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Int. J. Radiation Oncology Biol. Phys., Vol. 34, No. I, pp. 85-91, 1996 Copyright 0 1995 Elsevier Science Inc. Printed in the USA. All rights reserved 0360-30 I h/96 $ IS.00 + 00 ELSEVIER l Biology Original Contribution 0360-3016(95)02078-P EFFECT OF RADIATION DOSE RATE AND CYCLOPHOSPHAMIDE ON PULMONARY TOXICITY AFTER TOTAL BODY IRRADIATION IN A MOUSE MODEL A~AL SAFWAT, M.B.B.CH., M.Sc.,*+ OLE S. NIELSEN, M.D, D.M.Sc.,* SAMY EL-BADAWY, F.R.C.R.+ AND JENS OVERGAARD, M.D, D.M.Sc.* *Department of Oncology and Danish Cancer Society, Department of Experimental Clinical Oncology, Aarhus University Hospital, Aarhus, Denmark, ‘Department of Radiotherapy, National Cancer Institute, Fom El-Khalig, Cairo, Egypt Purpose: Interstitial pneumonitis (IP) is stiii a major complication after total body irradiation (TBI) and bone marrow transplantation (BMT). It is diilicult to determine the exact role of radiation in this multifacto- rial complication, especiaiiy because most of the experimental work on lung damage was done using localized lung irradiation and not TBI. We have thus tested the effect of radiation dose rate and combining cyciophosphamide (CTX) with single fraction TBI on lung damage in a mouse model for BMT. Methods and Materials: TBI was given as a single fraction at a high dose rate (HDR, 0.71 GyAnin) or a low dose rate (LDR, 0.08 Gy/min). CTX (250 mg/kg) was given 24 h before TBI. Bone marrow transpianta- tion (BMT) was performed 4-6 h after the last treatment. Lung damage was assessed using ventilation rate (VR) and IethaIity between 28 and 180 days (LD--&. Results: The LDso for lung damage, ? standard error (SE), increased from 12.0 (2 0.2) Gy using sit&e I&&it HDR to 15.8 (t 0.6) Gy using LDR. Adding CTX shii the dose-response curves towards lower doses. The LDsO values for the combined treatment were 5.3 (2 0.2) and 3.5 (2 0.2) Gy for HDR and LDR, respectively. This indicates that the combined effect of CTX and LDR was more toxic than that of combined CTX and HDR. Lung damage evaluated by VR demonstrated two waves of VR increase. The iirst wave of VR increase occurred after 6 weeks using TBI only and after 3 weeks in the combined CTX-TBI treatment, irrespective of total dose or dose rate. The second wave of VR elevation resembled the IP that follows locaiized thoracic irradiation in its time of occurrence. Conclusions: Lung damage foiiowhtg TBI could be spared using LDR. However, CTX markedly enhances TBI-induced lung damage. The combination of CTX and LDR is more toxic to the lungs than combining CTX and HDR. Whole body irradiation, Dose rate, Cyclophosphamide, Lung, Bone marrow transplantation. INTRODUCTION Bone marrow transplantation (BMT) is a life-saving treat- ment modality that is indicated in an increasing number of otherwise fatal malignancies and genetic disorders. How- ever, it is a very toxic procedure with a wide range of treatment-related acute and late side effects. One of the major life-threatening complications after BMT is interstitial pneumonitis (IQ. Its incidence ranges from 20-65% (l), and it is estimated that 25% of all BMT patients will die of Ip (2). About half of the occurrences of IF’ are caused by infectious agents, mainly cytomegalovirus (CMV). Tbe other half has been referred to as idiopathic interstitial pneu- monitis because its cause has not been defined. Many factors that may influence the incidence of IF’ have been identified, among them radiation alone as well as radiation combined with chemotherapy (2). Other factors may include drugs used to prevent graft-versus-host disease (GVHD) (2), the GVHD itself (l), old age (lo), and previous lung disease (4). Therefore, it is particularly difficult to ascertain the exact role of radiation in causing this problem. Unfortunately, most of the available clinical data are based on nonrandom- ized studies; moreover, most of the experimental studies, even those designed to address a specific problem related to TBI in BMT (such as lung damage), were performed using localized and not total body irradiation. The present Reprint requests to: Akmal Safwat, Danish Cancer Society, Department of Experimental Clinical Oncology, Aarhus Univer- sity Hospital, Nerrebrogade 44, DK-8000 Aarhus C, Denmark. Acknowledgements-The authors would like to thankP. Schjer- beck for her enthusiastic and skillful technicalhelp. This work is supported by a grant from the DanishCancerSociety. Accepted for publication 31 July 1995. 85

