shifted to the ambient points to give the best least square fit considering the error bars. A hypoxic fraction of 50% is obtained with this method. However, due to statistical errors and intrinsic methodological problems29 the accuracy of this result is very poor. The cellular composition ofthe tumor cell suspensions prepared for colony assay was analyzed by flow cytometry using DNA stains (Fig. 3). This analysis revealed that untreated R 1H rhabdomyosarcomas contained a substantial subpopulation of diploid host cells that can be clearly distinguished from the nearly octoploid tumor cells.6,‘9 Figure 4 shows the number of all tumor cells (clonogenie and non-clonogenic) and the number of nucleated host cells released per gram as a function of total dose. An exponential decrease with a rate of 0.071 + 0.003 Gy-’ and 0.026 + 0.003 Gyp’, respectively, is observed. No significant difference between the different fractionation schedules was found. As mentioned previously, the surviving fraction can give a misleading impression of the overall effect of ex-
$ Leitz, Wetzlar, F.R.G.
,y
H cells irradiated in vitro; (b) RlH cells irradiated in situ 5 minutes after irradiated in situ in air breathing animals (A). The solid lines were obtained to the data (in vitro: cy = 0.20 Gy-‘, p = 0.041 Gym2, in vivo hypoxic: a fraction of hypoxic cells under in situ air-breathing conditions was deterthe ambient (broken line) and the completely hypoxic population.
crease in cell yield, that is number of tumor cells (clonogenie and non-clonogenic) released per gram of tumor tissue, was found with increasing dose. However, the dissociation efficiency, that is the proportion of clonogenic tumor cells released per clonogenic tumor cell in the tissue, was shown to be approximately constant in the course of a 6 week fractionated irradiation treatment8
* Phywe, Giittingen, F.R.G. t Jenaer Glaswerke Schott, Mainz,
b
F.R.G.
I. J. Radiation
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1-a
Host cells
.I
-2_
-3
Tumour cells Pl 0
Gl/o
90
60
30
Relative
DNA content
120
per cell
Fig. 3. DNA histogram of the RI H tumor showing host and tumor cell population with their corresponding G l/GO and G2 peaks.
Total dose
Fig. 5. Surviving fraction of R 1H tumor cells in the course of two different fractionation schemes. (0) 3 fractions of 2.00, 2.50, and 2.67 Gy per week. (0) 10 fractions of 1.50, 2.00, and 2.50 Gy per week. Note that about the same dose of(O) is given in the threefold time in (0).
(Gy)
Fig. 4. Number of (a) tumor cells and (b) host cells suspended per gram of tumor tissue as a function of total dose. The number of tumor and of host cells decreases exponentially with a rate of 0.071 f 0.003 Gy-’ and 0.026 f 0.003 Gym’, respectively.
tended treatments due either to lysis of radiation sterilized cells or proliferation by survivors.’ The surviving fraction data (Fig. 5) may enable some assessment of the contributions of cell loss and proliferation during the different fractionation schedules. After an initial steep decrease. the surviving fraction plateaued at average doses of about 1 Gy per day, which suggests that an equilibrium between inactivation and removal ofdead tumor cells and proliferation of surviving cells may have been reached after about 30 days of treatment. For an average daily dose of about 3 Gy equilibrium was not reached, and the surviving fraction continued to fall with increasing total dose. To overcome the ambiguity of surviving fraction as an endpoint in fractionation experiments, we used the number of clonogenic tumor cells per tumor to assess the effect ofthe different fractionation treatments (Eq. 1). Figure 6 shows an example of the results obtained for a particular fractionation scheme (3 fractions per week and 2.00 Gy per fraction). The number of clonogenic tumor cells per tumor is plotted as a function of time after start of treatment. The solid line indicates the expected slope of the kinetics of clonogenic tumor cells in the course of a fractionated treatment: every fraction causes a reduction of clonogenic tumor cells (vertical segments) while in the time interval between the fractions, the surviving tumor cells proliferate (rising segments). As will be shown later on, the data of all fractionation schemes (Fig. 7) can be described by a common inactivation rate (l/Do) and a common proliferation rate (In 2/T,) in the course of treatment. Thus, the lines shown in Figures 6 and 7 are the result of a simultaneous fit of all data obtained for all fractionation schemes of this study. The simplest assumptions possible for the description of the inactivation and repopulation kinetics of clono-
Fractionated
irradiation
in the R 1H rhabdomyosarcoma
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scribes the number of clonogenic tumor cells per tumor during fractionated irradiation as a function of time t and total dose D is obtained: N(t, D) = N(0, 0) exp(t In 2/Td - D/Do). Equation 4 can be transformed into a linear that can be used to determine Do and Td: In2 y=---x Td
(4)
expression
1 Do
with y=ln(s)/tandx=D/t.
