Dose rate and fractionation of total body irradiation in dogs: Short and long term effects

Radiotherapy and Oncology, Suppl. 1 (1990) 51-59 Elsevier

Dose rate and fractionation of total body irradiation in dogs: Short and long term effects

Hans-Jochem Kolb1>6, Leena Kaisa Losslein*, Klaus Beiber*, Ekkehart Sch3ffer3, Ernst Hollera, Nimrod Schwella’** Eva Hochhaussei?, Walter Lehmacheg, Otto Balk4 and Stefan Thierfelderl llnstitut fur Immunologie, %stitut fir Klinische Hdmatologie, Gesellschafr fiir Strahlen- und Umweltforschung, Marchioninistr. 25, 8000 Miinchen 70, 3lnstitut fiir Pathologie, %stitut fiir Bioiogie, %stitut fir Medizinische Informatik und Systemforschung, Gesellschaf fur Strahlen- und Umweltforschung, Ingolstiidter Landstr. 1 I 8042 Miinchen-Neuherberg, 6Medizinische Klinik III, Klinikum Grophadern, Ludwig-Maximilians-Universitiit, Marchioninistr. 15, 8000 Miinchen 70, F.R.G.

Keywords: Total body irradiation; Bone marrow transplantation; Dose rate; Fractionation; Acute effects; Late effects

Summary

Variations of regimens of total body irradiation (TBI) were investigated in the dog as a preclinical model for bone marrow transplantation. Inactivation of hemopoietic precursor cells (CFU-GM) was studied following irradiation of marrow in vitro, following TBI at sublethal doses in vivo and following autologous transplantation of marrow obtained after sublethal TBI. Inactivation and recovery of CFU-GM as well as restoration of hemopoiesis following autologous transplantation was independent of the dose rate, but nadirs of blood counts were lower following sublethal TBI with the higher dose rate. Acute non-hemopoietic toxicity of TBI depended on the dose, the dose rate and the total treatment time and not on the fractionation regimen. At a total dose of 25 Gy acute mortality was prevented by prophylactic administration of oral, non-absorbable antibiotics. Late mortality was due to degenerative and autoimmune-like disorders with or without infections and to malignant tumors. Evaluation of long-term survival is still preliminary, since surviving dogs of two groups (10 Gy as single dose, 25 Gy as hyperfractionated TBI) have not yet reached the median survival time of their group. So far, long-term survival depended on the total dose (p = 0.05) and, possibly, the fractionation regimen (p = 0.12). The latency period until development of malignant tumors was influenced by the total doses given in the same treatment time (p = 0.05) and by the total treatment time for equal doses (p = 0.04). It was concluded that TBI at a low dose rate may give the best therapeutic ratio of inactivation of hemopoietic precursor cells to acute toxicity. A possible benefit of hyperfractionation on long-term survival due to less toxicity has to be weighed against less effective inactivation of clonogenic hemopoietic precursors and less effective immunosuppression seen in allogeneic transplantation.

Introduction

TBI is widely used as conditioning treatment for bone marrow transplantation in patients with hematological malignancy [ 151. The therapeutic goal of TBI is the elimination of malignant cells and the

0167-8140/90/$03.50 0 1990 Elsevier Science Publishers B.V. (Biomedical Division)

52

suppression of the host’s immune response against the allogeneic marrow graft. The therapeutic ratio of TBI can be defined by its effect on leukemia and on the host’s immune reactivity against allogeneic marrow in relation to its acute and chronic side effects. The therapeutic ratio may be increased by variations of dose rate and fractionation regimens as well as shielding of critical organs. The optimal dose, dose rate and fractionation schedule has not been defined. Some radiotherapists prefer large doses at low dose rates [15], others give rather small doses at higher dose rates [14]. Most centers use TBI in total doses between 5 and 15 Gy. However, experience with localised irradiation of extramedullary leukemic infiltrates [4] and prophylactic irradiation of the CNS [9] indicates that doses of at least 20 Gy are required to prevent recurrence of leukemia in the majority of cases.

Therefore, we investigated the influence of dose rate and fractionation of TBI on acute and late toxicity in dogs. Doses up to 30 Gy followed by infusion of autologous marrow were studied. Inactivation of hemopoietic precursor cells (CFU-GM) instead of clonogenic leukemic cells was taken as therapeutic goal and death due to acute or chronic radiation damage as toxic end point.

