- Email: [email protected]
Residents essay award: low-dose-rate total lymphoid irradiation: A new method of rapid immunosuppression
Inl. J. Radiation OncologyBid. Phys.. Vol. IS, pp. 547-552 Printed in the U.S.A. All rights reserved.
Copyright
0360-3016/88 $3.00 + .oO 0 1988 Pergamon Press plc
??Special Feature
RESIDENTS ESSAY AWARD: LOW-DOSE-RATE TOTAL LYMPHOID IRRADIATION: A NEW METHOD OF RAPID IMMUNOSUPPRESSION JULIA E. BLUM, M.D., SHARI M. DE SILVA, M.D., DANIEL B. DRACHMAN, M.D., AND STANLEY E. ORDER, M.D., Sc.D., F.A.C.R. Departments of Radiation Oncology and Neurology, Johns Hopkins University School of Medicine, Baltimore, MD
TotalLymphoidIrradiation (TLI)hasbeensuccessfulin inducingimmunosuppression in experimental and clinical applications. However, both the experimental and clinical utility of TLI are hampered by the prolonged treatment courses required (23 days in rats and 30-60 days in humans). Low-dose-rate TLI has the potential of reducing overall treatment time while achieving comparable immunosuppression. This study examines the immunosuppressive activity and treatment toxicity of conventional-dose-rate (23 days) vs lowdose-rate (2-7 days) TLI. Seven groups of Lewis rats were given TLI with 6oCo. One group was treated at conventional-dose-rates (80-110 cGy/ min) and received 3400 cGy in 17 fractions over 23 days. Six groups were treated at low-dose-rate (7 cGy/min) and received total doses of 800,1200,1800,2400,3000, and 3400 cGy over 2-7 days. Rats treated at conventionaldose-rates over 23 days and at low-dose-rate over 2-7 days tolerated radiation with minimal toxicity. The level of immunosuppression was tested using allogeneic (Brown-Norway) skin graft survival. Control animals retained allogeneic skin grafts for a mean of 14 days (range 8-21 days). Conventionaldose-rate treated animals (3400 cGy in 23 days) kept their grafts 60 days (range SO-66 days) (p < 801). Low-dose-rate treated rats (800 to 3400 cGy total dose over 2-7 days) also had prolongation of allogeneic graft survival times following TLI with a doseresponse curve established. The graft survival time for the 3400 cGy low-dose-rate group (66 days, range 52-78 days) was not significantly different from the 3400 cGy conventional-dose-rate group (p > 0.10). When the total dose given was equivalent, low-dose-rate TLI demonstrated an advantage of reduced overall treatment time compared to conventionaldose-rate TLI (7 days vs. 23 days) with no increase in toxicity. This was accomplished without compromise of the immunosuppressant activity of TLI as demonstrated by comparable allogeneic skin graft survival times between the two 3400 cGy treatment groups. This clinical advantage would prove to be beneficial where immediate suppression of the immune system is desirable. Total lymphoid irradiation, Low-dose-rate, Immunosuppression. INTRODUCXION Total-Lymphoid-Irradiation (TLI) has been shown to be an effective immunosuppressant in laboratory aniand in clinical practice.3~15~20*2’ Experiments mals 16-18*23 using TLI in animals have demonstrated prolonged survival of allogeneic skin,17*‘8,23bone marrow,16-18*23kidney, I3 and heart grafts.” In patients, TLI has resulted in improved survival of kidney4,12*24*25 and bone marrow” grafts. In addition, TLI treatment has resulted in clinical improvement in several presumed autoimmune disorders including rheumatoid arthritis,*‘,** lupus nephritis,*’ and multiple sclerosis.3 Despite the demonstrated efficacy of TLI as an immunosuppressant its clinical utility is hampered by the prolonged course of 30-60 days required to achieve radiation derived immunosuppression.3*12~20~2’ In~24 small laboratory animals, experimental TLI treatment as presently used requires 23 day~.‘~-‘~
Reprint requests to: Dr. Julia E. Blum, Department of Radiation Oncology, 600 N Wolfe St., Baltimore, MD 2 1205. Accepted for publication 1 April 1988.
Our study was designed to test a regimen based on radiobiologic data to be described which would shorten the experimental and ultimately the clinical course of TLI for immunosuppression. In this study the immunosuppressive activity and toxicity of conventional (23 days) vs. low-dose-rate (2-7 days) TLI were compared. Survival of allogenic skin grafts was used as the criterion to assess immunosuppression efficacy. Measures of treatment toxicity included diarrhea, weight loss, hematologic response and death. This study presents the results, the radiobiologic rationale, and the potential for future applications of low-dose rate TLI. METHODS
AND
MATERIALS
Adult female Lewis rats weighing 180-230 grams were given TLI with a Siemens Cobalt@’unit* at a treatment * Seimens Cob&’ unit; Seimens Aktiengesellschaft Medical Engineering Group: Erlangen, West Germany.
548
I.