n = 3ffw d = 2.0 Gy D/t
= 6.0
10
Days
after
Gy
20
start
30
LO
of treatment
Fig. 6. Illustration of the evaluation method for the colony assay data. The number of clonogenic tumor cells per tumor is plotted as a function of time during a treatment of 3 fractions of 2.00 Gy per week. The kinetics of cell inactivation in the course of a fractionated irradiation is assumed to depend on two main processes: inactivation due to irradiation (vertical segments) and proliferation of clonogenic tumor cells (rising segments).
genie tumor cells in the course of a fractionated irradiation treatment are a constant repopulation rate with time and a constant i’nactivation rate with dose. Then repopulation is given by:
t , N(t) = N(0) exp y ( d 1
(2)
where N(0) is the number of clonogenic tumor cells at start of treatment and N(t) the number of clonogenic tumor cells at time t. Td represents the doubling time for the number of clonogenic tumor cells N during treatment. If all fractions are assumed to be isoeffective, cell inactivation is described by: N(D) = N(0) exp(-D/Do),
Figure 8 shows all results of Figure 7 plotted as ln(N(t, D)/N(O, O))/t as a function of D/t. N(0, 0) was determined as the log mean value of all control tumors. The solid line shows the linear regression obtained for the data. The intercept with the y-axis yields In 2/Td and the slope yields 1/Do. All fractionation regimens can be fitted with one single set of parameters Td = 9 + 2 d and Do = 4.3 t 0.2 Gy. The solid lines in Figure 7 indicate the slopes calculated for each fractionation scheme using Eq. 5; the vertical lines represent the inactivation per fraction and the rising horizontal lines represent proliferation. There is a good agreement between fit and data, perhaps excepting the experiment with one fraction per week, where the inactivation rate seems to be higher as compared to the other regimens. The constant proliferation rate during treatment that was obtained from evaluation of the data shown in figure 7 seems to contradict the surviving fraction data of figure 5. As shown in figure 4, cell yield per gram tumor depends on total dose alone. Thus, the difference in surviving fraction indicates more repopulation in the 3 X 2.5 Gy per week schedule at later times in treatment (>30 days). Nevertheless, the general conclusion concerning a decelerated proliferation as compared to unirradiated tumors seems to be defendable. Additional experiments are necessary to elucidate the problem of proliferation during treatment. The cellular proliferation kinetics after end of treatment was investigated using a protocol where 2.50 Gy were delivered as one fraction per day for 10 days. From Figure 9, it is clear, that the number of clonogenic tumor cells remained fairly constant for the first 6 days after irradiation was finished. The data, however, are also consistent with a doubling time of 9 days as was determined for the clonogenic tumor cells in the course of the different fractionation schedules. By 10 days accelerated repopulation was apparent (doubling time 1.5 days).
