Br. J. Cancer (1978) 37, Suppl. III, 178
ENHANCEMENT OF LOCAL TUMOUR CONTROL BY MISONIDAZOLE AND HYPERTHERMIA H. B. STONE Fronm the Departmnent of Radiology and Radiation Biology, Colorado State University, Fort Collits, Colorado 80523, U.S.A.
Summary.-Combination treatment of a C3H mammary carcinoma with misonidazole, hyperthermia and radiation resulted in greater local tumour control than with any of these agents singly or in pairs. Dose modification factors for the 3-fold combinations were 3-28 when tumours were immersed in a 42 5°C waterbath for 1 h after irradiation, and 5.03 at 43-O0C. Cytotoxicity of misonidazole alone was slight, as reflected histologically and in tumour growth. Hyperthermia had a marked effect on these two parameters. Foot damage by hyperthermia was greatest when feet were taped.
MISONIDAZOLE, although it is selectively toxic to hypoxic cells (Hall and RoizinTowle, 1975; Moore, Palcic and Skarsgard, 1976; Sridhar, Koch and Sutherland, 1976) does not by itself produce local tumour control following a single dose. The doses of radiation required for a given level of local tumour control, however, have been shown to be reduced by factors of about 2 in combination with this drug (Brown, 1975; Peters, 1976; Sheldon, Foster and Fowler, 1974; Sheldon and Hill, 1977; Stone and Withers, 1975). In contrast, treatment of tumours by hyperthermia alone at temperatures of 41-5°-45-00C has been shown to produce local control (Cavaliere et al., 1967; Crile, 1963; Overgaard and Overgaard, 1972). Hyperthermia has also been shown to enhance local control of tumours by radiation (Crile, 1963; Hahn, Alfieri and Kim, 1974; Hahn et al., 1976; Thrall, Gillette and Dewey, 1975). In addition, it also has been shown to enhance the cytotoxicity of misonidazole (Bleehen, Honess and Morgan, 1977; George, Hirst and McNally, 1977; Hall et al., 1977; Sridhar and Sutherland, 1977; Stratford and Adams, 1977), and in combination with misonidazole and irradiation, to enhance significantly cellular radiosensitivity (Hofer et al., 1977). The pur-
pose of the present investigation was to determine the effect of the three agents, radiation, hyperthermia, and misonidazole, on local control of a C3H mouse mammary carcinoma. METHODS AND MATERIALS
Third generation implants of the C3H mammary carcinoma CSU-MCa were made i.m. into the right hind leg of male and female C3H/Mai mice (Microbiological Associates, Inc., Walkersville, MD, USA). At a diameter of 8 mm, when they were treated, these tumours contain an estimated 4000 hypoxic cells (Thrall et al., 1975). Misonidazole was generously donated by Dr Carey Smithen of Roche Products, Ltd., England. It was administered i.p. at a dose of 1 0 mg/g body wt 30 min before irradiation. A concentration of 20 mg/ml in Ringer's solution was used, and controls were given an equivalent volume of Ringer's solution. Tumours were irradiated with a General Electric Maxitron, operated at 300 kVp, 20 ma, with an HVL of 1-36 mm Cu, and a dose rate of 680 rad/min to a field 2-7 cm in diameter. The mice were breathing air during irradiation and were anaesthetized with pentobarbital (0.065 mg/g body wt, but mice treated with misonidazole were given 0-032 mg/g because of the interaction of the two drugs). The TCD50/120 (radiation dose required for local control at 120 days after irradiation in 500' of the mice) was deter-
179
IRRADIATION, MISONIDAZOLE AND HYPERTHERMIA
mined on the basis of 24-61 mice irradiated at 7-14 doses per determination. The procedure has been described previously by Stone (1974). For hyperthermia, mice were immersed to the groin in waterbaths at 42-5°C or 43 0°C for 1 h, starting within 2-3 min after irradiation. The order of irradiation and heating was based on the finding of Thrall et al. (1975) that irradiation followed by hyperthermia was more effective than heating followed by irradiation. Tumours were removed for histological studies at 1, 6, 24 or 48 h after treatment and equatorial cross-sections were stained with haematoxylin and eosin. The entire tumour cross-section was projected on to a grid using a slide projector, resulting in a magnification of 840 x. Areas occupied by grossly viable and necrotic tumour, haemorrhage, interspersed necrotic tumour and haemorrhage, normal tissue, and space were tallied with microscopic verification. Nine of 66 tumours were fragmented during preparation and were therefore evaluated microscopically by the point-intercept method of Chalkley (1943). Tumours scored by both methods gave similar results. In tumour control experiments, the foot was not included in the radiation field, but was immersed in the waterbath during heating of the leg, as illustrated in Fig. la, or, for larger mice, as in Fig. lb. Excessive foot damage was observed in these mice, and a separate experiment was performed to compare other methods of immobilization: a wire hook around the ankle (Fig. Ic) and instant-