J. Radiation Oncology 0 Biology 0 Physics
distance of 100 cm. We fabricated custom made lucite devices to hold the animals in an immobilized position in conjunction with focused ostalloy blocks during TLI (Fig. 1). Dosimetry was performed using a rat-shaped wax phantom in the lucite cage. Holes were drilled in the phantom and measurements of radiation doses were made at the midplane of the phantom with a solid-state detector (diode) to determine the absorbed radiation dose. The dose distribution was determined for the field at the treatment distance by measurement with the phantom and found to be homogeneous (+3.5%) within the field with minimal scatter under the ostalloy blocks (< 12%) (Fig. 2). Prior to each treatment rats were anesthetized with an intraperitoneal injection of 4% chloral hydrate (0.4 mg/gram body weight) and the ostalloy blocks were placed on the rat cages fluoroscopically to achieve accurate shielding. Mantle, paraaortic/splenic, and pelvic fields were treated in one ventral port for each fractional dose. Dose-rates were altered using lead transmission placed on a block tray near the machine head. Animals were separated into seven treatment groups. One group received conventional-dose-rates (80- 110 cGy/min) TLI at 200 cGy/day to a total of 3400 cGy. Six groups of rats were treated at low-dose-rate (7 cGy/min) to varying total doses (800 cGy in two fractions over 2 days, 1200 cGy in three fractions over 3 days, 1800 cGy in three fractions over 3 days, 2400 cGy in four fractions over 4 days, 3000 cGy in five fractions over 5 days, and 3400 cGy in seven fractions over 7 days). Matched control animals for each group of treated animals received anesthesia but no radiation. Toxic effects of therapy including diarrhea, loss of body weight, and death were monitored. Approximately 0.3 ml of venous blood was drawn from the orbital capil-
September 1988, Volume 15, Number 3
lary plexus of the rats while they were anesthetized with intraperitoneal chloral hydrate and methoxyflurane gas to measure hematocrits and white blood cell counts. Hematocrits were determined by measurements and white blood cells were counted using a hemocytometer capillary tube centrifuge. Four to seven days after TLI was completed both groups of treated animals as well as controls received isogeneic (Lewis) and allogeneic (BrownNorway) full thickness skin grafts to their proximal tails.” Animals received drinking water containing Neomycin Sulfate (500 mg/l) and Polymixin B Sulfate ( 125 mg/l) during and following TLI until the skin graft pins were removed. Table 1 shows the distribution of animals in each control and treated group. Animals were eliminated from the analysis if they died during treatment or during skin graft anesthesia, or if they lost their skin grafts secondary to infection or poor healing before day 7. Skin grafts were considered rejected when they sloughed off the animal. Graft survival times were observed and recorded for the control animals, conventional-dose-rate treated animals (3400 cGy), and the six sets of low-dose-rate treated animals (800,1200, 1800, 2400,3000, and 3400 cGy).
RESULTS Total Lymphoid Irradiation at both conventional and low-dose-rates was well tolerated by the Lewis rats. Mild diarrhea occurred in both groups, and hematocrits fell by less than 5% (Fig. 3). Body weight decreased during TLI in both the conventional and low-dose-rate treated animals ( 10% and 20% decrease respectively) but was regained after the completion of therapy (Fig. 4). Three of 30 animals in the control groups died during the experi-
Fig. 1. Anesthetized rats were irradiated after being placed in custom made lucite devices with focused ostalloy shielding blocks to protect non-lymphoid tissue.
Low-dose-rate total lymphoid irradiation 0 J. E. BLUM etal.
549
ments compared to 4 of 38 in the irradiated groups. Necropsies performed on animals which died during TLI showed evidence of liver and spleen laceration with peritoneal hemorrhage thought to have been caused by trauma from the IP needle stick for anesthesia. Total white blood cell counts fell to < 15% of pretreatment levels within 3 days afier the start of TLI in both the 3400 cGy conventional and 3400 cGy low-dose-rate treated animals and remained low for the duration of therapy (Fig. 5). After TLI was completed, the white blood cell counts increased to control levels in approximately 7- 14 days. Four to seven days after TLI was completed control and treated animals received isogeneic (Lewis) and allogeneic (Brown-Norway) skin grafts. Isogeneic skin grafts were accepted by both control and treated animals. Allogeneic skin grafts were rejected by control rats in 14 f 0.7 days (mean + SEM; range 8-21 days). In contrast, 3400 cGy conventional-dose-rate TLI animals retained their allogeneic skin grafts for 60 + 2.9 days (mean * SEM; range 50-66 days) (Fig. 6). Low-dose-rate TLI also resulted in prolongation of skin graft survival with the 3400 cGy group retaining their grafts for 66 f 5.7 days (mean + SEM; range 52-78 days). A dose response curve was established showing increased graft survival time with increasing total radiation dose (Fig. 6). A comparison of 3400 cGy conventional-dose-rate TLI and 3400 cGy low-dose-rate TLI shows no significant difference in graft survival time (p > 0.10, Students T-test and log rank test). DISCUSSION Fig. 2. Roentgenogram of anesthetized rat in lucite device.
Total-Lymphoid Irradiation has been used in the treatment of Hodgkin’s disease for over 20 years. As a single method of treatment it has not been associated
Opaque areas overlying animals are ostalloy shielding blocks. Numbers show the percent absorbed dose at midplane of the animal within the treatment field and under the blocks.
Table 1. Distribution
of animals in TLI and control groups Died *
Total Controls (no radiation) Conventional-dose-rate Low-dose-rate TLI Conventional-dose-rate (3400 cGy in 23 days) Low-dose-rate 800 cGy 12OOcGy 18OOcGy 2400 cGy 3000 cGy 3400 cGy * Died DT/PTG
DT/PTG
DGA
Graft infected
Included in analysis
5
1
25
2
4 23
6
1
5
1 1 1 :
= animals who died during TLI or prior to skin grafting; DGA = animals who died during graft anesthesia.