(3)
where D is the total dose delivered up to the time t. If these two equations are combined, an equation that de-
(5)
DISCUSSION In the present cell depopulation
study the kinetics of clonogenic tumor during the administration of one to ten
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Fig. 7. The number of clonogenic tumor cells per tumor as a function of time after start of treatment is shown for the different fractionation regimens. The data were simultaneously fitted by a common inactivation rate of 4.3 i 0.2 Gy and a common proliferation rate corresponding to a doubling time of 9 2 2 days. The solid lines show the corresponding fit.
doses per week of fractionated radiotherapy to an experimental rat tumor was examined. It was found that the tumor response does not depend on the number of fractions at a given total dose. This finding was unexpected, since from extrapolation of the results obtained by single-dose and split-dose experiments a decreasing tumor response with increasing number of fractions is predicted. As already mentioned in the introduction, studies on experimental tumors using “clinical” fractionation and hyperfractionation schedules are scarce. We are aware of only one other study’ of the type described here, in which the kinetics of cellular inactivation by fractionated irradiation in Lewis lung carcinoma was determined by means of an excision survival assay. Regimens of one and two fractions per day (dose range: 2.3 to 6.5 Gy per fraction) for 10 days were compared and it was found that the fraction of tumor cells inactivated per day was only dependent on the total dose per day, that is, the cellular response was the same whether the daily dose was given in one or two fractions. The results of the present study confirm this finding for a very different experimental tumor system.
This independence of the tumor response in the number of fractions might be explained either if the acute survival curve had no shoulder or if it had a very large shoulder with a substantial initial slope (flexure dose above 12.5 GY).~~ However, the pronounced shoulders on the single-dose survival curves in Figure 2 suggest that these interpretations are very unlikely. There are several possibilities to explain why the tumor response is independent of the number of fractions: 1. Cell cycle effects: Irradiation could induce partial synchrony. The results would require an increasing number of cells in the sensitive phases with decreasing interfraction intervals. 2. Reoxygenation: Increasing number of fractions could spare endothelial cells and thus, improve reoxygenation.5 3. Diminution of repair capacity during fractionated irradiation: This effect has been reported for cells in ~i/ro.~~,~~,~’On the other hand in a comprehensive survey of fractionated data for tumors irradiated in situ it was shown that there seems to be no diminution of repair up to 10 fractions.42 The results of the present paper also show that the repopulation of clonogenic tumor cells is considerably de-
Fractionated irradiation in the RlH rhabdomyosarcoma
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323
T&9 +2d -lO-
Do=4.3*0.2
+0
Gy
3
2
1
c
5
L
6
D/t P,/d-‘I Fig. 8. Data of Figure 7 transformed according to Eq. 5.ln(N(t,D)/N(O,O))/t is plotted as a function of D/t according to Eqn. 5. The solid line shows the linear regression obtained for the data. The y-intercept yields In 2/Td and the slope yields l/Do. Error bars (S.D.) are shown only for a few points.
celerated during the course of treatment. This finding seems to be contradictory to other studies that report a compensatory accelerated proliferation following irradiation or other types of cytotoxic treatments,2,22,24,33.34 whereby the speed of proliferation seems to depend on
t
/ -ii
f-i L
I
0
1
”
2
‘1
4
Days after
‘1
end60f
‘1’
8
10
treatment
Fig. 9. Number of clonogenic tumor cells per tumor as a function of time after end of a fractionated course of 10 fractions of 2.50 Gy given in IO days. The number of clonogenic tumor cells was fairly constant between day 0 and 6. After this lag period repopulation is accelerated (Td = 1.5 days). The line until day 6 corresponds to a Td of 9 days which was the result of the analysis from Figure 7.