bonding glue (Fig. Id, Super-Glue-3, WVoodhill Chemical Sales Corporation, Cleveland, Ohio 44128), which was carefully removed with acetone after treatment. The feet were scored for swelling, desquamation, and limb loss every 2-3 days for 2 weeks, then weekly through 90 days after treatment. RESULTS
Local control of 8 mm CSU-MCa mammary tumours following treatment with X-rays alone and in combination with misonidazole and hyperthermia are shown in Table I, along with their dose modification factors (DMF = TCD50 in controlTCD50 in experimental group). Both misonidazole and hyperthermia enhanced tumour control over that by radiation only, and the combination of three modalities was more effective than any two. Growth curves of unirradiated tumours treated with misonidazole, 43 0°C waterbath, or both, or neither are shown in Fig. 2. Although misonidazole appeared to have a slight effect, hyperthermia and misonidazole with hyperthermia significantly delayed tumour growth. There was considerable variation, however, between individual tumours in the groups treated with hyperthermia, as reflected in the wide error bars. There were no cures. The results of the tumour histology
TABLE I.-Local Control of Tumours Grotip
Cointrol (X only) Misoni(lazole (M + X)* Hyperthermia (X 4- A)t 42 -5°C 43 *0°C Misonidazole + Hyperthermia
(M + X + A)*t 42 * 50C 43 - O°C
TCD50 (95% confidtence limit) 6350 (6070-6640) 2530 (2460-2610)
No. of Mice 37s 51
DMF (95% confi(lence limit): (i's X only)
Other DAlFs (950' confi(lenice limit)
2-51 (2 40-2 62)
1.31 (1-15-1-48)
(01(l-B59-2-52) 3680 (3420-3970)
*3680 (3320-4080)
40 61
1*73 (161-1.84)-441.90 (l'-66-2.18) 1 * 73 (1 56-1 91) 91 (2 * 28-:33 73) -
1940 (1650-2280)
24 3 * 28 (2 * 88-3 7') 1260 (950-1680) 5 03 (3 99)6634) 50 * 1-0 mg misonidlazole/g body wt was given i.p. 30 min before irra(liation. t The leg was immersed in a temperature-controlled water-bath at the stated temperatuire within 2-3 min after completion of irradiation. t
TCD50controiITCD50experimentaI
for
I h. starting
180
H. B. STONE
a,
b
d. ..
.01.0
:.
FIG. 1.-Methods of immobilization of rnice during hyperthermia treat,ment: (a) Foot taped to a metal rod, with body either held in a plastic tube, or (b) taped to the rod; (c) ankle caught with a metal hook, and (d) toes affixed to a piece of X-ray film using instant bonding glue.
study are shown in Table II. In controls, the proportion of viable and necrotic tissue remained fairly constant for 24 h after treatment, but by 48 h there was a slight decrease in viable tissue and an increase in necrotic tissue. During this time interval, these tumours doubled in volume. In misonidazole-treated mice,
there was a slight transient decrease in the mean proportion of viable tumour and an increase in necrotic and haemorrhagicnecrotic tissue, but by 48 h the values were similar to those in controls, although the mean tumour volume was about 80% of that in controls. Tumours treated with hyperthermia, with and without misoni-
181
IRRADIATION, MISONIDAZOLE AND HYPERTHERMIA
TABLE II.-Tumour Histology % Cross-sectional area ± s.d.t t
__A
Misonidazole + 430, Misoni- 43°C, Time Histol1 h 1 h h* ogyt Control dazole 31 3 55 I 15 73 78± 14 78 7 28 -4-13 17 16 16 + 17 8 N 15 10 4 17 3 HRN 6± 5 14 V 6 77 ±- 6 55 35 37 27 50 9 24 35 i 13 32 11 13 ± 4 30 N 5 17 28 21 18 HN 10 ± 10 15 32 V 5 16 18 6 24 80± 10 67 10 54=127 7 56 12 J 5 12 N 8 30 17 23 38 HN 8± 8 21 6 35 4 5 27 67 ± 21 66 V 48 17 3 45 25 56 16 22 N 23 5 28±1140±13 10 ± 9 12 HN * Time of sample with respect to end of hvperthermia treatment. Misonidazole or Ringer's solution were given 40 min before the start of the ]-h hyperthermia treatment; or, for misonidazole alone and controls, 2 h 40 min before the "1-h" sample. This regimen was chosen to be comparable to that in tumout control studies, but without irradiation. t V = viable; N = necrotic (pyknotic, karyorrhectic nuclei); HN= haemorrhagic-necrotic: no sharp demarcation between haemorrhagic and necrotic tissue. The data have been normalized to exclude normal tissue, tissue spaces, and blood. I Mean of 3-5 tumours per group. 1
+1
E
E
w w
--
cr :D 0
DAYS AFTER TREATMENT FIG. 2.-Growth of CSU-MCa mammary tumours in controls, misonidazole-treated mice (1. Omg/g body weight, i.p.), hyperthermia-treated mnice (immersion of tumourbearing leg in 43 0°C waterbath for 1 h), and mice treated with both misonidazole and hyperthermia. There were 10 mice in MisoniControl; each group. A (43°C, dazole (1 mg/g, i.p.); ,.
