trimodality therapy (drug/hyperthermia/radiation) with bcnu or mitomycin c

In1 J. Radiakm Oncology Biol Pkys bol. 18, pp. 375-382 0360-3016/90 $3.00 + .OU Printed in the U.S.A. All rights reserved. Copyright 0 1990 Pergamon Press plc

??Original Contribution

TRIMODALITY THERAPY (DRUG/HYPERTHERMIA/RADIATION) WITH BCNU OR MITOMYCIN C

TERENCE S. HERMAN, M.D.,‘T~ BEVERLY A. TEICHER, PH.D.‘,~ AND SYLVIA A. HOLDEN, PH.D.*

‘Dana-Farber Cancer Institute, and ‘Joint Center for Radiation Therapy, 44 Binney Street, Boston, MA 02 115

To develop multimodality treatment combinations with high curative potential in advanced local disease, BCNU (N,N’-bis(2-chloroethyl)-N-nitro-sourea) and mitomycin C were tested with hyperthermia and radiation in the FSaIIC fibrosarcoma system. Growth delay experiments demonstrated that, while neither BCNU nor mitomycin C produced dose modification of the radiation response, and hyperthermia (43”C, 30 min) produced only a moderate dose modification (1.4 f 0.2), the combination of BCNU plus hyperthermia resulted in a radiation dose modifying factor (DMF) of 1.9 + 0.3, and mitomycin C plus hyperthermia a dose modifying factor of 2.1 + 0.4. Tumor cell survival over a range of BCNU doses administered i.p. immediately before hyperthermia resulted in a dose modifying factor of 1.8 + 0.2 versus drug alone. With mitomycin C however, giving the drug immediately prior to heating produced a dose modifying factor due to hyperthermia of only 1.2 + 0.10. Hoechst 33342 diffusion was used to separate tumor cells into predominately oxic and hypoxic subpopulations. Administration of the single, double and trimodality therapies showed that BCNU was 3.1-fold more toxic to the oxic versus the hypoxic cells whereas mitomycin C was 3.5fold more toxic to the hypoxic compared to the oxic cells. Hyperthermia was IA-fold more toxic to the hypoxic versus the oxic cells whereas 10 Gy of radiation was 2.0-fold more toxic to the oxic compared to the hypoxic cells. The combination of hyperthermia plus radiation increased killing in both Hoechst dye defined subpopulations but relatively more in the hypoxic cells in which killing was l&fold greater than in the oxic cells. When heat was delivered immediately after i.p. administration of the anticancer drugs, hyperthermia increased BCNU killing in the oxic cells by 17.2-fold versus 4.4-fold in the hypoxic cells and increased mitomycin-killing by 2.6-fold in the oxic cells versus 17-fold in the hypoxic cells. Use of the full trimodality treatment, given in the sequence drug (BCNU, 50 mg/kg or mitomycin-C 5 mg/kg) + heat (43”C, 30 min) * radiation (10 Gy) produced a 3 log kill in the oxic cells versus a 2 log kill in the hypoxic cells with BCNU and a 2 log kill in the oxic cells versus a 3 log kill in the hypoxic cells with mitomycin C. These results indicate that the use of selected anticancer drugs with hyperthermia and radiation can produce highly cytotoxic interactions which markedly modify the effect of radiation. Proper choice of the anticancer drug can shift the maximum cytotoxicity to either the oxic, presumably oxic or to the hypoxic, presumably hypoxic, cell populations.

BCNU, Mitomycin C, Hyperthermia, Radiation, Tumor subpopulations, Trimodality therapy.

INTNODUCTION

Radiation therapy is often the most appropriate treatment option for the local control of human malignancies ( 15), but advanced tumors often cannot be eradicated by radiation. The development of effective adjuvant treatments for use with radiation, therefore, is an important research question. In this study, we have examined the efficacy of adding the anticancer drugs BCNU (N,N’-bis[2-chloroethyl]-N-nitrosourea) or mitomycin C, to hyperthermia and radiation therapy in an effort to develop combination therapies with highly curative potential.