the dose that is, the amount of cell killing,22.35 and tumors which normally have a high cell loss seem to respond quicker.‘0,24 However, most of these experiments were single dose experiments, high doses were used and the repopulation response was measured after the end of treatment. In case of fractionated irradiation the response seems to be different. The repeated irradiations in short intervals within the fractionated treatment apparently delay the accelerated repopulation until the end of treatment. This delay seems to last for a considerable time period as is indicated by the results of the fractionation experiment shown in Figure 9, where the accelerated proliferation only starts after a delay of about 1 week. This phenomenon could be explained by radiation induced division delay.‘2~‘3,37~43 This delay roughly amounts to 2 hr/Gy,*’ which does not seem to be enough to explain the whole effect. More probable seems to be an explanation given by Suit et al.36 During fractionated radiotherapy of the C3H murine mammary carcinoma, the influence of the overall treatment time was found to be high under clamp conditions, lower under ambient conditions and nearly no influence was observed under hyperbaric oxygen. This result was interpreted by considering the local distribution of the surviving tumor cells. Under ambient irradiation conditions the cells of the hypoxic region show higher surviving fractions. Since these cells proliferate very slowly because of deprivation of other nutrients as well, the influence of overall treatment time on the tumor control dose is small. The data presented in this paper indicate that the extrapolation of results from single-dose and split-dose ex-
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periments to the situation during fractionated irradiation seems to be questionable. This view is supported for example by experiments showing that the oxygen enhancement ratio depends on the dose per fraction,9,3’ which in fractionated therapy is much smaller then in single dose experiments. Further, it is to be expected that in the course of a fractionated treatment repair, reoxygenation, repopulation, and redistribution interact with each other and are influenced by physiological changes caused by the irradiation. Thus, to find out the effect of a particular fractionation scheme with a high number of fractions it seems to be necessary to perform the complete scheme. Even top-up dose experiments can be misleading, because they are performed in a relatively short time not
February 1988. Volume 14, Number 2
including long-term physiological changes which may be of radiobiological importance. In this study no dose sparing in the tumor during hyperfractionation was found. Since dose sparing in normal tissues is seen during hyperfractionation, irradiation with 7 or 10 fractions per week should yield better results in treatment of the Rl H rhabdomyosarcoma. This does not seem to be a peculiarity of the RIH-tumor, since similar results have been reported for the Lewis lung carcinoma.’ To establish the generality of the superiority of hyperfractionation treatment, more studies with more fractions per day, using different methods. different tumor host systems, and different end points would be desirable.
REFERENCES 1. Barendsen,
G.W.: Dose fractionation, dose rate and isoeffect relationships for normal tissue responses. Int. J. Radiat. Oncol. Biol. Phys. 8: 1981-1997, 1982.
2. Barendsen,
3.
4.
5.
6.
7.
8.
9.
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. Europ. J. Cuncer 5: 373-393, 1969. Barendsen, G.W., Broerse, J.J.: Experimental radiotherapy of a rat rhabdomyosarcoma with 15 MeV neutrons and 300 kV x-rays. II. Effects of fractionated treatments, applied five times a week for several weeks. Europ. J. C’ancer 6: 89-109, 1970. Beck, H.-P.: Evaluation of flow cytometric data of human tumours. Correction procedures for background and cell aggregations. Cell Tissue Kinet. 13: 1’?3- I8 1, 1980. Beck-Bornholdt, H.-P.: Parameters affecting the response of experimental tumors to fractionated irradiation. Struhlentherapie (In press). Beck, H.-P., Omniczynski, M.: Comparison of the applicability of flow cytometry and autoradiography for cell kinetic studies. Radiotoxic effects of incorporated 3H-thymidin and tumour response to irradiation. Acta Puthol. Microbiol. Scund. A 274 (Suppl.): 327-330, 198 1. Beck-Bornholdt, H.-P., Peacock, J.H., Stephens, T.C.: Kinetics of cellular inactivation by fractionated and hyperfractionated irradiation in Lewis lung carcinoma. Znt. J. Radiat. Oncol. Biol. Phys. 11: 1171-I 179, 1985. Beck-Bornholdt, H.-P., Vogler, H.: The colony assay in dose fractionation experiments. In Rodent Tumor Models. In Experimental Cancer Therapy, Kallman, R.F. (Ed.). Oxford, Pergamon. 1987, pp. 106-107. Brosing, J.W., Palcic, B., Skarsgard, L.D.: Cell survival at low doses and the measurement of O.E.R. Radial. Res. 87:
501-507,198l. 10. Denekamp, J.: Cell Kinetics and Cancer Therapy. Springfield, IL, C. C. Thomas, 1982. 11. Denekamp, J., Stewart, F.A.: Evidence for repair capacity in mouse tumors relative to skin. Int. J. Rudiat. Oncol.