4 5 4 5 4 4
550
September 1988. Volume 15, Number 3
I. J. Radiation Oncology 0 Biology 0 Physics
20-
e-0 o--o
IO5
IO
15
20
25
30
35
40
TLI C0”w.l. 45
50
55
hYS
woo cty
LOW Do”ROM I+-TL,_ 60-
5
IO
15
20
25
30
35
Days
Fig. 3. Upper panel shows hematocrit of conventional-dose-rate TLI animals and controls. Lower panel shows hematocrit of a low-dose-rate TT .I animals and controls (mean + SEM).
with an increased incidence of a second malignancy or other sequelae such as severe bacterial infection or sepsis.” This radiotherapy regimen has been shown to induce marked changes in the immune status of treated patients.6 The prolonged immunosuppression induced
i .o, p
60 0
““““““’ 5 15 3400
25
Conventional-Des*-Role
35
Controls
45
55
65
TLI
Days
s
c 5 3400 cGy
Low-Dar-Rote
15
25
35
-
TLI
-
Controls
45
55
65
TLI
Days Fig. 4. Upper panel shows body weights of conventional-doserate TLI animals and controls. Lower panel shows body weights of lowdose-rate TLI animals and controls (mean + SEM).
by TLI19 has led to its use in the treatment mune diseases and organ transplantation.
of autoim-
Basis for immunosuppression During and after TLI a significant decrease in the peripheral blood lymphocyte count is seen. There is a gradual recovery of the count following TLI but it may take as long as 2 years to reach pretreatment levels. In addition to an absolute lymphocytopenia, changes in individual T-cell subsets are seen after TLI.2032’Prior to TLI the number of helper/inducer (T4 cells) is greater than the number of suppressor/cytotoxic (T8 cells) such that the T4/T8 ratio is > 1. Following TLI both subpopulations of T-cells are reduced.2032’With time there is a gradual recovery of the T-suppressor cells while the number of T-helper cells continues to remain decreased.79’3*24This results in a reversal of the T4/T8 ratio to < 1. The resultant lymphocytopenia and subsequent preferential Tsuppressor cell recovery are thought to account for the immunosuppressive action of TLI. This suppressor Tcell dominance may allow the induction of tolerance to foreign tissues or autoantigens. Low-dose-rate radiobiology As dose-rate is lowered radiation damage may be repaired during the prolonged exposure time, allowing survival of potentially damaged cells following radiation. A change in dose-rate affects cellular systems to differing degrees depending on their ability to repair intracellular damage. ‘**The capacity of cells to repair sublethal damage is reflected in the shape of their survival curves’; systems capable of a large amount of repair have large shoulders on the initial part of the survival curves.’ Differences in the ability of cells to repair sublethal damage may be exploited therapeutically by using a
Low-dose-rate total lymphoid irradiation 0
J. E. BLUM
5
IO
15
2b
25
30
35
5
IO
15
20
25
30
35
40
etal.
45
551
50
Fig. 5. Upper panel shows white blood cell counts of conventionaldose-rate TLI animals and controls. Lower panel shows white blood cell counts of low-dose-rate TLI animals and controls (mean + SEM).
change in dose-rate to selectively protect some cellular systems while keeping other systems sensitive to the effects of radiation.’ In our experimental model the target cellular system is the lymphocyte population. Lymphocytes are not able to repair sublethal damage as evidenced by the lack of a shoulder on their radiation survival curve99’4;they remain sensitive to ionizing radiation despite a decrease in dose-rate. By contrast gastrointestinal cells exhibit a broad shoulder on their survival curve and can be expected to repair a large amount of sublethal damage as the dose-rate is lowered. Therefore
a lowered dose-rate should allow continued sensitivity of lymphocytes while selectively sparing the GI tract resulting in an improved therapeutic ratio for a given dose of radiation. Because of the decreased radiation effects seen on the GI tract at low-dose-rates, higher doses may be given daily and the overall treatment time shortened without adverse consequences. Thus, the unique radiobiologic features of low-doserate irradiation which permit repair of sublethal damage of non-target tissues while producing cytotoxic damage to lymphocytes during a shorter period of treatment time make low-dose-rate TLI a modality of experimental and clinical interest.
Conventional vs. low-dose-rate, toxicity and immunosuppression In these experiments conventional-dose-rate TLI and low-dose-rate TLI were well tolerated by the Lewis rats. Acute side effects such as diarrhea and decreased body weight were transient. The effects of TLI on white blood cell counts was evidenced by a rapid decrease in levels immediately after treatment started. Despite the return of the leukocyte count to normal the effects of TLI persisted for extended periods as demonstrated by prolonged survival of allogeneic skin grafts in treated animals. The duration of graft survival seen with our 3400 cGy conventional-dose-rate TLI group compares favorably to that reported by others.” Low-dose-rate TLI also extended allogeneic skin graft survival time and a dose response curve was evident. DosekGy)
Fig. 6. Comparison of skin graft survival in control animals, 3400 cGy conventional-dose-rate TLI animals, and 800-3400 cGy low-dose-rate TLI animals (mean + SD).
Future application, clinical and experimental Low-dose-rate TLI offers a potential clinical advantage over conventional-dose-rate TLI. The overall treatment time was reduced in treated animals from 23 to 7
552
I. J. Radiation Oncology 0 Biology 0 Physics
days. Duration of therapy is an important consideration especially in autoimmune diseases characterized by chronic debilitating side effects where a prolonged course of therapy would impose an emotional, physical, and financial strain on the patient and family as well as allowing possible progression of disease. In organ transplantation short course TLI could also be used to advantage in
September 1988, Volume 15. Number 3
achieving rapid immunosuppression. A short course of low-dose-rate TLI accomplished immunosuppression adequately as demonstrated by comparable allogeneic skin graft survival times between the two 3400 cGy treatment groups. This clinical advantage of shortened treatment time may prove to be beneficial where immunosuppression is desired as immediately as possible.