h);
---
misonidazole + A.
dazole, showed a marked decrease in the 80 proportion of viable tissue and an increase _ _ in necrotic and haemorrhagic-necrotic F 60 -- t X,TAPE,TUBE,A - - - - - - - - NO X,TAPE,TUBE,A 0 tissue. However, as observed in tumour - ( X,TAPE,A growth, there was considerable variability 00 40 X ,HOOK, A between tumours in the same group, as /,GLUE,A .g 20 _.1/ ,TAPE ,TUBE, NO A reflected in the large standard deviation. Loss of toes in mice immobilized various .................................................................... 0 ways is shown in Fig. 3. Damage was 20 30 70 80 90 10 40 50 60 greatest in those mice whose feet were DAYS AFTER TREATMENT and were also given hyperthermia taped Fie. 3. Toe loss in mice immobilized as in treatment, but minimal in those imFig. 1. The abbreviations are as follows: X =4000 rad to the leg, not including the mobilized with the hook or glue. Swelling foot; Tape, tube = as in Fig. 1(a); Tape of the feet was observed in all groups, but as in Fig. 1(b); Hook = as in Fig. (1); Glue =as in Fig. 1(d); A=immersion of leg and subsided in about a month in mice given foot in a 42-5°C waterbath for 1 h immedieither hyperthermia or irradiation only. ately after irradiation. In mice treated with both hyperthermia and irradiation, swelling persisted in other tumours with the same dosage and about half the feet for 90 days after timing of drug administration (Brown, treatment. 1975; Peters, 1976; Sheldon et al., 1974; and Hill, 1977; Stone, 1974). This Sheldon DISCUSSION value probably reflects both sensitization The DMF of 2-51 for misonidazole in of hypoxic cells by misonidazole and this tumour is higher than that found for cytotoxicity. If the reduction in proporO
=
182
H. B. STONE
tion of viable cells observed histologically in the 6-h sample represents a loss from the hypoxic compartment only, then about 4 of the hypoxic cells were killed. The maximal reduction in volume would be about 30%, or to about 7 mm in diameter. The delay of approximately 1 day observed in tumour growth, if indeed it is significant, is of this order of magnitude. Thrall et al. (1975), found a DMF of 1P39 when this tumour was treated in a 44 5°C waterbath for 15 min following irradiation in air. The DMF of 1-73 for heating at 42-5°C or 43 0°C for 1 h suggests that these doses of heat treatment are more effective than 44 5°C for 15 min. The finding that the TCD50 for irradiation plus heating for 1 h in a waterbath was the same at 42 5°C and at 43 0°C was not expected. Preliminary measurements of tumour core temperatures with a thermistor probe mounted in a 24-gauge needle indicated individual differences between tumours, and considerable overlap between the two groups, with final core temperatures ranging from 0-1 to 0 8°C below waterbath temperatures. Further investigations are in progress. This may also explain the differences in response of individual tumours observed in heattreated tumours in the histological and tumour growth studies. The product of the DMF values of 2-51 for misonidazole plus radiotherapy and 1-73 for heat-plus-radiotherapy predicts a DMF for combination therapy of 4-33 (3.40-5.49) for 42-50C and of 4-33 (3-305.68) for 43-00C if hyperthermia and misonidazole were acting independently in enhancing the radioresponse of the tumours. The observed values were 3.28 (2-88-3-73) and 5-03 (3.99-6-34), respectively. This suggests that at 42-5°C the two agents may have been interacting with radiation either partially by similar mechanisms, or on the same tumour subpopulation of cells, perhaps hypoxic cells. The DMF at 43-0OC is consistent with a hypothesis of independence, but may also reflect enhanced cytotoxicity of misonidazole at the higher temperature (Bleehen,