Biol. Phys. 5: 2003-20 10, 1979. 12. Dewey, W.C., Highfield, D.P.: G2-block in Chinese Hamster cells induced by x-irradiation, hyperthermia. cycloheximide, or actinomycin D. Radiut. Res. 65: 51 l-528, 1976. 13. Ehmann. U.K., Nagasawa, K., Petersen, D.F., Lett, J.T.: Symptoms of x-ray damage to radiosensitive mouse leukemic cells: asynchronous populations. Rudiut. Res. 60:
453-472,
1974.
14. Embleton. M.J.: How to determine tumourimmunogenicity. In Rodent Tumor Models in Experimental Cancer Therapy>. Kallman, R.F. (Ed.). New York, Pergamon Press. 1987, pp. 19-22. radiotherapy? Br. 15. Fowler, J.F.: What next in fractionated .I. Cancer 49 (Suppl. VI): 285-300. 1984. 16. Fowler, J.F., Sheldon, P.W., Harris, S.R., Hill, S.A., Ayres, S.E.: Relative effectiveness of 12-hourly fractionation and a non-uniform X-ray schedule in the optimum fractionation ofC3H mouse mammary tumours. Br. J. Radiol. 48:
58 1-589. 1975. 17. Gonzalez, D., Haveman, J.: Effects of irradiation by single or multiple fractions per day on a transplantable murine mamma carcinoma. Br. J. Rudiol. 55: 9 16-92 1, 1982. for normal tissue dam18. Hornsey, S.: Isoeffect relationships age: high and low linear energy transfer radiations. In The Biological Busis ofRadiotherapy>. New York, Elsevier Publishers. 1983, pp. 167- 197. 19. Jung, H., Beck, H.-P., Brammer, I., Zywietz, F.: Depopulation and repopulation of the RI H rhabdomyosarcoma of the rat after X-irradiation. Europ. .I Cancer 17: 375-386,
1981. Kal, H.B.: Proliferation behaviour of P and Q cells in a rat rhabdomyosarcoma after irradiation as determined by DNA measurements. Europ. J. Cuncer9: 753-757, 1973. 21. Kob, D.. Kloetzer, K.H., Arndt, J., Kriester, A., Magdon, E.: Ergebnisse tierexperimenteller Untersuchungen bei Anwendung unterschiedlicher Fraktionierungsrhythmen und Strahlenarten niedriger LET. Arch. Geschwulstforsch. 20
46: 529-537, 1976. und Repopulierung des 22. Kruger, H.-J.: Depopulierung transplantablen Rhabdomyosarkoms R 1H der Ratte nach Rontgenbestrahlung. Thesis, University of Hamburg, 1984. J.: Regeneration in tumours. Presented at 23. Kummermehr, the 33rd Annual Meeting of the Radiation Research Society. Los Angeles, 1985, (Abstr). 24. Kummermehr, J., Trott, K.-R.: Rate of repopulation in a slow and a fast growing mouse tumor. In Progress in Radio-Oncology, Karcher, K.H., Kogelnik, H.D., Reinhartz, G. (Eds.). New York, Raven Press. 1982, pp. 299-307. 25. Looney, W.B., Hopkins, H.A.: Solid tumor modells for the assessment of different treatment modalities: XXV. Comparison of the effect of one radiation fraction per day with multiple fractions per day (MFD) given either continuously or intermittently on tumor response and normal tis-
Fractionated irradiation in the R 1H rhabdomyosarcoma sue reaction,
Int. J. Radiat. Oncol. Biol. Ph~x 12: 203-
210, 1986. 26. McNally, N.J., de Ronde, J.: The effect of repeated small doses of radiation on recovery from sub-lethal damage by Chinese hamster cells irradiated in the plateau phase of growth. Int. J. Radiat. Biol. 29: 221-234, 1976. 21. Mitchell, J.B., Bedford, J.S., Bailey, SM.: Dose-rate effects in mammalian cells in culture. III. comparison of cell killing and cell proliferation during irradiation for six different cell lines. Radial. Res. 19: 537-555, 1979. 28. Moulder, J.E., Fischer, J.J., Milardo, R.: Time-dose relationships for the cure of an experimental rat tumor with fractionated radiation. Int. J. Radiat. Oncol. Biol. Phys. 1: 43 l-438, 1976. 29. Moulder, J.E.. Rockwell, S.: Hypoxic fractions of solid tumors: Experimental techniques, methods of analysis and a survey of existing data. Int. J. Rudiat. Oncol. Bioi. Phys.