Effect of radiation dose rate and cyclophosphamide on pulmonary toxicity after total body irradiation in a mouse model

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Int. J. Radiation Oncology Biol. Phys., Vol. 34, No. I, pp. 85-91, 1996 Copyright 0 1995 Elsevier Science Inc.

Printed in the USA. All rights reserved 0360-30 I h/96 $ IS.00 + 00

ELSEVIER

l Biology Original Contribution

0360-3016(95)02078-P

EFFECT OF RADIATION DOSE RATE AND CYCLOPHOSPHAMIDE ON PULMONARY TOXICITY AFTER TOTAL BODY IRRADIATION

IN A MOUSE MODEL

A~AL SAFWAT, M.B.B.CH., M.Sc.,*+ OLE S. NIELSEN, M.D, D.M.Sc.,* SAMY EL-BADAWY, F.R.C.R.+ AND JENS OVERGAARD, M.D, D.M.Sc.*

*Department of Oncology and Danish Cancer Society, Department of Experimental Clinical Oncology, Aarhus University Hospital, Aarhus, Denmark, ‘Department of Radiotherapy,

National Cancer Institute, Fom El-Khalig, Cairo, Egypt

Purpose: Interstitial pneumonitis (IP) is stiii a major complication after total body irradiation (TBI) and bone marrow transplantation (BMT). It is diilicult to determine the exact role of radiation in this multifacto- rial complication, especiaiiy because most of the experimental work on lung damage was done using localized lung irradiation and not TBI. We have thus tested the effect of radiation dose rate and combining cyciophosphamide (CTX) with single fraction TBI on lung damage in a mouse model for BMT. Methods and Materials: TBI was given as a single fraction at a high dose rate (HDR, 0.71 GyAnin) or a low dose rate (LDR, 0.08 Gy/min). CTX (250 mg/kg) was given 24 h before TBI. Bone marrow transpianta- tion (BMT) was performed 4-6 h after the last treatment. Lung damage was assessed using ventilation rate (VR) and IethaIity between 28 and 180 days (LD--&. Results: The LDso for lung damage, ? standard error (SE), increased from 12.0 (2 0.2) Gy using sit&e I&&it HDR to 15.8 (t 0.6) Gy using LDR. Adding CTX shii the dose-response curves towards lower doses. The LDsO values for the combined treatment were 5.3 (2 0.2) and 3.5 (2 0.2) Gy for HDR and LDR, respectively. This indicates that the combined effect of CTX and LDR was more toxic than that of combined CTX and HDR. Lung damage evaluated by VR demonstrated two waves of VR increase. The iirst wave of VR increase occurred after 6 weeks using TBI only and after 3 weeks in the combined CTX-TBI treatment, irrespective of total dose or dose rate. The second wave of VR elevation resembled the IP that follows locaiized thoracic irradiation in its time of occurrence. Conclusions: Lung damage foiiowhtg TBI could be spared using LDR. However, CTX markedly enhances TBI-induced lung damage. The combination of CTX and LDR is more toxic to the lungs than combining CTX and HDR.

Whole body irradiation, Dose rate, Cyclophosphamide, Lung, Bone marrow transplantation.

INTRODUCTION

Bone marrow transplantation (BMT) is a life-saving treat- ment modality that is indicated in an increasing number of otherwise fatal malignancies and genetic disorders. How- ever, it is a very toxic procedure with a wide range of treatment-related acute and late side effects. One of the major life-threatening complications after BMT is interstitial pneumonitis (IQ. Its incidence ranges from 20-65% (l), and it is estimated that 25% of all BMT patients will die of Ip (2). About half of the occurrences of IF’ are caused by infectious agents, mainly cytomegalovirus (CMV). Tbe other half has been referred to as idiopathic interstitial pneu-

monitis because its cause has not been defined. Many factors that may influence the incidence of IF’ have been identified, among them radiation alone as well as radiation combined with chemotherapy (2). Other factors may include drugs used to prevent graft-versus-host disease (GVHD) (2), the GVHD itself (l), old age (lo), and previous lung disease (4). Therefore, it is particularly difficult to ascertain the exact role of radiation in causing this problem. Unfortunately, most of the available clinical data are based on nonrandom- ized studies; moreover, most of the experimental studies, even those designed to address a specific problem related to TBI in BMT (such as lung damage), were performed using localized and not total body irradiation. The present