et al., 1977; George et al., 1977; Hall et al., 1977; Sridhar et al., 1976; Sridhar and
Sutherland, 1977; Stratford and Adams, 1977). Hofer et al. (1977) reported significant potentiation of cell killing by combining metronidazole or misonidazole with irradiation during hyperthermia. As with their results, the enhancement of effect in the present experiments was greater than the maximal enhancement expected if oxygen were fully sensitizing all hypoxic cells, a maximal DMF of 2-5-3-0. An important consideration for clinical application of such combination treatment will be assessment of critical normal tissue damage in order to determine whether there will be a therapeutic gain, i.e., less enhancement of normal tissue response than tumour response. This will depend on inherent differences in tumour and normal tissue response in a given tumour and on the development of technology for heating tumours to higher temperatures than the surrounding tissues. CONCLUSIONS
(1) Significant enhancement of tumour control was observed in tumours treated with a combination of misonidazole, radiation, and hyperthermia, compared to any of the treatments alone or paired. The latter two agents did not by themselves or together produce local tumour control. The dose modification factor of the 3-fold combination was greater than the oxygen enhancement ratio. (2) The amount of damage to the foot, which was heated but not irradiated, was markedly enhanced in mice whose feet were immobilized by tape during hyperthermia treatment. This damage could be greatly reduced, however, by gluing the foot or attaching it with a hook. (3) Toxicity by hyperthermia and by misonidazole and hyperthermia combined was reflected in tumour growth and histology. This investigation was supported by Grant Numbers CA 17831 and CA 20344, awarded by the National Cancer Institute, DHEW.
IRRADIATION, MISONIDAZOLE AND HYPERTHERMIA I would like to thank Vicki Woodard and Beverly Ensley for excellent technical assistance, and Dr William C. Dewey for criticism of the manuscript. Tumour histology was examined at the suggestion of Dr Ralph E. Durand.
REFERENCES BLEEHEN, N. M., HONESS, D. J. & MORGAN, J. E. (1977) Interaction of Hyperthermia and the Hypoxic Cell Sensitizer Ro-07-0582 on the EMT6 Mouse Tumour. Br. J. Cancer, 35, 299. BROWN, J. M. (1975) Selective Radiosensitization of the Hypoxic Cells of Mouse Tumors With the Nitroimidazoles Metronidazole and Ro-07-0582. Radiat. Res., 64, 633. CAVALIERE, R., CIOCATTO, E. C., GIOVANELLA, B. C., HEIDELBERGER, C., JOHNSON, R. O., MARGOTTINI, M., MONDOVi, B., MORICCA, G. & Rossi-FANELLI, A. (1967) Selective Heat Sensitivity of Cancer Cells. Cancer, 20, 1351. CHALKLEY, H. W. (1943) Method for the Quantitative Morphologic Analysis of Tissues. J. natn. Cancer Inst., 4, 47. CRILE, G.. JR. (1963) The Effect of Heat and Radiation on Cancers Implanited on the Feet of Mice. Cancer Res., 23, 372. GEORGE, K. C., HIRST, D. G. & MCNAL,LY, N. J. (1977) Effect of Hyperthermia on Cytotoxicity of the Radiosensitizer Ro-07-0582 in a Solid Mouse Tumour. Br. J. Cancer, 35, 372. HAHN, E. W., ALFIERI, A. A. & KIM, J. H. (1974) Increased