REFERENCES 1. Broerse, J.J., Zoetelief, J.: The occurrence ofradiation
syndromes in rodents and monkeys in dependence on dose rate and radiation quality. In Response of D&erent Species to Total Body Irradiation. Boston. Martinus Nijhoff Pub. 1984, pp. 175-187. 2. Cassady, J. R., Order, S., Camitta, B., Marck, A.: Modification of gastrointestinal symptoms following irradiation by low dose rate technique. Znt. J. Radiat. Oncol. Biol. Phys. 1( l-2): 15-20, 1975. 3. Cook, S.D., Devereux, C., Troiano, R., Hafstein, M.P., Zito, G., Hemandez, E., Lavenhar, M., Vidaver, R., Dowling, P.C.: Effect of total lymphoid irradiation in chronic progressive multiple sclerosis. Lancet 1: 1405- 1409, 1986. 4. Cortesini, R., Malogoni, E.R., Monari, C., Berloco, P., Capua, A., Marinucci, G., Alfani, D.: Total lymphoid irradiation in clinical transplantation: Experience in 30 high-risk patients. Trans. Proc. 17: 1291-1293, 1985. 5. Fu, K., Phillips, T.L., Kane, L.J., Smith, V.: Tumor and normal tissue response to irradiation in vivo: Variation with decreasing dose rates. Radiology 114: 709-7 16, 1975. 6. Fuks, Z., Strober, S., Bobrove, A.M., Sasazuki, T., McMichael, A., Kaplan, H.S.: Long term effects of radiation on T and B lymphocytes in peripheral blood of patients with Hodgkin’s disease. J. Clin. Invest. 58: 803-8 14, 1976. 7. Haas, G.S., Halperin, E., Doseretz, D., Linggood, R., Russess, P., Colvin, R., Barrett, L., Cosimi, A.B.: Differential recovery of circulating T cell subsets after nodal irradiation for Hodgkin’s disease. J. Zmmunol. 132: 1026-1030, 1984. 8. Hall, E.J.: Radiation dose rate: A factor of importance in radiobiology and radiotherapy. Br. J. Radiol. 45: 81-97, 1972. 9. Hall, E.J.: Radiobiology for the Radiologist. Philadelphia, Harper and Row Pub., 1978. 10. Hudson, L., Hay, R.: Practical Immunology. Edinburgh, Blackwell Scientific Pub., 1980. 11. Kaplan, H.: Hodgkin’s Disease. Cambridge, Harvard University Press, 1972. 12. Myburgh, J.A., Smit, J.A., Meyers, A.M., Botha, J.R., Browde, S., Thomson, M.B.: Total lymphoid irradiation in renal transplantation. World J. Surg. 10: 369-380, 1986. 13. Myburgh, J.A., Smit, J.A., Stark, J.H., Browde, S.: Total lymphoid irradiation in kidney and liver transplantation in the baboon: Prolonged graft survival and alterations in T cell subsets with low cumulative dose regimens. J. Zmmunol. 132: 1019-1025,1984. 14. Order, S.E.: The effects of therapeutic irradiation on lymphocytes and immunity. Cancer 39: 737-743, 1977.
15. Ramsay, N.K.C., Taehwan, K., Nesbit, M.E., Krivit, W., Coccia, P.F., Levitt, S.H., Woods, W.G., Kersey, J.H.: Total lymphoid irradiation and cyclophosphamide as preparation for bone marrow transplantation in severe aplastic anemia. Blood 55(2): 344-346, 1980. 16. Slavin, S., Fuks, Z., Kaplan, H.S., Stroker, S.: Transplantation of allogenic bone marrow without graft-versus-host disease using total lymphoid irradiation. J. Exp. Med. 147: 963-972,1978. 17. Slavin, S., Reitz, B., Bieber, C.P., Kaplan, H.S., Strobe& S.: Transplantation tolerance in adult rats using total lymphoid irradiation: permanent survival of skin, heart, and marrow allografts. J. Exp. Med. 147: 700-707, 1978. 18. Slavin, S., Strober, S., Fuks, Z., Kaplan, H.S.: Induction of specific tissue transplantation tolerance in adult mice: long-term survival of allogenic bone skin grafts. J. Exp. Med. 146: 34-48,1977. 19. Strober, S.: “Managing” the immune system with TLI. Hosp. Prac. 16: 77-86, 1977. 20. Strober, S., Filed, E., Hoppe, R.T., Kotzin, B.L., Shemesh, O., Engleman, E., Ross, J.C., Myers, B.D.: Treatment of intractable lupus nephritis with total lymphoid irradiation. Ann. Znt.Med. 102: 450-458,1985. 21. Strober, S., Tanay, A., Field, E., Hoppe, R.T., Calin, A., Engleman, E., Kotzin, B., Brown, B., Kaplan, H.S.: Efficacy of total lymphoid irradiation in intractable rheumatoid arthritis. Ann. Znt.Med. 102: 44 l-449, 1985. 22. Tanay, A., Field, E.H., Hoppe, R.T., Strobe& S.: Longterm followup of rheumatoid arthritis patients treated with total lymphoid irradiation. Arth. Rheum. 30: I-IO, 1987. 23. Vaiman, M., Daburon, F., Remy, J., Villiers, P.A., de Riberolles, C., Lecompte, Y., Mahouy, G., Fradelizi, D.: Allograft tolerance in pigs after fractionated lymphoid irradiation. Transplantation31: 358-364, 198 1. Y., Ang, K.K., van der 24. Waer, M., Vanrenterghem, Schueren, E., Michielsen, P., Vandeputte, M.: Comparison of the immunosuppressive effect of fractionated total lymphoid irradiation (TLI) vs conventional immunosup pression (CI) in renal cadaveric Allotransplantation. J. Zmmunol. 132: 1041-1048, 1984. 25. Waer, M., Vanrenterghem, Y., Roels, L., Ang, K.K., Bouillom, R., Lerut, T., Gruwez, L., Michielsen, P., Van der Schueren, E., Vandeputte, M.: Total lymphoid irradiation in renal cadeveric transplantation in diabetics. Lancet 2: 1354, 1985.