Materials and Methods

Dogs were Beagles, Labrador retrievers and mongrels of both sexes which were raised in the kennels of the Gesellschaft ftir Strahlen- und Umweltforschung (GSF). They were vaccinated against distemper, leptospirosis, canine hepatitis and, more recently, also against parvovirus infection. The median age was 18 months at the time of irradiation. They were kept off food and water from the day before TBI until 5 days after autologous marrow infusion. They were given parenteral fluids prophylactically and were treated with antibiotics, blood and platelet transfusions as clinically indicated. Bone marrow was aspirated from humerus’, femur’s and the pelvic crests in general anesthesia and frozen with 10% dimethylsulfoxide as previously described [ 11. It was thawed rapidly and infused Whout further dilution. Complete autopsies and histological evaluations were done on dogs that died and biopsies were taken from tumors. The evaluation of the incidence of malignant tumors was based on autopsy findings.

TBI was applied from two opposing 60Cobalt sources 8 m apart to the dog placed midway between the sources. The dose rate of 46 cGy/min was applied by adjusting the distance of the sources to 2.7 m, that of 0.4 cGy/min by lead attenuators and aluminum screens. Dosimetry was performed with ionisation chambers at 17 different points including esophagus, stomach, rectum and colon via indwelling rubber tubes in the anesthesised dog. Tissue air ratios between 0.75 and 1.03 were determined and the value of 0.87 was used for dose calculations. TBI was given in the following regimens:

2.1 Gy without marrow rescue as single dose with 4.6 cGy/min (7 dogs) and 46 cGy/min (8 dogs); 3.3 Gy without marrow rescue as single dose with 4.6 cGy/rnin (8 dogs): doses of 10-30 Gy were given with autologous marrow rescue; 10 Gy as single dose, dose rate 4.6 cGy/rnin, 10 dogs; 13.3 Gy as single dose with 4.6 cGy/min (6 dogs) and with 46 cGy/min (6 dogs); 20 Gy as single dose with 4.6 cGy/min (7 dogs) and with 0.4 cGy/min (8 dogs), marrow of DLA-identical littermates was used for rescue; 20 Gy fractionated into 10 Gy day -1 and 5 Gy either on days -2 and -3 (6 dogs), or on days -3 and -5 (11 dogs), or on days -4 and -7 (9 dogs); 30 Gy fractionated into 10 Gy day -1 and 5 Gy each on days -9, -7, -5 and -3 (4 dogs); 25 Gy fractionated within 7 days fractionated into 10 Gy day -1 and either 5 Gy on days -7, -5 and -3 (16 dogs), or 2.5 Gy every day (6 dogs), or 1.25 Gy twice per day (15 dogs); 25 Gy fractionated and accelerated with 10 Gy day -1 and either 1.25 Gy three times per day from day -5 until day -2 (4 dogs), or 1.25 Gy four times per day from day -4 until day -2 (4 dogs).

53

The growth of hemopoietic precursor cells (CFU-GM) was studied in semisolid agar culture using serum of a neutropenic dog for stimulation as previously described [l].

Statistical tests for comparing the number of CFU-GM and nadirs of blood counts were the Wilcoxon ranking test, short term survival by the Fisher’s exact test and long term survival by a generalised Wilcoxon-Breslow test.

RC?SUltS

Effect of dose rate on canine hemopoiesis

Aspirated bone marrow was irradiated in vitro with 0.5, 1.0 and 2.0 Gy and the growth of CFU-GM measured. The dose effect curve was character&d by a Do of 0.4 Gy and an extrapolation number of n = 1.12 (data not shown). These findings indicate that canine hemopoietic precursor cells are highly radiosensitive and have a poor capacity for repair of sublethal radiation damage. This conclusion was confirmed by irradiation with different dose rates (Table 1). Inactivation of CFU-GM was not different whether 1.0 Gy was given with 53 cGy/min, 5.3 cGy/min or 0.48 cGy/min.

The recovery of the CFU-GM concentration in the marrow of 23 dogs was studied weekly following sublethal TBI in doses of 2.1 Gy and 3.3 Gy (Fig. 1). Variation of the dose rate did not change significantly inactivation or recovery of CFU-GM, whereas the higher dose inactivated more CFU-GM at 1 week and delayed recovery after 3 and 4 weeks (~~0.01). The nadirs of leucocyte, lymphocyte and platelet counts were lower in dogs given the higher radiation dose (p<O.Ol), but also lower in dogs treated with the higher dose rate (~~0.05).