Reprint requests to: Akmal Safwat, Danish Cancer Society, Department of Experimental Clinical Oncology, Aarhus Univer- sity Hospital, Nerrebrogade 44, DK-8000 Aarhus C, Denmark. Acknowledgements-The authors would like to thank P. Schjer-

beck for her enthusiastic and skillful technical help. This work is supported by a grant from the Danish Cancer Society.

Accepted for publication 31 July 1995.

85

86 1. J. Radiation Oncology 0 Biology 0 Physics Volume 34, Number 1, 1996

7

- I

7 6 GY 10 Gy

: HOR HOR

4 4 0 12 24 36 46 60 72 0 12 24 36 48 60 72

0 12 24 36 48 60 72

Weeks oflsr treatment

Fig. 1. The change in the average ventilation rate (VR) after single fraction TBI at HDR (0) compared to untreated controls (0). Error bars represent 1 SE.

study examined the effect of dose rate and combining CTX with TBI on lung damage in a mouse model for BMT. This study is part of a larger work investigating late normal tissue complications after TBI and BMT. The effect of combining fractionated and hyperfmctionated TBI with CTX on lung damage will be presented separately.

METHODS AND MATERIALS

Animals Adult male C3D2Fl/BOM mice were used in all experi-

ments and were treated when 14-16 weeks of age. They were kept eight per cage under normal laboratory conditions and given tap water and food ad lib. Light and darkness were adjusted to a 12-h cycle. The animals were ear marked, and each animal was given a unique number. They were

11 Gy

LDR

checked daily, and dead animals were recorded. Necmpsies were not done routinely on dead animals.

Treatment Unanesthetized mice were restrained in acrylic jigs

placed in a specially constructed acrylic box as previously described (18). The mice were irradiated to the whole body with a 250 kV x-ray unit (10 mA, 2.2 mm Cu HVL). The animals were treated with a single fraction at a high dose rate (0.71 Gylmin) or low dose rate (0.08 Gylmin) as described before (9).

Intravenous injection (into the tail vein) of 2 X 10h syngeneic bone marrow ceils 4-6 h after the last treat- ment was done in all animals. The marrow was obtained from the femurs of normal mice and the marrow ceil suspension was diluted in balanced salt solution (8).

Cyciophosphamide was dissolved in saline and given intraperitonealiy. The dose was 250 mg/kg, which is the maximum tolerated dose, i.e., the dose needed to kill 1% of the animals within 150 days (19).

Assessment of lung damage Lung damage was assessed by the use of the lethality

(LD) within 28- 180 days after treatment and by ventiia- tion rate (VR). Deaths were recorded as they occurred and dose-response curves of lethality were constructed. LDsO values (the dose needed to kill 50% of the animals within 28- 180 days) were computed by a iogit analysis.

The ventilation rate (VR) was measured by a whole-body mouse piethysmograph as described by Travis et al. (14). The mice were placed unrestrained in a 132 cm-’ chamber through which a constant air flow was passed. When the mouse settled, the VR was recorded. After two to four mea- surements, the mice became accustomed to the chamber. The settling period was reached in about 30 s.

Experimental design and data analysis Two series of experiments were performed. In the first

series, the animals received TBI only. In the second series,

7

14 Gy

t

17 Gy

LDR LDR

6 T

0 122436486072 0 122436486072 0 12 24 36 48 60 72

Weeks after treatment Weeks after treatment Weeks after treatment

Fig. 2. The change in the average ventilation rate (VR) after single fraction TFH at LDR (0) compared to untreated controls (0). Error bars represent 1 SE.