Copyright
0360-3016/88 $3.00 + .oO 0 1988 Pergamon Press plc
??Special Feature
RESIDENTS ESSAY AWARD: LOW-DOSE-RATE TOTAL LYMPHOID IRRADIATION: A NEW METHOD OF RAPID IMMUNOSUPPRESSION JULIA E. BLUM, M.D., SHARI M. DE SILVA, M.D., DANIEL B. DRACHMAN, M.D., AND STANLEY E. ORDER, M.D., Sc.D., F.A.C.R. Departments of Radiation Oncology and Neurology, Johns Hopkins University School of Medicine, Baltimore, MD
TotalLymphoidIrradiation (TLI)hasbeensuccessfulin inducingimmunosuppression in experimental and clinical applications. However, both the experimental and clinical utility of TLI are hampered by the prolonged treatment courses required (23 days in rats and 30-60 days in humans). Low-dose-rate TLI has the potential of reducing overall treatment time while achieving comparable immunosuppression. This study examines the immunosuppressive activity and treatment toxicity of conventional-dose-rate (23 days) vs lowdose-rate (2-7 days) TLI. Seven groups of Lewis rats were given TLI with 6oCo. One group was treated at conventional-dose-rates (80-110 cGy/ min) and received 3400 cGy in 17 fractions over 23 days. Six groups were treated at low-dose-rate (7 cGy/min) and received total doses of 800,1200,1800,2400,3000, and 3400 cGy over 2-7 days. Rats treated at conventionaldose-rates over 23 days and at low-dose-rate over 2-7 days tolerated radiation with minimal toxicity. The level of immunosuppression was tested using allogeneic (Brown-Norway) skin graft survival. Control animals retained allogeneic skin grafts for a mean of 14 days (range 8-21 days). Conventionaldose-rate treated animals (3400 cGy in 23 days) kept their grafts 60 days (range SO-66 days) (p < 801). Low-dose-rate treated rats (800 to 3400 cGy total dose over 2-7 days) also had prolongation of allogeneic graft survival times following TLI with a doseresponse curve established. The graft survival time for the 3400 cGy low-dose-rate group (66 days, range 52-78 days) was not significantly different from the 3400 cGy conventional-dose-rate group (p > 0.10). When the total dose given was equivalent, low-dose-rate TLI demonstrated an advantage of reduced overall treatment time compared to conventionaldose-rate TLI (7 days vs. 23 days) with no increase in toxicity. This was accomplished without compromise of the immunosuppressant activity of TLI as demonstrated by comparable allogeneic skin graft survival times between the two 3400 cGy treatment groups. This clinical advantage would prove to be beneficial where immediate suppression of the immune system is desirable. Total lymphoid irradiation, Low-dose-rate, Immunosuppression. INTRODUCXION Total-Lymphoid-Irradiation (TLI) has been shown to be an effective immunosuppressant in laboratory aniand in clinical practice.3~15~20*2’ Experiments mals 16-18*23 using TLI in animals have demonstrated prolonged survival of allogeneic skin,17*‘8,23bone marrow,16-18*23kidney, I3 and heart grafts.” In patients, TLI has resulted in improved survival of kidney4,12*24*25 and bone marrow” grafts. In addition, TLI treatment has resulted in clinical improvement in several presumed autoimmune disorders including rheumatoid arthritis,*‘,** lupus nephritis,*’ and multiple sclerosis.3 Despite the demonstrated efficacy of TLI as an immunosuppressant its clinical utility is hampered by the prolonged course of 30-60 days required to achieve radiation derived immunosuppression.3*12~20~2’ In~24 small laboratory animals, experimental TLI treatment as presently used requires 23 day~.‘~-‘~
Reprint requests to: Dr. Julia E. Blum, Department of Radiation Oncology, 600 N Wolfe St., Baltimore, MD 2 1205. Accepted for publication 1 April 1988.
Our study was designed to test a regimen based on radiobiologic data to be described which would shorten the experimental and ultimately the clinical course of TLI for immunosuppression. In this study the immunosuppressive activity and toxicity of conventional (23 days) vs. low-dose-rate (2-7 days) TLI were compared. Survival of allogenic skin grafts was used as the criterion to assess immunosuppression efficacy. Measures of treatment toxicity included diarrhea, weight loss, hematologic response and death. This study presents the results, the radiobiologic rationale, and the potential for future applications of low-dose rate TLI. METHODS
AND
MATERIALS
Adult female Lewis rats weighing 180-230 grams were given TLI with a Siemens Cobalt@’unit* at a treatment * Seimens Cob&’ unit; Seimens Aktiengesellschaft Medical Engineering Group: Erlangen, West Germany.
548
I.