Significantly increased killing of cells in culture by BCNU at hyperthermic temperatures has been demon-

strated (6, 10, 27). The mechanism of this effect appears to be an increase in the level of alkylation by the drug under hyperthermic conditions (6). BCNU also exhibits even greater cytotoxicity at elevated temperature under acidotic conditions (7, 8) such as those which have been found in regions of solid tumors (40). In addition, treatment of both the EMT6 tumor and a rat glioma tumor in vivo demonstrated a higher cure rate when hyperthermia was added to BCNU (3, 36).

Mitomycin C has been described as the prototype bio- reductive alkylating agent available for clinical use (28). It has been shown to be selectively toxic to hypoxic cells in a variety of systems in vivo and in vitro (17-2 1,24, 28, 34). The interaction of mitomycin C with hyperthermia

This work was presented in part at the 30th ASTRO Meeting in New Orleans, October, 1988.

Reprint requests to: Dr. Terence S. Herman, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02 115.

This work was supported by NC1 grant ROl-CA47379-01 and a grant from Bristol Myers Company, Wallingford, CT.

Accepted for publication 19 July 1989.

375

376 I. J. Radiation Oncology 0 Biology 0 Physics

has been examined in vitro in normally oxygenated (33, 38) and hypoxic cells (33). Although the cytotoxicities of the two treatments were greater than additive in both oxic (33, 38) and hypoxic cells (33), the cytotoxic interaction appeared significantly greater in hypoxic cells (33). Ad- ditionally, the rate of formation of reactive metabolites of mitomycin C under hypoxic conditions was measured in cell-free preparations and a 30 to 50% increase in alkylating activity was observed at elevated temperatures, suggesting that the enhanced cytotoxicity of mitomycin C under hypoxic conditions at hypetthermic temperatures was likely due, at least in part, to an increased rate of activation of the drug (33).

Both BCNU and mitomycin C, therefore, appear to be appropriate agents for use with hyperthermia. In addition, treatment with BCNU has been shown to increase the survival duration in patients with malignant gliomas treated with radiation therapy (37) and mitomycin C has been shown to increase both local control and survival in patients with advanced head and neck cancers treated with radiation therapy (23, 39). Thus, both drugs also appear to be appropriate anticancer drugs for use with hyperthermia and radiation from the standpoint of the radiation-chemotherapy interaction.

METHODS AND MATERIALS

Drugs BCNU (carmustine) was obtained from the Dana-Far-

ber Cancer Institute pharmacy and mitomycin C was purchased.*

Tumor The FSaII fibrosarcoma (26) adapted for growth in cul-

ture (FSaIIC) (35) was carried in C3H/FeJ male mice.+ For the experiments, 2 X 106 tumor cells prepared from a brei of several stock tumors were implanted S.C. into the legs of C3H/FeJ male mice 8-10 weeks of age.

Tumor growth delay experiments When the tumors were approximately 100 mm3 in vol-

ume, treatment was initiated. In those groups receiving the drug BCNU (50 mg/kg) or mitomycin C (5mg/kg) in 0.9% phosphate-buffered saline (0.2 ml) was injected as a single dose i.p. on the first day of treatment. In those groups receiving hyperthermia, heat was delivered locally as a single dose on day 1 of the treatment to the tumor- bearing limb by immersion in a specially designed plexi- glas water bath at 44°C which allowed the centers of tumors to reach 43 -+ 0.2”C as measured by a digital readout thermistoti placed into the center of the tumor in selected control animals as previously described (13). In those groups receiving radiation, X rays were delivered with a

February 1990, Volume 18, Number 2

‘37Cs radiation units at a dose rate of approximately 0.88 Gy/min locally to the tumor-bearing limb (the whole body received less than 2% of the total dose) as single doses of 10, 20 or 30 Gy. No anesthetic was used. The progress of each tumor was measured thrice weekly until it reached a volume of 500 mm3. Two measurements were made on each tumor using calipers and tumor volumes were calculated assuming the tumors to be hemiellipsoids. Tu- mor growth delay was calculated as the days taken by each individual tumor to reach 500 mm3 compared to the untreated controls. Each treatment group had seven animals and the experiment was repeated three times. Days of tumor growth delay are the mean + SE for the treatment group compared to the control.