Cures Using Fractionated Exposures of X irradiation and Hyperthermnia in Local Treatment of the Ridgway Osteogenic Sarcoma in Mice. Radiology, 113, 199. HAHN, E. W., CANADA, T. R., ALFIERI, A. A. & McDONALD, J. C. (1976) The Interaction of Hyperthermia with Fast Neutrons or X-rays on Local Tumor Response. Radiat. Res., 68, 39. HALL, E. J., ASTOR, M., GEARD, C. & BIAGLOW, J. (1977) Cytotoxicity of Ro-07-0582; Enhancement by Hyperthermia and Protection by Cysteamine. Br. J. Cancer, 35, 809. HALL, E. J. & RoIzIN-TOWLE, L. (1975) Hypoxic Sensitizers: Radiobiological Studies at the Cellular Level. Radiology, 117, 453. HOFER, K. G., HOFER, M. G., IERACITANO, J. &
183
McLAUGHLIN, W. H. (1977) Radiosensitization of Hypoxic Tumor Cells by Simultaneous Administration of Hyperthermia and Nitroimidazoles. Radiat. Res., 70, 362. MOORE, B. A., PALCIC, B. & SKARSGARD, L. D. (1976) Radiosensitizing and Toxic Effects of the 2nitroimidazole Ro-07-0582 in Hypoxic Mammalian Cells. Radiat. Res., 67, 459. OVERGAARD, K. & OVERGAARD, J. (1972) Investigations on the Possibility of a Thermic Tumour Therapy-I. Short-wave Treatment of a Transplanted Isologous Mouse Mammary Carcinoma. Eur. J. Cancer, 8, 65. PETERS, L. J. (1976) Modification of the Radiocurability of a Syngeneic Murine Squamous Carcinoma by its Site of Growth, by Electron-affinic Drugs, and by ICRF 159. Br. J. Radiology, 49, 708. SHELDON, P. W., FOSTER, J. L. & FOWLER, J. F. (1974) Radiosensitization of C3H Mouse Mammary Tumours by a 2-nitroimidazole Drug. Br. J. Cancer, 30, 560. SHELDON, P. W. & HILL, S. A. (1977) Hypoxic Cell Radiosensitizers and Local Control by X-ray of a Transplanted Tumour in Mice. Br. J. Cancer, 35, 795. SRIDHAR, R., KOCH, C. & SUTHERLAND, R. (1976) Cytotoxicity of Two Nitroimidazole Radiosensitizers in an in vitro Tumour Model. Int. J. Radiat. Oncol. Biol. Phys., 1, 1149. SRIDHAR, R. & SUTHERLAND, R. (1977) Hyperthermic Potentiation of Cytotoxicity of Ro-07-0582 in Multicell Spheroids. Int. J. Radiat. Oncol. Fiol. Phys., 2, 531. STONE, H. B. (1974) Radiotherapy of a MVTouse Mamnmary Carcinoma Following Treatment With 5-iodo-2'-deoxyuridine. Radiology, 112, 719. STONE, H. B. & WITHERS, H. R. (1975) Enhancement of the Radioresponse of a Murine Tumour by a Nitroimidazole. Br. J. Radiology, 48, 411. STRATFORD, I. J. & ADAMS, G. E. (1977) Effect of Hyperthermia on Differential Cytotoxicity of a Hypoxic Cell Radiosensitizer, Ro-07-0582, on Mammalian Cells in vitro. Br. J. Cancer, 35, 307. THRALL, D. E., GILLETTE, E. L. & DEWEY, W. C. (1975) Effect of Heat and Ionizing Radiation on Normal and Neoplastic Tissue of the C3H Mouse. Radiat. Res., 63, 363.
ENHANCEMENT OF LOCAL TUMOUR CONTROL BY MISONIDAZOLE AND HYPERTHERMIA H. B. STONE Fronm the Departmnent of Radiology and Radiation Biology, Colorado State University, Fort Collits, Colorado 80523, U.S.A.
Summary.-Combination treatment of a C3H mammary carcinoma with misonidazole, hyperthermia and radiation resulted in greater local tumour control than with any of these agents singly or in pairs. Dose modification factors for the 3-fold combinations were 3-28 when tumours were immersed in a 42 5°C waterbath for 1 h after irradiation, and 5.03 at 43-O0C. Cytotoxicity of misonidazole alone was slight, as reflected histologically and in tumour growth. Hyperthermia had a marked effect on these two parameters. Foot damage by hyperthermia was greatest when feet were taped.