J. Radiation Oncology 0 Biology 0 Physics
distance of 100 cm. We fabricated custom made lucite devices to hold the animals in an immobilized position in conjunction with focused ostalloy blocks during TLI (Fig. 1). Dosimetry was performed using a rat-shaped wax phantom in the lucite cage. Holes were drilled in the phantom and measurements of radiation doses were made at the midplane of the phantom with a solid-state detector (diode) to determine the absorbed radiation dose. The dose distribution was determined for the field at the treatment distance by measurement with the phantom and found to be homogeneous (+3.5%) within the field with minimal scatter under the ostalloy blocks (< 12%) (Fig. 2). Prior to each treatment rats were anesthetized with an intraperitoneal injection of 4% chloral hydrate (0.4 mg/gram body weight) and the ostalloy blocks were placed on the rat cages fluoroscopically to achieve accurate shielding. Mantle, paraaortic/splenic, and pelvic fields were treated in one ventral port for each fractional dose. Dose-rates were altered using lead transmission placed on a block tray near the machine head. Animals were separated into seven treatment groups. One group received conventional-dose-rates (80- 110 cGy/min) TLI at 200 cGy/day to a total of 3400 cGy. Six groups of rats were treated at low-dose-rate (7 cGy/min) to varying total doses (800 cGy in two fractions over 2 days, 1200 cGy in three fractions over 3 days, 1800 cGy in three fractions over 3 days, 2400 cGy in four fractions over 4 days, 3000 cGy in five fractions over 5 days, and 3400 cGy in seven fractions over 7 days). Matched control animals for each group of treated animals received anesthesia but no radiation. Toxic effects of therapy including diarrhea, loss of body weight, and death were monitored. Approximately 0.3 ml of venous blood was drawn from the orbital capil-
September 1988, Volume 15, Number 3
lary plexus of the rats while they were anesthetized with intraperitoneal chloral hydrate and methoxyflurane gas to measure hematocrits and white blood cell counts. Hematocrits were determined by measurements and white blood cells were counted using a hemocytometer capillary tube centrifuge. Four to seven days after TLI was completed both groups of treated animals as well as controls received isogeneic (Lewis) and allogeneic (BrownNorway) full thickness skin grafts to their proximal tails.” Animals received drinking water containing Neomycin Sulfate (500 mg/l) and Polymixin B Sulfate ( 125 mg/l) during and following TLI until the skin graft pins were removed. Table 1 shows the distribution of animals in each control and treated group. Animals were eliminated from the analysis if they died during treatment or during skin graft anesthesia, or if they lost their skin grafts secondary to infection or poor healing before day 7. Skin grafts were considered rejected when they sloughed off the animal. Graft survival times were observed and recorded for the control animals, conventional-dose-rate treated animals (3400 cGy), and the six sets of low-dose-rate treated animals (800,1200, 1800, 2400,3000, and 3400 cGy).
RESULTS Total Lymphoid Irradiation at both conventional and low-dose-rates was well tolerated by the Lewis rats. Mild diarrhea occurred in both groups, and hematocrits fell by less than 5% (Fig. 3). Body weight decreased during TLI in both the conventional and low-dose-rate treated animals ( 10% and 20% decrease respectively) but was regained after the completion of therapy (Fig. 4). Three of 30 animals in the control groups died during the experi-
Fig. 1. Anesthetized rats were irradiated after being placed in custom made lucite devices with focused ostalloy shielding blocks to protect non-lymphoid tissue.
Low-dose-rate total lymphoid irradiation 0 J. E. BLUM etal.
549
ments compared to 4 of 38 in the irradiated groups. Necropsies performed on animals which died during TLI showed evidence of liver and spleen laceration with peritoneal hemorrhage thought to have been caused by trauma from the IP needle stick for anesthesia. Total white blood cell counts fell to < 15% of pretreatment levels within 3 days afier the start of TLI in both the 3400 cGy conventional and 3400 cGy low-dose-rate treated animals and remained low for the duration of therapy (Fig. 5). After TLI was completed, the white blood cell counts increased to control levels in approximately 7- 14 days. Four to seven days after TLI was completed control and treated animals received isogeneic (Lewis) and allogeneic (Brown-Norway) skin grafts. Isogeneic skin grafts were accepted by both control and treated animals. Allogeneic skin grafts were rejected by control rats in 14 f 0.7 days (mean + SEM; range 8-21 days). In contrast, 3400 cGy conventional-dose-rate TLI animals retained their allogeneic skin grafts for 60 + 2.9 days (mean * SEM; range 50-66 days) (Fig. 6). Low-dose-rate TLI also resulted in prolongation of skin graft survival with the 3400 cGy group retaining their grafts for 66 f 5.7 days (mean + SEM; range 52-78 days). A dose response curve was established showing increased graft survival time with increasing total radiation dose (Fig. 6). A comparison of 3400 cGy conventional-dose-rate TLI and 3400 cGy low-dose-rate TLI shows no significant difference in graft survival time (p > 0.10, Students T-test and log rank test). DISCUSSION Fig. 2. Roentgenogram of anesthetized rat in lucite device.
Total-Lymphoid Irradiation has been used in the treatment of Hodgkin’s disease for over 20 years. As a single method of treatment it has not been associated
Opaque areas overlying animals are ostalloy shielding blocks. Numbers show the percent absorbed dose at midplane of the animal within the treatment field and under the blocks.
Table 1. Distribution
of animals in TLI and control groups Died *
Total Controls (no radiation) Conventional-dose-rate Low-dose-rate TLI Conventional-dose-rate (3400 cGy in 23 days) Low-dose-rate 800 cGy 12OOcGy 18OOcGy 2400 cGy 3000 cGy 3400 cGy * Died DT/PTG
DT/PTG
DGA
Graft infected
Included in analysis
5
1
25
2
4 23
6
1
5
1 1 1 :
= animals who died during TLI or prior to skin grafting; DGA = animals who died during graft anesthesia.