Sequence of therapy Studies with cisplatin (12) and bleomycin (3 1) in com-

bination with hyperthermia and radiation specifically examined the effect of treatment sequence and drug + heat --f radiation provided the greatest antitumor effects. Because of this prior data from our laboratory as well as previous studies of BCNU (3, 36) and mitomycin C (38) with hyperthermia addressing the question of optimal treatment sequence, in these studies when drug was given with hyperthermia, the heat treatment immediately followed i.p. injection of drug. When radiation was used, radiation always followed heating. When only drug and radiation were tested, radiation followed i.p. injection of drug by 30 min (i.e. as it would have if hyperthermia had been used).

Tumor excision assay When the tumors were approximately 100 mm3 in

volume (about 1 week after tumor cell implantation), the animals were given injections i.p. of BCNU (0, 10, 30, 50, 100, 150 or 200 mg/kg) or mitomycin C (0, 5, 10, 15, or 20 mg/kg) alone, or immediately followed by hyperthermia (43°C 30 min), as described above, to the tumor- bearing limb. Mice were sacrificed 24 hr after treatment to allow for full expression of drug cytotoxicity and repair of potentially lethal damage and then soaked in 95% ethanol. The tumors were excised under sterile conditions and single cell suspensions were prepared for the colony forming assay (32). There was no significant difference in cell yields from tumors of the various treatment groups compared to the cell yields obtained from untreated control tumors. Overall, for these experiments from four pooled tumors the cell yield was 20.5 + 3.5 X lo6 cells. One week later the plates were stained with crystal violet and colonies of more than 50 cells were counted. The untreated tumor cell suspensions had a plating efficiency of 8- 12%. The results are expressed as the surviving frac-

* Sigma Chemical Co., St. Louis, MO. + Jackson Laboratories, Bar Harbor, ME.

$ Sensortech, Inc., Clifton, NJ. p Gammacell 40, Atomic Energy of Canada, Ltd.

Trimodality therapy with BCNU/mitomycin C 0 T. S. HERMAN et al. 311

tion f SE of cells from treated groups compared to untreated controls.

Tumor subpopulation studies: tumor growth and Hoechst 33342 labeling

FSaIIC fibrosarcoma tumors were grown as described above. Animals received the various drug, hyperthermia and radiation treatments described above for the tumor growth delay experiments. Hoechst 33342* (2 mg/kg) dissolved in PBS (phosphate buffered 0.9% saline) was administered by tail vein injection (0.25 ml) to tumor- bearing mice, and tumor cell suspensions were prepared by excising the tumor 20 min after i.v. administration of the dye (1, 2, 22, 29). Single cell suspensions of tumor cells were prepared as described for the tumor excision assay. The cell yields for the various treatment groups (4 pooled tumors) were: controls, 23.6 X 106; 10 Gy, 2 1.7 X 106; 43°C (30 min), 22.2 X 106; BCNU (50 mg/kg), 20.7 X 106; mitomycin C (5 mg/kg), 21.3 X 106; BCNU/ heat, 23.5 X 106; BCNlJ/lO Gy, 19.7 X 106; mitomycin C/heat, 25.1 X 106; mitomycin/ 10 Gy, 22.1 X 106; BCNU/ heat/ 10 Gy, 22.7 X 10” and mitomycin C/heat/ 10 Gy, 23.7 X lo6 cells. There was no detectable cell lysis from the treatments and no statistically significant trends in cell numbers with the various treatments. To remove contaminating erythrocytes, 0.17 M NH3Cl was added to the tumor cell pellets for 3 min at room temperature just after filtering through gauze. The cells were washed once with aMEM, (alpha minimal essential medium)** sup- plemented with 10% FBS (fetal bovine serum).