10: 695-7 12, 1984. 30. Ngo, F.Q.H., Youngman. K., Suzuki, S., Koumoundouros, J.. Iliakis, G.: Evidence for reduced repair capacity for damage accumulation and repair in plateau phase C3H lOT1/2 cells following multiple dose irradiation with Xrays. Radiat. Rex 106: 380-395, 1986. 31. Palcic, B., Brosing, J.W., Skarsgard, L.D.: Survival measurements at low doses: Oxygen enhancement ratio. Br. J. Cancer 46: 980-984, 1982. 32. Reinhold, H.S.: A cell dispersion technique for use in quantitative transplantation studies with solid tumours. Europ. J. Cancer 1: 61-7 1, 1965. 33. Rowley, R., Hopkins, H.A.. Betsill, W.L., Ritenour, E.R., Looney, W.B.: Response and recovery kinetics of a solid tumor after irradiation. Br. J. Cancer 42: 586-595, 1980. 34. Stephens, T.C., Currie, G.A.. Peacock. J.H.: Repopulation of x-irradiated Lewis lung carcinoma by malignant cells
0 H. VOGLER AND H.-P. BECK-B• RNHOLDT and host macrophage
progenitors.
325
Br. J. Cancer 38: 573-
582, 1978. 35. Stephens, cytotoxic
T.C., Steel, G.G.: Regeneration of tumors after treatment. In Radiation Biology in Cancer Reseurch. New York, Raven Press. 1980, pp. 385-395. 36. Suit, H.D., Howes, A.E., Hunter, N.: Dependence of response of a C3H mammary carcinoma to fractionated irradiation on fraction number and intertreatment interval.
Radiat. Res. 72: 440-454,
1977.
prolifera37. Szczepanski, L.V., Trot& K.R.: Post-irradiation tion kinetics of a serially transplanted murine adenocarcinoma. Br. J. Radio/ 48: 200-208, 1975. 38. Thames, H.D., Withers, H.R., Peters, J.P., Fletcher, G.H.: Changes in early and late radiation responses with altered dose fractionation: implications for dose-survival relationships. Znt. J. Radiat. Oncol. Biol. Phys. 8: 219-226, 1982. 39. Tucker, S.L., Thames, H.D.: Flexure dose: the low-dose limit of effective fractionation. Int. J. Radiat. Oncol. Biol. Phys. 9: 1373-l 383, 1983. 40. Van Putten, L.M., Kallman, R.F.: Oxygenation status of a transplantable tumor during fractionated radiotherapy. J. Nut/. Cancer Inst. 40: 44 l-45 1, 1968. 41. Van Rongen, E.: Analysis of cell survival after multiple fractions per day and low-dose irradiation of two in-vitro cultured rat tumor cell lines. Radiat. Rex 104: 28-46,
1985. 42. Williams, M.V., Denekamp, J., Fowler, J.F.: A review ofd/ s ratios for experimental tumors: Implications for clinical Znt. J. Radiat. Oncol. Biol. studies of altered fractionation. Phvsics 11: 87-96, 1985. 43. Zywietz, F., Jung, H.: Partial synchronisation ofthree solid animal tumours by X-rays. Europ. J. Cancer 16: 13811388, 1980.