4 5 4 5 4 4
550
September 1988. Volume 15, Number 3
I. J. Radiation Oncology 0 Biology 0 Physics
20-
e-0 o--o
IO5
IO
15
20
25
30
35
40
TLI C0”w.l. 45
50
55
hYS
woo cty
LOW Do”ROM I+-TL,_ 60-
5
IO
15
20
25
30
35
Days
Fig. 3. Upper panel shows hematocrit of conventional-dose-rate TLI animals and controls. Lower panel shows hematocrit of a low-dose-rate TT .I animals and controls (mean + SEM).
with an increased incidence of a second malignancy or other sequelae such as severe bacterial infection or sepsis.” This radiotherapy regimen has been shown to induce marked changes in the immune status of treated patients.6 The prolonged immunosuppression induced
i .o, p
60 0
““““““’ 5 15 3400
25
Conventional-Des*-Role
35
Controls
45
55
65
TLI
Days
s
c 5 3400 cGy
Low-Dar-Rote
15
25
35
-
TLI
-
Controls
45
55
65
TLI
Days Fig. 4. Upper panel shows body weights of conventional-doserate TLI animals and controls. Lower panel shows body weights of lowdose-rate TLI animals and controls (mean + SEM).
by TLI19 has led to its use in the treatment mune diseases and organ transplantation.
of autoim-
Basis for immunosuppression During and after TLI a significant decrease in the peripheral blood lymphocyte count is seen. There is a gradual recovery of the count following TLI but it may take as long as 2 years to reach pretreatment levels. In addition to an absolute lymphocytopenia, changes in individual T-cell subsets are seen after TLI.2032’Prior to TLI the number of helper/inducer (T4 cells) is greater than the number of suppressor/cytotoxic (T8 cells) such that the T4/T8 ratio is > 1. Following TLI both subpopulations of T-cells are reduced.2032’With time there is a gradual recovery of the T-suppressor cells while the number of T-helper cells continues to remain decreased.79’3*24This results in a reversal of the T4/T8 ratio to < 1. The resultant lymphocytopenia and subsequent preferential Tsuppressor cell recovery are thought to account for the immunosuppressive action of TLI. This suppressor Tcell dominance may allow the induction of tolerance to foreign tissues or autoantigens. Low-dose-rate radiobiology As dose-rate is lowered radiation damage may be repaired during the prolonged exposure time, allowing survival of potentially damaged cells following radiation. A change in dose-rate affects cellular systems to differing degrees depending on their ability to repair intracellular damage. ‘**The capacity of cells to repair sublethal damage is reflected in the shape of their survival curves’; systems capable of a large amount of repair have large shoulders on the initial part of the survival curves.’ Differences in the ability of cells to repair sublethal damage may be exploited therapeutically by using a
Low-dose-rate total lymphoid irradiation 0
J. E. BLUM
5
IO
15
2b
25
30
35
5
IO
15
20
25
30
35
40
etal.
45
551
50
Fig. 5. Upper panel shows white blood cell counts of conventionaldose-rate TLI animals and controls. Lower panel shows white blood cell counts of low-dose-rate TLI animals and controls (mean + SEM).
change in dose-rate to selectively protect some cellular systems while keeping other systems sensitive to the effects of radiation.’ In our experimental model the target cellular system is the lymphocyte population. Lymphocytes are not able to repair sublethal damage as evidenced by the lack of a shoulder on their radiation survival curve99’4;they remain sensitive to ionizing radiation despite a decrease in dose-rate. By contrast gastrointestinal cells exhibit a broad shoulder on their survival curve and can be expected to repair a large amount of sublethal damage as the dose-rate is lowered. Therefore
a lowered dose-rate should allow continued sensitivity of lymphocytes while selectively sparing the GI tract resulting in an improved therapeutic ratio for a given dose of radiation. Because of the decreased radiation effects seen on the GI tract at low-dose-rates, higher doses may be given daily and the overall treatment time shortened without adverse consequences. Thus, the unique radiobiologic features of low-doserate irradiation which permit repair of sublethal damage of non-target tissues while producing cytotoxic damage to lymphocytes during a shorter period of treatment time make low-dose-rate TLI a modality of experimental and clinical interest.
Conventional vs. low-dose-rate, toxicity and immunosuppression In these experiments conventional-dose-rate TLI and low-dose-rate TLI were well tolerated by the Lewis rats. Acute side effects such as diarrhea and decreased body weight were transient. The effects of TLI on white blood cell counts was evidenced by a rapid decrease in levels immediately after treatment started. Despite the return of the leukocyte count to normal the effects of TLI persisted for extended periods as demonstrated by prolonged survival of allogeneic skin grafts in treated animals. The duration of graft survival seen with our 3400 cGy conventional-dose-rate TLI group compares favorably to that reported by others.” Low-dose-rate TLI also extended allogeneic skin graft survival time and a dose response curve was evident. DosekGy)
Fig. 6. Comparison of skin graft survival in control animals, 3400 cGy conventional-dose-rate TLI animals, and 800-3400 cGy low-dose-rate TLI animals (mean + SD).
Future application, clinical and experimental Low-dose-rate TLI offers a potential clinical advantage over conventional-dose-rate TLI. The overall treatment time was reduced in treated animals from 23 to 7
552
I. J. Radiation Oncology 0 Biology 0 Physics
days. Duration of therapy is an important consideration especially in autoimmune diseases characterized by chronic debilitating side effects where a prolonged course of therapy would impose an emotional, physical, and financial strain on the patient and family as well as allowing possible progression of disease. In organ transplantation short course TLI could also be used to advantage in
September 1988, Volume 15. Number 3
achieving rapid immunosuppression. A short course of low-dose-rate TLI accomplished immunosuppression adequately as demonstrated by comparable allogeneic skin graft survival times between the two 3400 cGy treatment groups. This clinical advantage of shortened treatment time may prove to be beneficial where immunosuppression is desired as immediately as possible.