Flow cytometry and sorting The fluorescence of the cells from tumors was analyzed

and the cells were sorted using the instrument.++ Hoechst 33342 intensity was measured using an argon ion laser with excitation at 350--360 nm (40 mW power) with emission monitored through a 457 nm long pass and 530 nm short pass filter. The fluorescence distribution of the tumor cells based on Hoechst 33342 intensity extended over four logs and two sorted fractions of cells were col- lected, one which contained the brighest 10% of cells and the other containing thle dimmest 20% of cells. These brightest and dimmest cells are the two ends of the fluorescence distribution and therefore represent subpopulations of the tumor near the vasculature and distal to the vasculature at the time the dye was in circulation. The cells were washed once with arMEM containing 10% FBS for colony formation. Alfter 1 week, colonies were stained with crystal violet and colonies of >50 cells were counted manually. The plating efficiency for the unsorted population was 15.5 + 2.7%. For the 10% brightest cells, the plating efficiency was 9.2 + 1.6% and for the 20% dimmest cells the plating efficiency was 5.5 * 1.4%. The results are

expressed as the surviving fraction + SE of the treated bright and dim fractions compared to the bright and dim untreated controls, respectively (14, 16, 3 1).

Data analysis Data on the delay of tumor growth were analyzed with

a BASIC program for a microcomputer.ss The program derived the best fit curve for each set of data, then calculated the median, mean, and standard error of the mean for individual tumor volumes and the day on which each tumor reached 500 mm3. Statistical comparisons were made with the Dunn multiple comparisons test (41).

The dose modifying factors (DMF’s) were calculated by subtracting the growth delay produced by drug, hyperthermia or drug plus hyperthermia at 0 radiation dose from the overall growth delay then calculating the factor that related the slope of the curve for radiation alone to that for radiation plus the other treatments. Additivity is defined here as the product of the surviving fractions for the individual treatments.

RESULTS

The effect on tumor growth delay of adding BCNU or mitomycin C and/or hyperthermia to treatment of the FSaIIC fibrosarcoma with single dose radiation therapy is shown in Figure 1. Preceding radiation delivery with hyperthermia (43°C 30 min) resulted in a dose modifying factor of 1.4 * 0.2. A single dose of BCNU (50 mg/kg) was effective therapy in this tumor and produced a growth delay of 8.7 f 0.9 days. The slope of the drug/radiation growth delay curve was less than that of radiation alone indicating some overlap in the populations of tumor cells being killed by this drug and radiation. When BCNU was followed by hyperthermia, tumor growth delay from the combination was 10.4 + 1.2 days. The trimodality therapy of BCNU then hyperthermia followed by radiation resulted in a dose modifying factor of 1.9 + 0.3 indicating that the drug plus hyperthermia is a more effective radio- sensitizer than hyperthermia or BCNU alone.

Mitomycin C (5 mg/kg) produced a growth delay of 5.3 + 0.5 days in the FSaIIC fibrosarcoma. The administration of mitomycin C prior to single dose radiation treatment resulted in a growth delay curve with a shallower slope than the radiation alone curve. Therefore, there also appeared to be some overlap in the tumor cells populations which were killed by radiation and by mitomycin C as there was for BCNU. When hyperthermia (43°C 30 min) was administered following mitomycin C injection a tumor growth delay of 8.6 + 1.2 days was observed. Radiation following the drug plus hyperthermia treatment resulted in a large enhancement of tumor growth delay and a dose modifying factor of 2.1 + 0.4. As was the case

** Grand Island Biological Company, Grand Island, NY. ++ Coulter Epics V, Hialeaha, FL.

*# Apple II+ microcomputer.

378 1. J. Radiation Oncology 0 Biology 0 Physics February 1990, Volume 18, Number 2

BCNU MITOMYCIN C

Radiation Dose, Gray

Fig. 1. Growth delay of the FSaIIC fibrosarcoma produced by radiation and BCNU (50 mg/kg) or mitomycin C (5 mg/kg) with or without hypetthermia (43°C 30 min) administered after drug injection and prior to radiation treatment. The treatment groups were: radiation (lo,20 or 30 Gy) (0); hyperthermia then radiation (0); drug then radiation (m); and drug followed by hyperthermia then radiation (0). Bars are S.E.M.

with BCNU, mitomycin C followed by hyperthermia was a more effective radiosensitizing treatment than hyperthermia or mitomycin C alone.