Table I. Surviving fraction of CFU-GM following irradiation of canine marrow in vitro at various dose rates

Total dose Dose rate (GY) (cGy/min)

Surviving fraction (Mean rf: 1 SD)

None None 1.0 53 1.0 5.3 1.0 0.48 2.0 5.3

1 (367 f 141)* 0.13 + 0.12 0.15 + 0.12 0.11 f 0.08 0.026 zk 0.043

* 2 x I@ mononuclear cells were plated.

The restorative capacity of bone marrow of 6 sublethaliy irradiated dogs was evaluated by autologous transplantation following a lethal dose of TBI. Marrow was cryopreserved 3 and 4 months following sublethal TBI and 1 x log/kg mononuclear cells were reinfused following 10 Gy TBI. The recovery of leucocyte and platelet counts was delayed in all pre-irradiated dogs, but the delay was not different in dogs given the higher dose rate prior to the marrow harvest.

Effect of dose rate and fractionation on acute mortality

The effect of TBI on organs other than hemopoiesis was studied in dogs given autologous marrow for rescue. Acute toxicity was primarily due to damage to the gastrointestinal tract with loss of fluids and electrolytes and with infections. A single dose of 13.3 Gy was tolerated at a dose rate of 4.6 cGy/min, but not at a dose rate of 46 cGy/min (Table 2). A further protraction to 0.4 cGy/min did not improve survival after 20 Gy.

100

1 1 0

??iiiiiii 2.1 Cy 4.6 cCy/min

??2.1 Gy 46.0 cGy I min

??3.3 Cy 4.6 cGy/min

I /ll!L :.:: ‘:‘:;;

._: ;ij; :,.; :_:: <. .:.. :::: ::j; :::: .<. lil!LA . . . ..,. .

.(,. ;.t . 2.

1 2 3

time after irradiation ( week )

Fig. 1. Comparison of the content of CFW-GM per l@ mononuclear marrow cells before and after a TBI with a sublethal dose. Marrow was aspirated from pelvic crests in a volume of l-2 ml, columns are mean, bars 1 standard deviation. Differences between 2.1 Gy and 3.3 Gy were significant at 1, 3 and 4 weeks (p&.01).

Fractionation of TBI into a fraction of 10 Gy and two fractions of 5 Gy within 3 days was not better than the protracted TBI (Table 2). Only the prolongation of the total treatment time to 5 and 7 days

Table 2. Acute mortality of dogs following total body irradiation with 20 Gy and marrow infusion effect of fractionation and treatment time

TBI-regimen No. of dogs surviving Survival time of decedents 30 days per no. studied @aY s)

Single dose 20 Gy in 3.3 days / 0.4 cGy/min 518 4, 4, 4

Fractionated 5 Gy days -3. -2, 10 Gy day -1 316 1, 2, 5 5 Gy days -5. -3. 10 Gy day -1 8/l 1 0, 5, 6 5 Gy days -7,+ 10 Gy day -1 9l9 -3 -9 -

Marrow was infused on day 0, dogs given single dose ‘IBI were given marrOw of a DL.A-identical littermate instead of autologous marrow.

55

improved survival (~~0.05). TBI in a dose of 30 Gy was not tolerated. Various fractionation regimens were compared at a total dose of 25 Gy in 7 days. Acute mortality was not different whether TBI was fractionated into one or two fractions per day or one fraction every other day. The importance of the total treatment time was further substantiated by unsuccessful attempts of acceleration of TBI to 5 days with 3 fractions per day and 4 days with 4 fractions per day: only one out of 8 dogs survived.

Effect of oral, non-absorbable antibiotics on acute mortality

The role of oral, non-absorbable antibiotics in acute toxicity was evaluated in dogs given 25 Gy within 7 days. Fifteen dogs received twice daily oral, non-absorbable antibiotics for prevention of infection (100 mg amphotericin B, 165 mg neomycin, 500 000 E colistin) from 10 days before transplantation until the day of recovery of leukocytes to 1 G/l. In contrast to 9 of 21 dogs not given antibiotics these dogs survived the period of acute toxicity (p<O.Ol).