MISONIDAZOLE, although it is selectively toxic to hypoxic cells (Hall and RoizinTowle, 1975; Moore, Palcic and Skarsgard, 1976; Sridhar, Koch and Sutherland, 1976) does not by itself produce local tumour control following a single dose. The doses of radiation required for a given level of local tumour control, however, have been shown to be reduced by factors of about 2 in combination with this drug (Brown, 1975; Peters, 1976; Sheldon, Foster and Fowler, 1974; Sheldon and Hill, 1977; Stone and Withers, 1975). In contrast, treatment of tumours by hyperthermia alone at temperatures of 41-5°-45-00C has been shown to produce local control (Cavaliere et al., 1967; Crile, 1963; Overgaard and Overgaard, 1972). Hyperthermia has also been shown to enhance local control of tumours by radiation (Crile, 1963; Hahn, Alfieri and Kim, 1974; Hahn et al., 1976; Thrall, Gillette and Dewey, 1975). In addition, it also has been shown to enhance the cytotoxicity of misonidazole (Bleehen, Honess and Morgan, 1977; George, Hirst and McNally, 1977; Hall et al., 1977; Sridhar and Sutherland, 1977; Stratford and Adams, 1977), and in combination with misonidazole and irradiation, to enhance significantly cellular radiosensitivity (Hofer et al., 1977). The pur-
pose of the present investigation was to determine the effect of the three agents, radiation, hyperthermia, and misonidazole, on local control of a C3H mouse mammary carcinoma. METHODS AND MATERIALS
Third generation implants of the C3H mammary carcinoma CSU-MCa were made i.m. into the right hind leg of male and female C3H/Mai mice (Microbiological Associates, Inc., Walkersville, MD, USA). At a diameter of 8 mm, when they were treated, these tumours contain an estimated 4000 hypoxic cells (Thrall et al., 1975). Misonidazole was generously donated by Dr Carey Smithen of Roche Products, Ltd., England. It was administered i.p. at a dose of 1 0 mg/g body wt 30 min before irradiation. A concentration of 20 mg/ml in Ringer's solution was used, and controls were given an equivalent volume of Ringer's solution. Tumours were irradiated with a General Electric Maxitron, operated at 300 kVp, 20 ma, with an HVL of 1-36 mm Cu, and a dose rate of 680 rad/min to a field 2-7 cm in diameter. The mice were breathing air during irradiation and were anaesthetized with pentobarbital (0.065 mg/g body wt, but mice treated with misonidazole were given 0-032 mg/g because of the interaction of the two drugs). The TCD50/120 (radiation dose required for local control at 120 days after irradiation in 500' of the mice) was deter-
179
IRRADIATION, MISONIDAZOLE AND HYPERTHERMIA
mined on the basis of 24-61 mice irradiated at 7-14 doses per determination. The procedure has been described previously by Stone (1974). For hyperthermia, mice were immersed to the groin in waterbaths at 42-5°C or 43 0°C for 1 h, starting within 2-3 min after irradiation. The order of irradiation and heating was based on the finding of Thrall et al. (1975) that irradiation followed by hyperthermia was more effective than heating followed by irradiation. Tumours were removed for histological studies at 1, 6, 24 or 48 h after treatment and equatorial cross-sections were stained with haematoxylin and eosin. The entire tumour cross-section was projected on to a grid using a slide projector, resulting in a magnification of 840 x. Areas occupied by grossly viable and necrotic tumour, haemorrhage, interspersed necrotic tumour and haemorrhage, normal tissue, and space were tallied with microscopic verification. Nine of 66 tumours were fragmented during preparation and were therefore evaluated microscopically by the point-intercept method of Chalkley (1943). Tumours scored by both methods gave similar results. In tumour control experiments, the foot was not included in the radiation field, but was immersed in the waterbath during heating of the leg, as illustrated in Fig. la, or, for larger mice, as in Fig. lb. Excessive foot damage was observed in these mice, and a separate experiment was performed to compare other methods of immobilization: a wire hook around the ankle (Fig. Ic) and instant-
bonding glue (Fig. Id, Super-Glue-3, WVoodhill Chemical Sales Corporation, Cleveland, Ohio 44128), which was carefully removed with acetone after treatment. The feet were scored for swelling, desquamation, and limb loss every 2-3 days for 2 weeks, then weekly through 90 days after treatment. RESULTS
Local control of 8 mm CSU-MCa mammary tumours following treatment with X-rays alone and in combination with misonidazole and hyperthermia are shown in Table I, along with their dose modification factors (DMF = TCD50 in controlTCD50 in experimental group). Both misonidazole and hyperthermia enhanced tumour control over that by radiation only, and the combination of three modalities was more effective than any two. Growth curves of unirradiated tumours treated with misonidazole, 43 0°C waterbath, or both, or neither are shown in Fig. 2. Although misonidazole appeared to have a slight effect, hyperthermia and misonidazole with hyperthermia significantly delayed tumour growth. There was considerable variation, however, between individual tumours in the groups treated with hyperthermia, as reflected in the wide error bars. There were no cures. The results of the tumour histology
TABLE I.-Local Control of Tumours Grotip
Cointrol (X only) Misoni(lazole (M + X)* Hyperthermia (X 4- A)t 42 -5°C 43 *0°C Misonidazole + Hyperthermia
(M + X + A)*t 42 * 50C 43 - O°C
TCD50 (95% confidtence limit) 6350 (6070-6640) 2530 (2460-2610)
No. of Mice 37s 51
DMF (95% confi(lence limit): (i's X only)
Other DAlFs (950' confi(lenice limit)
2-51 (2 40-2 62)
1.31 (1-15-1-48)
(01(l-B59-2-52) 3680 (3420-3970)
*3680 (3320-4080)
40 61
1*73 (161-1.84)-441.90 (l'-66-2.18) 1 * 73 (1 56-1 91) 91 (2 * 28-:33 73) -
1940 (1650-2280)
24 3 * 28 (2 * 88-3 7') 1260 (950-1680) 5 03 (3 99)6634) 50 * 1-0 mg misonidlazole/g body wt was given i.p. 30 min before irra(liation. t The leg was immersed in a temperature-controlled water-bath at the stated temperatuire within 2-3 min after completion of irradiation. t
TCD50controiITCD50experimentaI
for
I h. starting
180
H. B. STONE
a,
b
d. ..