When animals bearing FSaIIC fibrosarcoma tumors were treated with a range of doses of BCNU and tumor

0.0

:E o.oo’t b\ L v) I \

0.000 1

t

BCNU+43°(30Min)

0.0000 1 I .L

I 1 I I 1 50 100 150 200

BCNU Dose, mg/kg

Fig. 2. Survival of FSaIIC cells from tumors treated in vivo with various doses of BCNU (0) or with BCNU followed by hyperthermia (43°C 30 min) (0). Points are the means of three experiments; bars are S.E.M.

cell survival measured in vitro, the results shown in Figure 2 were obtained. Tumor cell kill by BCNU continued to increase in a log-linear manner over the dosage range examined. Hyperthermia (43°C 30 min) killed about 30% of the tumor cells and had a dose modifying effect on the toxicity of BCNU to the FSaIIC tumor cells. Whereas at 50 mg/kg of BCNU, hyperthermia increased tumor cell kill by 5-fold; at 200 mg/kg of BCNU, hyperthermia increased tumor cell kill by 2 logs. The dose modifying factor obtained by the addition of hyperthermia to treatment with BCNU was 1.8 + 0.2.

Using Hoechst 33342 diffusion as a marker of cell dis- tance from vascular supply (1, 2, 22, 29), the survival of tumor cells from FSaIIC tumors treated in vivo with BCNU, hyperthermia and radiation alone and in combination was examined and the results appear in Figure 3. Hoechst 3342 was administered i.p. 24 hr after the various treatments and the animals were sacrificed 20 min post dye injection. Single cell suspensions were prepared from the tumors and fractions representing the 10% brightest cells referred to hereafter as the oxic cells, and the 20% dimmest cells referred to hereafter as the hypoxic cells, were sorted and plated for colony formation. As we

BRIGHT DIM

Fig. 3. Survival of subpopulations based on Hoechst 33342 fluorescence intensity of FSaIIC cells from tumors treated in vivo with hyperthermia (43”C, 30 min), radiation (10 Gy) or BCNU (50 mg/kg) alone or in combination. Each experiment was repeated three times. Bars are S.E.M.

Trimodality therapy with BCNU/mitomycin C 0 T. S. HERMAN et al.

have described previously, hyperthermia (43°C 30 min) was about 1.4-fold more cytotoxic toward the hypoxic subpopulation and radiation (10 Gy) was about 2.0-fold more toxic toward the oxic cell subpopulation (14, 16, 3 1). The combination treatment of hyperthermia followed by radiation was about 1.8-fold more toxic toward the hypoxic subpopulation indicating a positive interaction of these two modalities in the presumed hypoxic tumor regions (14). BCNU (50 mg/kg) was about 3. l-fold more toxic toward the oxic cells than toward the hypoxic cells which may indicate a di:fference in the ability of this drug to diffuse into those tumor regions which are more distal from the vasculature. Hyperthermia, administered immediately after the drug injection increased the toxicity of the BCNU 17.2-fold in the oxic cell subpopulation and 4.4-fold in the hypoxic cell subpopulation. Therefore, the increase in the toxicity of BCNU by hyperthermia in the oxic cell subpopulation was greater than additive and the combination was approximately additive in the hypoxic cells. BCNU (50 mg/kg) followed by radiation ( 10 Gy) was 5.0-fold more toxic toward the oxic subpopulation than toward the hypoxic subpopulation. This level of cytotoxicity represents approximately additive toxicity of the two modalities in each of the subpopulations. Finally, treatment with BCNU followed by hyperthermia and then radiation killed nearly 3-logs of oxic cells and approximately 2-logs of hypoxic cells resulting in about a 9.0- fold sparing of the hypoxic relative to the oxic cell subpopulation with this trimodality treatment combination.

As has been observed with other alkylating agents, mitomycin C showed incmasing kill of FSaIIC tumor cells with increasing dose of the drug in a log-linear manner (Fig. 4). As opposed to HCNU, only a very limited degree of dose modification was seen when mitomycin C administration was followed by hyperthermia (43°C 30 min) (dose modifying factor = 1.2 + 0.1) and the action of this combined treatment in the whole tumor survival assay appeared to be ad’ditive.