Effect of dose, treatment time and fractionation regimen on late mortality

Seventy-seven dogs were observed between 2 months and more than 10 years following TBI with doses of between 10 and 25 Gy and autologous marrow transplantation for the development of late effects. The total doses and treatment times were as follows: 10 Gy as single dose (10 dogs), 13.3 Gy as single dose (6

E 3 k n

ii > ._ ? J w

100

50

0

-p.-a.-.

L. Ll- . I-.-_..-__,

L-l c *---I__ L,

L-a 1 L --W-1

? 25 Cy / 7 days LO,

(n=33) 5 i

? -1

. . sacrificed dogs

1 2 3 4 5 6 7 8 9 10

time post transplantation ( year )

Fig. 2. Kaplan-Meier plot of survival time after fractionated TBI with 25 Gy and 20 Gy; dogs; 17 dogs were killed because of lack of kennel space or succumbed in fights and were censored from evaluation.

56

Table 3. Causes of deaths following total body irradiation and autologous marrow transplantation in dogs

TBI-regimen Observation time (months) median (range)

No. of dogs No. of deaths with No. of dogs studied alive

Degen. dis./ Malignancy Other* infections

Single dose 10 Gy 13.3 Gy

60 (7-93) 10 2 2 6 14.5 (7-71) 6 1 1 4 0

Fractionated 20 Gy 3-5 day 75 (28-95) 11 2 6 3 0 7 days 69 (2-122) 17 3 6 8 0

25 Gy 2.5-5 Gy 48 (5-98) 17 7 6 3 1 1.25 Gy 46.5 (7-90) 16 3 3 4 6

Other causes of deaths were: 11 dogs sacrificed because of lack of kennel space, 12 dogs succumbed to dog fights, 1 dog died of an unknown cause (no autopsy was performed).

dogs), 20 fractionated in 3 days (3 dogs), 5 days (8 dogs) or 7 days (17 dogs), 25 Gy fractionated within 7 days with a fraction 10 Gy on day -1 and fractions of 5 Gy every other day (16 dogs), or fractions of 2.5 Gy every day (1 dog) or fractions of 1.25 Gy twice per day (16 dogs). Twenty dogs were killed because of lack of kennel space between 6.5 and 17 months after TBI. They were evaluated until the time of death.

The actuarial survival time was evaluated by the method of Kaplan and Meier [lo]. The median survival time of dogs given 25 Gy was 62 months as compared to 86 months of dogs given 20 Gy (Fig. 2). Only 2 dogs died after 70 and 89 months of malignant tumors after a single dose of 10 Gy, but the observation time of less than 5 years in 5 surviving dogs is still too short for comparison. Only 2 of 6 dogs given 13.3 Gy as a single dose were observed until their death of pneumonia (44.5 months) and lymphoma (70.5 months). The median survival following 20 Gy in 7 days was 92 months as compared to 77 months following 20 Gy in 3-5 days (data not shown). Dogs given 25 Gy in a hyperfractioned regimen survived slightly better than those given the same dose in the same time in fractions of 5 Gy (p=O. 12).

The causes of death were mainly degenerative (lung fibrosis, interstitial nephritis, fibrosis and atrophy of the pancreas) of autoimmune-like diseases (vasculitis, thyroiditis, hepatitis) with or without infections within four years of treatment and malignant tumors after that time (Table 3). Interstitial pneumonitis as seen in human patients was not observed. The latency period until death with a tumor was influenced by the total dose and treatment time. It was 77.5 months following 25 Gy and 87 months following 20 Gy (Fig. 3). It was shorter in dogs given 20 Gy in 3-5 days (median: 77 months) than in those given 20 Gy in 7 days (median: 104 months; p = 0.04). An influence of the fractionation regimen cannot be evaluated presently, because only 3 dogs of the group treated with the hyperfractionated regimen died of tumors and 6 are still alive between 3 and 8 years after TBI.

57

loo- I

r--o

ok alive 20Gy (n=27)

. . death due to other causes

50 - 25Cy (n=32)

-- l

I-J

0 +# , ,.._._.:::z: 0 1 2 3 4 5 6 7 8 9 10

time post transplantation ( year )

Fig. 3. Kaplan-Meier plot of latency until death with cancer; deaths of causes, other than cancer, were censored.