Murine lung damage after TBI l A. SAFWAT et al. 87

100 r vv . . . a 00 0

10 177 I/ 0 LDR

0 . ..rn.O.rnO1-).~,.,.,.,.,

0 2 4 6 0 10 12 14 16 18 20 22

Dose (Cy)

Fig. 3. Lethality dose-response curves for lung damage after TBI (with and without CTX) and BMT. Error bars represent 1 SE.

animals were treated with a combination of CTX (250 mg/kg) followed 24 h later by TBI. TBI was given as a single fraction at either a high dose rate (HDR), or low dose rate (LDR). In each of these TBI schedules the ani- mals were distributed between different dose levels. Con- trols included a group receiving only CTX plus BMT, and a group to which only BMT was performed. There were 32 animals in total in the control groups, and from 8 to 16 animals in each dose level of the treated groups. VR was measured at Weeks 0, 3, 6, 9, 12 posttreatment and every 6 weeks until the end of first year, then every 12 weeks thereafter.

For the VR test a mean value of each group was calcu- lated at each point of follow-up and plotted against time. The resultant curves served to illustrate the presence of functional damage and its degree, but these curves were not suitable for numerical analysis of the data. Instead, the data were converted to quantal response (all or noth- ing) data. In each experimental group, animals were di- vided into responders and nonresponders. Responders were defined as those animals in which the VR values exceeded a predetermined level. This threshold level was chosen to be “values more than three times the standard deviation of the control mean.”

The dose-response data were used to calculate the iso- effective doses for the different schedules. The error bars in the curves represent one standard error (SE). The dose

enhancement factor (DEF) was defined as the ratio be- tween the dose needed to cause 50% lethality from lung damage using TBI only and that causing the same effect using combined CTX and TBI. The dose modifying factor (DMF) was defined as the ratio between the dose needed to cause 50% lethality (LD,,) using a LDR TBI and the dose needed to cause the same effect when TBI was given as a single fraction at HDR.

RESULTS

Single fraction total body irradiation To study the effect of TBI on lung function, the average

values of ventilation rate (VR) of mice treated with a given dose were plotted against time after treatment. Ani- mals receiving a single fraction of TBI at HDR (Fig. 1) or LDR (Fig. 2) showed an early increase in VR in the first 12 weeks after treatment. This early damage was not seen with doses 18 Gy using single-fraction HDR. With LDR, this early damage though less clear was seen at 14 Gy. The peak of this early increase was not related to dose. It was reached at week 6 after treatment, irrespective of the dose level or dose rate and was resolved completely by week 12. By tracing individual animals, this was shown to be a true resolution and not due to death of the animals with high values of VR (data not shown). Ani- mals that received doses s 10 Gy HDR or 5 14 Gy LDR continued with normal VR in the rest of the follow-up period. On the other hand, animals that received 2 12 Gy HDR or 17 Gy LDR showed a second elevation of VR that was followed by death within 24 weeks after treatment in most of the animals. The few animals surviving beyond 180 days (the period dominated by IF’) showed an unre- solved elevation of VR, and none of them survived be- yond 30 weeks after treatment.

The percentage of animals expressing a predetermined degree of lung damage (VR values >3 standard devia- tions above the control mean) did not reveal a dose- response relationship. Decreasing the cut-off level for response to VR values >2 standard deviations above the control mean, did not help in this matter as the resulting values were heterogeneous. Consequently, only lethality

Table 1. Dose-response data for lung damage after total body irradiation (TBI) and bone marrow transplantation (BMT) with and without cyclophosphamide in mice

Treatment schedule

TBI only TBI + CTX

L&o (GY) DME value LDso (GY) DMF value DEF values

High dose rate 12.1 t 0.2* - 5.2 + 0.3* - 2.3 + 0.14* Low dose rate 15.8 + 0.6* 1.3 + o.os* 3.5 + 0.2* 0.7 t 0.06* 4.5 2 0.3*

The data are generated using a logit program. LDw : The dose needed to cause 50% lethality from lung damage. DMF: Dose modifying factor. DEF: Dose enhancement factor. * 2 I SE.

I. J. Radiation Oncology 0 Biology 0 Physics Volume 34, Number I, 1996

CTX only

(250 m/kg)

5 6-

al L .