Using Hoechst 33342 diffusion as a marker of cell dis- tance from the vascular supply as described above, the effect of mitomycin C (5 mg/kg), hyperthermia (43°C 30 min) and radiation (10 Gy) was examined on the survival of oxic and hypoxic FS,aIIC tumor subpopulations (Fig. 5). Mitomycin was about 3.5-fold more toxic toward the hypoxic cells than toward the oxic cells ( 16). This finding is consistent with the observation in many cell lines that mitomycin C is more toxic toward hypoxic cells (presumably dim cells) than toward normally oxygenated cells (presumably bright cells) (17-2 1, 24, 34). When mitomycin C administration was followed by hyperthermia treatment there was a 2.6-fold increase in cytotoxicity toward the oxic cell subpopulation and a 17-fold increase in cytotoxicity toward the hypoxic cell subpopulation. These changes represent approximate additivity of the two modalities in the oxic cells and a greater than additive toxicity of the two modalities in the hypoxic cells. The overall result of the combination of mitomycin C and

0.0010 5 10 15 20

Mitomycin C Dose, mg/kg

Fig. 4. Survival of FSaIIC cells from tumors treated in vivo with various doses of mitomycin C (0) or with mitomycin C followed by hyperthermia (43”C, 30 min) (0). Points are the means of three experiments; bars are S.E.M.

hyperthermia was a 2 1.5-fold greater kill of hypoxic cells compared to the oxic cells.

Mitomycin C followed by radiation produced approximately additive cytotoxicity in the oxic cells and slightly less than additive cytotoxicity in the hypoxic cell subpopulation resulting in an overall 1 S-fold relative sparing of the hypoxic cells. When mitomycin C was followed by hyperthermia and then radiation, nearly 2-logs of cell kill was observed in the oxic tumor cells and nearly 3-logs in the hypoxic tumor cell subpopulation. Overall, therefore, there was a relative 7.2-fold greater kill of the hypoxic cells by this trimodality combination treatment than of the oxic cells.

DISCUSSION

Animal and human tumors have been shown to contain areas which are relatively hypoxic and acidotic as compared with normal tissues (40) presumably because of inadequate blood supply. In recent years, interest in the study of physiological parameters relevant to the mi- croenvironments of tumors has increased. Both pH and the level of oxygenation have been studied in relation to the cytotoxicity of many anticancer drugs, as they have for radiation.

Hahn and Shiu studied the effect of both pH and temperature elevation on the cytotoxicity of BCNU. A major finding was that, in cells both acutely exposed to low pH and chronically adapted to low pH, a marked increase in sensitivity to BCNU was seen at 42°C in the acidotic en- vironment (7, 8). By tumor growth delay we observed a

380 I. J. Radiation Oncology 0 Biology 0 Physics February 1990, Volume 18, Number 2

BRIGHT DIM 1 .o

T T

g 01 ._ . 0 2 IL cn c .- > .-

g 0.01 v)

0.00 1

I

Fig. 5. Survival of subpopulations based on Hoechst 33342 fluorescence intensity of FSaIIC cells from tumors treated in viva with hyperthermia (43”C, 30 min), radiation (10 Gy) or mitomycin C (5 mg/kg) alone or in combination.

dose modifying effect of BCNU plus hyperthermia on radiation therapy of the FSaIIC fibrosarcoma which was not evident in the absence of the hyperthermia treatment. Using a single 50 mg/kg dose of BCNU with hyperthermia we observed a marked increase in the killing of the oxic (bright) tumor cell subpopulation, which would be expected to be at normal pH, and a lesser increase in killing of the hypoxic (dim) tumor cell subpopulation, which may include acidotic cells, as compared to the effect of BCNU alone. This finding probably reflects a lower level of drug reaching the hypoxic cells and effective activation of the drug present in both subpopulations by the hyperthermia. The addition of hyperthermia (43’C, 30 min) probably was not able to significantly increase the amount of BCNU reaching the hypoxic cell population.