REFERENCES 1. Broerse, J.J., Zoetelief, J.: The occurrence ofradiation
syndromes in rodents and monkeys in dependence on dose rate and radiation quality. In Response of D&erent Species to Total Body Irradiation. Boston. Martinus Nijhoff Pub. 1984, pp. 175-187. 2. Cassady, J. R., Order, S., Camitta, B., Marck, A.: Modification of gastrointestinal symptoms following irradiation by low dose rate technique. Znt. J. Radiat. Oncol. Biol. Phys. 1( l-2): 15-20, 1975. 3. Cook, S.D., Devereux, C., Troiano, R., Hafstein, M.P., Zito, G., Hemandez, E., Lavenhar, M., Vidaver, R., Dowling, P.C.: Effect of total lymphoid irradiation in chronic progressive multiple sclerosis. Lancet 1: 1405- 1409, 1986. 4. Cortesini, R., Malogoni, E.R., Monari, C., Berloco, P., Capua, A., Marinucci, G., Alfani, D.: Total lymphoid irradiation in clinical transplantation: Experience in 30 high-risk patients. Trans. Proc. 17: 1291-1293, 1985. 5. Fu, K., Phillips, T.L., Kane, L.J., Smith, V.: Tumor and normal tissue response to irradiation in vivo: Variation with decreasing dose rates. Radiology 114: 709-7 16, 1975. 6. Fuks, Z., Strober, S., Bobrove, A.M., Sasazuki, T., McMichael, A., Kaplan, H.S.: Long term effects of radiation on T and B lymphocytes in peripheral blood of patients with Hodgkin’s disease. J. Clin. Invest. 58: 803-8 14, 1976. 7. Haas, G.S., Halperin, E., Doseretz, D., Linggood, R., Russess, P., Colvin, R., Barrett, L., Cosimi, A.B.: Differential recovery of circulating T cell subsets after nodal irradiation for Hodgkin’s disease. J. Zmmunol. 132: 1026-1030, 1984. 8. Hall, E.J.: Radiation dose rate: A factor of importance in radiobiology and radiotherapy. Br. J. Radiol. 45: 81-97, 1972. 9. Hall, E.J.: Radiobiology for the Radiologist. Philadelphia, Harper and Row Pub., 1978. 10. Hudson, L., Hay, R.: Practical Immunology. Edinburgh, Blackwell Scientific Pub., 1980. 11. Kaplan, H.: Hodgkin’s Disease. Cambridge, Harvard University Press, 1972. 12. Myburgh, J.A., Smit, J.A., Meyers, A.M., Botha, J.R., Browde, S., Thomson, M.B.: Total lymphoid irradiation in renal transplantation. World J. Surg. 10: 369-380, 1986. 13. Myburgh, J.A., Smit, J.A., Stark, J.H., Browde, S.: Total lymphoid irradiation in kidney and liver transplantation in the baboon: Prolonged graft survival and alterations in T cell subsets with low cumulative dose regimens. J. Zmmunol. 132: 1019-1025,1984. 14. Order, S.E.: The effects of therapeutic irradiation on lymphocytes and immunity. Cancer 39: 737-743, 1977.
15. Ramsay, N.K.C., Taehwan, K., Nesbit, M.E., Krivit, W., Coccia, P.F., Levitt, S.H., Woods, W.G., Kersey, J.H.: Total lymphoid irradiation and cyclophosphamide as preparation for bone marrow transplantation in severe aplastic anemia. Blood 55(2): 344-346, 1980. 16. Slavin, S., Fuks, Z., Kaplan, H.S., Stroker, S.: Transplantation of allogenic bone marrow without graft-versus-host disease using total lymphoid irradiation. J. Exp. Med. 147: 963-972,1978. 17. Slavin, S., Reitz, B., Bieber, C.P., Kaplan, H.S., Strobe& S.: Transplantation tolerance in adult rats using total lymphoid irradiation: permanent survival of skin, heart, and marrow allografts. J. Exp. Med. 147: 700-707, 1978. 18. Slavin, S., Strober, S., Fuks, Z., Kaplan, H.S.: Induction of specific tissue transplantation tolerance in adult mice: long-term survival of allogenic bone skin grafts. J. Exp. Med. 146: 34-48,1977. 19. Strober, S.: “Managing” the immune system with TLI. Hosp. Prac. 16: 77-86, 1977. 20. Strober, S., Filed, E., Hoppe, R.T., Kotzin, B.L., Shemesh, O., Engleman, E., Ross, J.C., Myers, B.D.: Treatment of intractable lupus nephritis with total lymphoid irradiation. Ann. Znt.Med. 102: 450-458,1985. 21. Strober, S., Tanay, A., Field, E., Hoppe, R.T., Calin, A., Engleman, E., Kotzin, B., Brown, B., Kaplan, H.S.: Efficacy of total lymphoid irradiation in intractable rheumatoid arthritis. Ann. Znt.Med. 102: 44 l-449, 1985. 22. Tanay, A., Field, E.H., Hoppe, R.T., Strobe& S.: Longterm followup of rheumatoid arthritis patients treated with total lymphoid irradiation. Arth. Rheum. 30: I-IO, 1987. 23. Vaiman, M., Daburon, F., Remy, J., Villiers, P.A., de Riberolles, C., Lecompte, Y., Mahouy, G., Fradelizi, D.: Allograft tolerance in pigs after fractionated lymphoid irradiation. Transplantation31: 358-364, 198 1. Y., Ang, K.K., van der 24. Waer, M., Vanrenterghem, Schueren, E., Michielsen, P., Vandeputte, M.: Comparison of the immunosuppressive effect of fractionated total lymphoid irradiation (TLI) vs conventional immunosup pression (CI) in renal cadaveric Allotransplantation. J. Zmmunol. 132: 1041-1048, 1984. 25. Waer, M., Vanrenterghem, Y., Roels, L., Ang, K.K., Bouillom, R., Lerut, T., Gruwez, L., Michielsen, P., Van der Schueren, E., Vandeputte, M.: Total lymphoid irradiation in renal cadeveric transplantation in diabetics. Lancet 2: 1354, 1985.