Discussion

TBI is a major constituent of most conditioning treatment regimens for marrow transplantation, since it exerts a long-lasting myelosuppressive effect and its antileukemic effect is believed to be similarly long-lasting. The doses presently applied are far below those necessary for treatment of extramedullary leukemic infiltrates [4] and effective CNS-prophylaxis [9]. Dogs serve as a valuable animal model for preclinical studies, because they can be examined repeatedly, treated with supportive therapy and observed for many years for the development of late effects in a similar way as human patients. In our experiments with increasing doses of TBI, we find that dogs can tolerate single doses of 10 and 13.3 Gy at a low dose rate of 4.6 cGy/min, 20 Gy as fractionated TBI in 7 days and 25 Gy as fractionated TBI in 7 days with oral, non-absorbable antibiotics without severe acute toxicity. In our experiments a decrease of the dose rate below 4.6 cGy/min did not improve the short term survival rate. Similar to our previous experience [ 111, Deeg et al. [5] found a single dose of 14 Gy as a maximally tolerated dose in dogs and no improvement of the short term survival rate by fractionation within 2-3 days.

Unfortunately, the impact of dose rate on long term survival after TBI with single doses could not be evaluated. In the study of Deeg et al. [5], dogs given single doses of TBI died within 7 months of wasting, hepatitis, pancreatic fibrosis and anemia. In our study, comparing dose rates of 4.6 cGy/min and 0.4 cGy/min at a dose of 20 Gy allogeneic, DLA-identical marrow was given instead of autologous marrow. However, dogs treated with the lower dose rate survived between 10 and 12 months, when they were killed because of lack of kennel space, whereas dogs treated with the higher dose rate died with severe graft-versus-host disease (data not shown). Long term survival of dogs with autologous grafts was influenced by the total dose of TBI, the total treatment time and, possibly, the fractionation regimen. It was limited by degenerative and autoimmune-like disorders with or without infections and, in the

58

majority of dogs, by malignant tumors. However, interstitial pneumonitis as seen in human patients was not observed. Interstitial pneumonitis in patients has a multifactorial pathogenesis in which irradiation plays only a minor role. Deeg et al. reported an increased cancer incidence in canine radiation chimeras as compared to control dogs [6]. Broerse et al. [2] observed malignant tumors in 16 out of 29 irradiated rhesus monkeys as compared to none in 21 untreated controls observed for a similar time period of more than 10 years. Our experience in dogs with autologous marrow grafts adds to their reports evidence of the role of the total dose and the treatment time in the tumor development in larger animals. There is a suggestive improvement of survival with hyperfractionated TBI as compared to TBI in larger fractions, but the role of hyperfractionation on tumor development cannot be evaluated yet. In allogeneic transplantation between DLA-haploidentical littermate dogs that were conditioned with hyperfrac- tionated TBI rejected the marrow graft more frequently than those conditioned with larger fractions and some of them recovered their own hemopoiesis [12]. In this situation, hyperfractionated TBI is not sufficiently immunosuppressive nor does it preclude autochthonous recovery of hemopoietic stem cells.

Acute and chronic toxicity has to be weighed against the therapeutic goal of inactivating hemopoietic stem cells. Similar to CFU-S of mice [7,16] canine CFU-GM were inactivated independently of the dose rate of irradiation. Moreover, regeneration of hemopoiesis following sublethal TBI and autologous transplantation of sublethally irradiated marrow was not significantly influenced by the dose rate, but the nadirs of blood counts were lower in dogs treated with the higher dose rate. This discrepancy can best be explained by assuming different abilities of stem cells and more mature cells for repairing sublethal radiation damage. Contrary to variations of dose rate fractionation may allow considerable repair of the hemopoietic system by repopulation [3]. Our results of hyperfractionated TBI and autochthonous hemopoietic recovery in dogs following DLA-haploidentical transplantation confirm these observations [ 121. Unlike the myeloid leukemia of the BN-rat [8], and leukemic cell lines [ 171, spontaneous human leukemias have a small shoulder (extrapolation number less than 1.7) indicating a poor capacity for sublethal repair [13]. Following fractionated TBI mixed chime&m as evidence of hemopoietic repopulation has been described, but an increased rate of leukemic recurrence has not been observed.

In conclusion of our studies in dogs, we use fractionated TBI in a dose of 12 Gy with large fractions (3 x 4 Gy) and a low dose rate (4.5 cGy/min) for marrow transplantation in human patients. At present, it cannot be excluded that this regimen produces more late effects than the same dose given in a hyperfractionated regimen, but it is likely to be more effective.

Acknowledgements

This work was supported by the Wilhelm-Sander-Foundation.