The cytotoxicities of many drugs have also been studied as a function of the level of oxygenation of in vitro cell cultures (5,9,34). With mitomycin C important increases in cytotoxicity were apparent under hypoxic conditions (17-2 1, 24, 34). Teicher et al. (33) have also shown that the cytotoxicity of mitomycin C towards hypoxic cells was increased at 42°C. By the tumor growth delay assay in the FSaIIC fibrosarcoma, the combination of mitomycin C and hyperthermia produced a dose modification

of radiation which did not occur in the absence of hyperthermia treatment. Although the action of mitomycin C and hyperthermia in combination in the whole tumor excision experiments appeared to be additive, the Hoechst dye tumor cell subpopulation assay indicated a greater than additive effect of the drug plus hyperthermia in the hypoxic cell subpopulation. The advantage of the mitomycin C/hyperthermia treatment in the hypoxic cells also carried over to the trimodality treatment and resulted in a 9-fold increase in killing of the hypoxic versus the oxic cells. If hypoxic cells do contribute significantly to the failure to control human malignancies with radiation then knowledge of the relative cytotoxic potential of these treatments within putative oxic versus hypoxic tumor cell populations should be important.

We have made extensive use of the Hoechst 33342 dye methodology (1, 2, 14, 22). This technique allows separation of tumor cells into dim (presumably hypoxic) and bright (presumably euoxic) subpopulations based on ex- travascular diffusion of the dye. Solid tumor vasculature is in constant flux (25) so that giving the dye 24 hr post radiation may have given a different separation of bright and dim cells than if the dye had been given during radiation. The 24 hr time point was chosen, however, to allow adequate time for potentially lethal radiation damage repair and chemotherapeutic cytotoxicity to be fully expressed. Moreover, we have previously studied the effect of giving the dye directly after irradiation as opposed to 24 hr following treatment and found no difference in the relative radioresistance of the dim cells at these time points (14). Hyperthermia also causes changes in the tumor vasculature (4). This is one reason why we chose a mild heat treatment of 43°C for 30 min which caused only a barely detectable growth delay of 1.4 days and no detectable lysis at 24 hr post treatment. We have not directly measured changes in tumor blood flow in the FSaIIC tumor, but, if the heat treatment had radically altered the tumor circulation, one would have expected greater levels of cell kill and growth delay than were observed because this effect is probably one of the major cytotoxic mechanisms of hyperthermia in vivo (30).

We have extensively examined the interactions of hyperthermia and radiation alone (14) and in combination with cisplatin (12) and bleomycin (3 1) as well as mitomycin C and BCNU in the laboratory. In all of the whole tumor and tumor subpopulation cell survival studies single doses of chemotherapy, hyperthermia and radiation were used. The doses of chemotherapy selected for the subpopulation studies were in the preclinical single dose therapeutic range. The hyperthermia dose (43°C 30 min) is also in the therapeutic range. In clinical treatment maintenance of 42”C-43°C for 1 hr is often the treatment goal (15). The radiation dose of 10 Gy was selected to produce about 1 log of tumor cell kill alone. Clinically, patients receiving trimodality therapy (cisplatin/hyperthermia/radiation) in our Phase I/II trial receive 0.4 Gy of radiation on the day of trimodality treatment (11) to

Trimodality therapy with BCNU/mitomycin C 0 T. S. HERMAN et al. 381

maximize the interaction of the cisplatin with the radia- ever, may require the use of drug combinations ( 15). Ad- tion. In the current studies, only in the case of mitomycin dition of a second drug such as mitomycin C, C did the combination treatment yield a greater cell kill misonidazole or SR2508 might be necessary to produce in the hypoxic cells, andl, in fact, each of the other treat- equally efficient killing of tumor cells existing over the ments was at least 1 log less effective in the hypoxic cells. range of physiological conditions present in solid tumors. Preliminary studies with SR2508 and misonidazole, The feasibility of these treatment combinations, however, however, indicated that these agents also produce greater will depend on the ability to heat tumors effectively and killing in the hypoxic cells when used with hyperthermia on the normal tissue toxicities of the drugs to be used + radiation. and, unfortunately, drugs such as BCNU and mitomycin

Our preliminary clinical data (11) indicate that the C at commonly used doses produce prolonged bone mar- combination of CDDP, lhyperthermia and radiation is ef- row suppression which will prevent their frequent admin- fective. Developing the most effective chemotherapy, hy- istration as might be desirable during a course of frac- perthermia and radiation treatments for the clinic, how- tionated radiation therapy.

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trimodality therapy (drug/hyperthermia/radiation) with bcnu or mitomycin c

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