.01.0
:.
FIG. 1.-Methods of immobilization of rnice during hyperthermia treat,ment: (a) Foot taped to a metal rod, with body either held in a plastic tube, or (b) taped to the rod; (c) ankle caught with a metal hook, and (d) toes affixed to a piece of X-ray film using instant bonding glue.
study are shown in Table II. In controls, the proportion of viable and necrotic tissue remained fairly constant for 24 h after treatment, but by 48 h there was a slight decrease in viable tissue and an increase in necrotic tissue. During this time interval, these tumours doubled in volume. In misonidazole-treated mice,
there was a slight transient decrease in the mean proportion of viable tumour and an increase in necrotic and haemorrhagicnecrotic tissue, but by 48 h the values were similar to those in controls, although the mean tumour volume was about 80% of that in controls. Tumours treated with hyperthermia, with and without misoni-
181
IRRADIATION, MISONIDAZOLE AND HYPERTHERMIA
TABLE II.-Tumour Histology % Cross-sectional area ± s.d.t t
__A
Misonidazole + 430, Misoni- 43°C, Time Histol1 h 1 h h* ogyt Control dazole 31 3 55 I 15 73 78± 14 78 7 28 -4-13 17 16 16 + 17 8 N 15 10 4 17 3 HRN 6± 5 14 V 6 77 ±- 6 55 35 37 27 50 9 24 35 i 13 32 11 13 ± 4 30 N 5 17 28 21 18 HN 10 ± 10 15 32 V 5 16 18 6 24 80± 10 67 10 54=127 7 56 12 J 5 12 N 8 30 17 23 38 HN 8± 8 21 6 35 4 5 27 67 ± 21 66 V 48 17 3 45 25 56 16 22 N 23 5 28±1140±13 10 ± 9 12 HN * Time of sample with respect to end of hvperthermia treatment. Misonidazole or Ringer's solution were given 40 min before the start of the ]-h hyperthermia treatment; or, for misonidazole alone and controls, 2 h 40 min before the "1-h" sample. This regimen was chosen to be comparable to that in tumout control studies, but without irradiation. t V = viable; N = necrotic (pyknotic, karyorrhectic nuclei); HN= haemorrhagic-necrotic: no sharp demarcation between haemorrhagic and necrotic tissue. The data have been normalized to exclude normal tissue, tissue spaces, and blood. I Mean of 3-5 tumours per group. 1
+1
E
E
w w
--
cr :D 0
DAYS AFTER TREATMENT FIG. 2.-Growth of CSU-MCa mammary tumours in controls, misonidazole-treated mice (1. Omg/g body weight, i.p.), hyperthermia-treated mnice (immersion of tumourbearing leg in 43 0°C waterbath for 1 h), and mice treated with both misonidazole and hyperthermia. There were 10 mice in MisoniControl; each group. A (43°C, dazole (1 mg/g, i.p.); ,.
h);
---
misonidazole + A.