References

1. Bodenberger, U.. Kolb, H.J., Rieder, I., Netzel, B., Schaffer. E.. Kolb, H. and Thierfelder, S. Fractionated total body irradiation and autologous marrow transplantation - Recovery after various marrow cell doses. Exp. Hematol., 8, 384-394, 1980.

2. Broerse, J.J., Hollander, C.F. and van Zwieten, M.J. Tumour induction in Rhesus monkeys after total body hradiation with X-rays and fission neutrons. Int. J. Radiat. Biol., 40, 671-676, 1981.

3. Chaffey, J.T. and Hellman, S. Radiation fractionation as applied to murine colony-forming cells in differing proliferative states. Radiology, 93, 1167-1172, 1969.

4. Chak, L.Y., Sapozink, M.D. and Cox, R.S. Extramedullary lesions in non-lymphocytic leukemia: Results of radiation therapy. Int. J. Radiat. Oncol. Biol. Phys., 9, 1173-1176. 1983.

59

5. Deeg, H.J., Storb, R., Weiden, P.L., Schumacher, D., Shuiman, H., Graham, T. and Thomas, E.D. High-dose total-body irradiation and autologous marrow reconstitution in dogs: Dose-rate-related acute toxicity and fractionation-dependent long-term survival. Radiat. Res., 88, 385-391, 1981.

6. Deeg, H.J., Prentice, R., Fritz, T.E., Sale, G.E., Lombard, L.S., Thomas, E.D. and Storb, R. Increased incidence of malignant tumours in dogs after total body irradiation and marrow transplantation. Int. J. Radiat. Oncol. Biol. Phys., 9, 1505-1511, 1983.

7. Fu, K.K., Phillips, T.L. Kane, L.J and Smith, V. Tumor and normal tissue response to irradiation in vivo: Variation with decreasing dose rates. Radiology, 114, 709-716, 1975.

8. Hagenbeek, A. and Martens, A.C.M. The effect of fractionated versus unfractionated total body irradiation on the growth of the BN acute myelocytic leukemia. Int. J. Radiat. Oncol. Biol. Phys., 7, 1075-1079. 1983.

9. Hustu, H.O. and Aur, R.J.A. Extramedullary leukaemia. Clinics in Haematology, 7, 313-337, 1978. 10. Kaplan, E.L. and Meier, P. Nonparametric estimation from incomplete observations. J. Am. Statist. Assoc., 53, 457, 1958. 11. Kolb, H.J., Rieder, I., Bodenberger, U., Netzel, B., Schaffer, E., Kolb, H., Thierfelder, S. and the Munich Cooperative Group for

Bone Marrow Transplantation. Dose rate and fractionation studies of total body irradiation in dogs. Pathologie Biologie, 27, 370-372, 1979.

12. Loesslein, L.K., Kolb, H.J., Porzsolt, S., Sch%ffer, E., Scholz, S., Meissner, H., Holler, E., Wilmanns, W. and Thierfelder, S. Hyperfractionation of total body irradiation and engraftment of marrow from DLA-haploidentical littermates. Transpl. Proc., 19, 2707-2708, 1987.

13. Ozawa, K., Miura, Y., Motoyoshi, K. and Takaku, F. Radiation sensitivity of leukemia progenitor cells in acute nonlymphocytic leukemia. Cancer Res., 43, 2339-2341, 1983.

14. Rider, W.D. and Messner, H.A. Magna-Field irradiation: Work in progress in bone marrow transplantation at the Princess Margret Hospital, Toronto. Intl. J. Radiat. Oncol. Biol. Phys., 9, 1167, 1983.

15. Thomas, E.D., Storb, R., Clift, R.A., Fefer, A., Johnson, F.L., Neiman, P.E., Lemer, K.G., Glucksberg, H. and Bucker, C.D. Bone marrow transplantation, N. Engl. J. Med., 292, 832-843, 1975. Cancer Res., 43, 2339-2341, 1983.

16. Travis, E.L., Peters, L.J., McNeill, J., Thames, H.D. and Karolis, C. Effect of dose-rate on total body irradiation: Lethality and pathologic findings. Radiother. Oncol., 4, 341-351, 1985.

17. Weichselbaum, R.R., Greenberger, J.S., Schmidt, A., Karpas, A., Moloney, W.C. and Little, J.B. In vitro radiosensitivity of human leukemia cells lines. Radiology, 139, 485-487, 1981.