4J 1 ” ‘a ” ‘*I’ ’ 0 12 24 36 48 60 72

Weeks after treatment

Fig. 4. The change in the average ventilation rate (VR) after 250 mg/kg CTX (0) compared to untreated controls (0). Error bars represent 1 SE.

was used for calculating the dose-response data of lung damage.

The dose-response curves for lung damage using le- thality between 28 and 180 days (LDs,,,zxm ,& are seen in Fig. 3. The LD,,, for lung damage using single-fraction HDR was 12.0 Gy (SE = & 0.2 Gy), while that using

LDR was 15.8 Gy (SE = ? 0.6 Gy), resulting in a DMF value of 1.3 (SE = t- 0.1). There was a tendency for the

8

2 GY

CTX-HDR

dose-response curve of HDR to be steeper than that for LDR. The dose range from 10 to 90% response rate was around 2 Gy for HDR, and 4.7 Gy for LDR. The dose- response data for the single fraction TBI schedules are shown in Table I.

Combined CTX and single fruction TBI When CTX alone was administered, the average values

of VR plotted against time after treatment showed a small elevation of the VR that started around week 36 (Fig. 4) and continued for the rest of the follow-up period. When CTX was combined with a single fraction at HDR (Fig. 5). even doses as small as 2 Gy showed evidence of early lung damage. This early increase in VR reflected a fatal lung damage at doses 28 Gy. In the dose range of 2-h Gy, this initial increase in VR was resolved and immedi- ately followed by a second increase in VR that persisted up to the last point of follow-up. The level of this second elevation of VR was not related to dose.

Combining CTX and LDR was the most toxic of the regimens studied. Figure 6 shows that all the animals receiving doses 29 Gy did not survive beyond week 9 after treatment. i.e., just following the peak of the initial increase in VR. Animals that received lower doses in the range of 2-7 Gy were not examined for increase in VR in the first 12 weeks after treatment. However, following

8

4 GY

7 CTX-HDR

0 12 24 36 48 60 72 0 12 24 36 48 60 72

T 6 GY

CTX-HDR

8

6

5

J 4

i 8 GY

0 12 24 36 48 60 72 0 12 24 36 48 60 72

Weeks after treatment Weeks after treatment

Fig. 5. The change in the average ventilation rate (VR) after 250 mg/kg CTX and single-fraction (0) compared to untreated controls (0). Error bars represent I SE.

7‘81 at HDR

89

8 8-

7 GY 7

7 CTX-LDR

$ 7- CTX-LDR

2. 9)

P 6

6 .- z .= 2 5

>”

:iL

4 4’ ’ ’ . ” ’ ’ ’ ’ ‘1

Murine lung damage after TBI 0 A. SAFWAT et al.

0 12 24 36 48 60 72 0 12 24 36 48 60 72

8 a

7 CTX-LDR CTX-LDR iii 7 7

s al

5 6 6

s ,- 5 .Z E 5 5

3

4 4 0 12 24 36 48 60 72 0 12 24 36 48 60 72

Weeks after treatment Weeks after treatment

Fig. 6. The change in the average ventilation rate (VR) after 250 mg/Kg CTX and single fraction TBI at LDR (0) compared to untreated controls (0). Error bars represent 1 SE.

week 12 after treatment they showed persistent increase in the VR that was associated with the lethality of all the animals that received doses 25 Gy within the first 18 weeks after treatment.

8

correlation coefficient 0.922

0 I I I , I I # I I I

0 3 6 9 12 15 18 21 24 27 30

Week of maximum VR

Fig. 7. The correlation between the week of maximum VR and day of death. Each point represents one animal. Dashed lines represent 95% confidence limits.

Comparing the dose-response curves for the lethality of animals receiving the combined treatment with the single-fraction TBI showed that the dose-response curves for the combined treatment were shifted to the left (Fig. 3). The dose enhancement factor (DEF) for combining CTX and HDR was 2.3 (SE = 2 0.1) while it was 4.5 (SE = -f 0.3) for LDR. Hence, combining CTX and LDR not only counteracted the sparing effect of LDR, but was even more toxic than combining CTX and HDR. The LD5,, for the CTX-HDR regimen was 5.3 Gy (SE = -+ 0.2 Gy), while it was 3.5 (SE = + 0.2 Gy) for the CTX- LDR regimen.