dazole, showed a marked decrease in the 80 proportion of viable tissue and an increase _ _ in necrotic and haemorrhagic-necrotic F 60 -- t X,TAPE,TUBE,A - - - - - - - - NO X,TAPE,TUBE,A 0 tissue. However, as observed in tumour - ( X,TAPE,A growth, there was considerable variability 00 40 X ,HOOK, A between tumours in the same group, as /,GLUE,A .g 20 _.1/ ,TAPE ,TUBE, NO A reflected in the large standard deviation. Loss of toes in mice immobilized various .................................................................... 0 ways is shown in Fig. 3. Damage was 20 30 70 80 90 10 40 50 60 greatest in those mice whose feet were DAYS AFTER TREATMENT and were also given hyperthermia taped Fie. 3. Toe loss in mice immobilized as in treatment, but minimal in those imFig. 1. The abbreviations are as follows: X =4000 rad to the leg, not including the mobilized with the hook or glue. Swelling foot; Tape, tube = as in Fig. 1(a); Tape of the feet was observed in all groups, but as in Fig. 1(b); Hook = as in Fig. (1); Glue =as in Fig. 1(d); A=immersion of leg and subsided in about a month in mice given foot in a 42-5°C waterbath for 1 h immedieither hyperthermia or irradiation only. ately after irradiation. In mice treated with both hyperthermia and irradiation, swelling persisted in other tumours with the same dosage and about half the feet for 90 days after timing of drug administration (Brown, treatment. 1975; Peters, 1976; Sheldon et al., 1974; and Hill, 1977; Stone, 1974). This Sheldon DISCUSSION value probably reflects both sensitization The DMF of 2-51 for misonidazole in of hypoxic cells by misonidazole and this tumour is higher than that found for cytotoxicity. If the reduction in proporO
=
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H. B. STONE
tion of viable cells observed histologically in the 6-h sample represents a loss from the hypoxic compartment only, then about 4 of the hypoxic cells were killed. The maximal reduction in volume would be about 30%, or to about 7 mm in diameter. The delay of approximately 1 day observed in tumour growth, if indeed it is significant, is of this order of magnitude. Thrall et al. (1975), found a DMF of 1P39 when this tumour was treated in a 44 5°C waterbath for 15 min following irradiation in air. The DMF of 1-73 for heating at 42-5°C or 43 0°C for 1 h suggests that these doses of heat treatment are more effective than 44 5°C for 15 min. The finding that the TCD50 for irradiation plus heating for 1 h in a waterbath was the same at 42 5°C and at 43 0°C was not expected. Preliminary measurements of tumour core temperatures with a thermistor probe mounted in a 24-gauge needle indicated individual differences between tumours, and considerable overlap between the two groups, with final core temperatures ranging from 0-1 to 0 8°C below waterbath temperatures. Further investigations are in progress. This may also explain the differences in response of individual tumours observed in heattreated tumours in the histological and tumour growth studies. The product of the DMF values of 2-51 for misonidazole plus radiotherapy and 1-73 for heat-plus-radiotherapy predicts a DMF for combination therapy of 4-33 (3.40-5.49) for 42-50C and of 4-33 (3-305.68) for 43-00C if hyperthermia and misonidazole were acting independently in enhancing the radioresponse of the tumours. The observed values were 3.28 (2-88-3-73) and 5-03 (3.99-6-34), respectively. This suggests that at 42-5°C the two agents may have been interacting with radiation either partially by similar mechanisms, or on the same tumour subpopulation of cells, perhaps hypoxic cells. The DMF at 43-0OC is consistent with a hypothesis of independence, but may also reflect enhanced cytotoxicity of misonidazole at the higher temperature (Bleehen,
et al., 1977; George et al., 1977; Hall et al., 1977; Sridhar et al., 1976; Sridhar and
Sutherland, 1977; Stratford and Adams, 1977). Hofer et al. (1977) reported significant potentiation of cell killing by combining metronidazole or misonidazole with irradiation during hyperthermia. As with their results, the enhancement of effect in the present experiments was greater than the maximal enhancement expected if oxygen were fully sensitizing all hypoxic cells, a maximal DMF of 2-5-3-0. An important consideration for clinical application of such combination treatment will be assessment of critical normal tissue damage in order to determine whether there will be a therapeutic gain, i.e., less enhancement of normal tissue response than tumour response. This will depend on inherent differences in tumour and normal tissue response in a given tumour and on the development of technology for heating tumours to higher temperatures than the surrounding tissues. CONCLUSIONS
(1) Significant enhancement of tumour control was observed in tumours treated with a combination of misonidazole, radiation, and hyperthermia, compared to any of the treatments alone or paired. The latter two agents did not by themselves or together produce local tumour control. The dose modification factor of the 3-fold combination was greater than the oxygen enhancement ratio. (2) The amount of damage to the foot, which was heated but not irradiated, was markedly enhanced in mice whose feet were immobilized by tape during hyperthermia treatment. This damage could be greatly reduced, however, by gluing the foot or attaching it with a hook. (3) Toxicity by hyperthermia and by misonidazole and hyperthermia combined was reflected in tumour growth and histology. This investigation was supported by Grant Numbers CA 17831 and CA 20344, awarded by the National Cancer Institute, DHEW.
IRRADIATION, MISONIDAZOLE AND HYPERTHERMIA I would like to thank Vicki Woodard and Beverly Ensley for excellent technical assistance, and Dr William C. Dewey for criticism of the manuscript. Tumour histology was examined at the suggestion of Dr Ralph E. Durand.
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