The dose-response curves were again very steep for the combined CTX-TBI treatment. The dose-response data for the combined CTX and single-fraction TBI schedules are shown in Table 1.

DISCUSSION

The results of ventilation rate (VR) used to estimate lung damage in this model for TBI and BMT have shown two waves of VR increase in the period dominated by interstitial pneumonitis (IP). The peak of the early wave of increased VR occurred at 6 weeks irrespective of total dose or dose rate. Such an early increase in VR has been reported before, following localized thoracic irradiation using different end points other than VR ( 1 1 - 13). It has

90 I. J. Radiation Oncology l Biology l Physics Volume 34, Number I, 1996

also been reported after the administration of CTX only (3), or after combined drugs and radiation (19). The lack of a relationship between TBI dose and the level of VR increase is probably the result of a limitation of the VR technique.

The onset of the second wave of VR increase resembled that of the IP that follows localized thoracic irradiation. However, contrary to localized thoracic irradiation we could not elicit a dose range at which a dose-dependent elevation of VR could be seen. The fact that the measure- ments of VR were made only every 6 weeks during the relevant period means that transient elevations could have been missed. This partly explains why we could not con- struct a dose-response curve for the VR. However, for individual animals the time of maximum VR was strongly correlated with the time of death within 28- 180 days after treatment (Fig. 7).

different pathologic entities. They differ with regard to latency, pathogenesis, dose dependence and probably dose distribution” (16). These low LDSo values are not an artifact caused by lethality from incisor damage re- ported by Down et al. (5), as this complication has been checked regularly in our mice and was found to be virtu- ally nonexistent.

The sparing effect of LDR in this model is similar to that reported by other studies using both localized lung irradiation (17) and TBI ( 15). Reducing the dose rate below 0.08 Gy/min should produce even greater sparing of lung damage (15), but this would not be practical from the clinical point of view. In our study, both the LD5” values for the single fraction at HDR and the DEF values for the combined CTX and HDR were slightly lower than those obtained in previous studies performed in our laboratory using localized thoracic irradiation (18, 19), but similar to another study using the same TBI and BMT model (7). This may indicate that TBI is more toxic to the lungs than localized thoracic irradiation. In a recent review, Trott stated that “radiation pneumonitis occurring after lung irradiation and IP occurring after TBI are two

Giving CTX before TBI advanced the early peak in VR to 3 weeks. This was reflected on lethality from lung damage within 28- 180 days. Similar results using mouse models for TBI and BMT have been reported by Yan et al. (20) and Nielsen et al. (7). The most striking result was the severe toxicity of combining CTX and LDR. Previously published results in rats (17) and mice (6) have suggested that CTX may defeat the sparing effect of LDR on the lungs. Adriamycin administered before LDR local- ized lung irradiation was also reported to exhibit the same effect (12). In our study, the combination with LDR was even more toxic than the combination with HDR. The difference may be the result of our use of a higher CTX dose and TBI instead of localized lung irradiation.

In conclusion, radiation-induced interstitial pneumoni- tis after TBI and BMT is an important problem that could be spared using LDR. CTX markedly enhanced TBI-in- duced lung damage, and its combination with LDR was more toxic to the lung than its combination with HDR. Although extrapolating these experimental results to the clinical situation is difficult, they indicate that although the use of LDR can spare lung from TBI-induced damage, its combination with CTX should be regarded with care. Whether this phenomenon is valid for other drugs and chemotherapeutic agents used in the BMT procedure is still to be seen.

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12. Sherman, D.; Carbell, S. C.; Belli, J. A.; Hellman, S. The effect of dose rate and adriamycin on the tolerance to tho-

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13. Siemann, D. W.; Hill, R. P.; Bush, R. S. Analysis of blood gas values in ice following pulmonary irradiation. Radiat. Res. 81:303-310; 1980.

14. Travis, E. L.; Down, J. D.; Holmes, S. J.; Hobson, B. Radia- tion pneumonitis and fibrosis in mouse lung assayed by respiratory frequency and histology. Radiat. Res. 84:133- 143; 1980.

15. Travis, E. L.; Peters, L. J.; McNeill, J.; Thames, H. D.; Karolis, Jr.; Karolis, C. Effect of dose-rate on total body irradiation: Lethality and pathologic finding. Radiother. On- col. 4:341-351; 1985.

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