INT. J. RADIAT. BIOL ., 1991, VOL . 60, NO . 5, 833-845

Hyperthermia promotes the incidence of tumours followingX-irradiation of the rat cervical cord region

P. SMINIAt, J . HAVEMANt, W. JANSEN$,J . J. G. W. HENDRIKSt and J. D. P. VAN DIJKt

t Department of Radiotherapy, University of Amsterdam,Academisch Medisch Centrum, Meibergdreef 9, 1105 AZ Amsterdam,The Netherlands$Laboratory for Pathological Anatomy, H . G. Gooszenstraat 1,7415 CL Deventer, The Netherlands

(Received 8 January 1991 ; revision received 24 April 1991 ;accepted 1 May 1991)

The cervical region of the rat, including the spinal cord (cervical 5-thoracic 2)was irradiated with single doses of 15-32 Gy 250 kV X-rays . Hyperthermia, attemperatures of 42-, 43- and 44±0 .1 ° C for 30min was applied to the cervicalvertebral column and immediate adjacent tissues for 5-10 min or 7 h afterX-irradiation. Over a period of 18-21 months, animals were followed up tomonitor neurological complications occurring as a result of damage to the spinalcord (Sminia et al . 1991). We also noted the development of neoplasms eitherinside or outside the cervical region . The data on tumour incidence wereanalysed retrospectively using the actuarial method. Although hyperthermiaalone was not carcinogenic, it led to a significant increase of radiation-inducedtumours. This increase of radiation carcinogenesis was observed both withhyperthermia applied 5-10 min after X-rays and with an interval of 7h betweenX-rays and heat . Cancer induction was highest after the lower radiation doses(16 Gy) combined with high heat doses (30 min 44 °C). The latent period forinduction of tumours by X-rays was 472+19 days (mean+ SEM ; n=24) .Latency was significantly shortened by hyperthermia to 404±34 days (n = 22) ifapplied 5-10 min after X-rays and to 348±6 days (n = 33) with an interval of 7 h .Histology revealed that 86% (38/44) of the examined tumours found inside thevolume treated with hyperthermia and irradiation were sarcomas . The percent-age of animals with a tumour outside the treated volume was almost the samefor all treatment groups. Most of these tumours were cf the mammary glandtype .

1 . IntroductionAs the success of modern cancer therapy has increased the duration of

survival and curability of many patients, so the recognition of long-term complica-tions of therapy has increased (Coleman and Tucker 1989). In the therapy of severalforms of cancer, such as melanomas, head-and-neck and breast malignancies, thecombination of radiotherapy and hyperthermia seems to achieve a better localcontrol than radiation alone (Gonzalez Gonzalez et al. 1986, 1988, Arcangeli et al.1988). Recently, Berdov and Menteshashvili (1990) reported that thermoradio-therapy of patients with locally advanced carcinoma of the rectum significantlyincreased the 5-year survival relative to the control group . As a consequence ofincreased duration of survival after initial cancer therapy, secondary neoplasms maybecome apparent .

0020-7616/91 $3 .00 © 1991 Taylor & Francis Ltd

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In vitro studies show that hyperthermia alone does not lead to cell transforma-tion (Harisiadis et al . 1980, Raaphorst et al. 1981, Watanabe et al . 1984, Hall andHei 1985) . Animal studies also show that hyperthermia alone does not inducecancer (Urano 1981, Baker et al . 1988, Sminia et al . 1990) . In vitro results on theeffects of hyperthermia on X-ray-induced cell transformation are not consistent .Several studies have shown that heat, either applied immediately before or afterX-rays, reduced cell transformation frequency (Harisiadis et al. 1980, Raaphorstet al. 1981, Hall and Hei 1985). However, Clark et al . (1981), Raaphorst et al . (1986)and Raaphorst and Azzam (1988) showed that incubation at 37°C and recoverybetween heat treatment and irradiation, or vice-versa, resulted in elevated celltransformation compared with irradiation alone . Discrepancy with respect to theoncogenic potential of X-rays and heat also exists in animal data . Baker et al . (1988)did not observe any effect of hyperthermia on radiation carcinogenesis in the mouseleg, whereas a significant increase was observed by Urano et al . (1989) . In aprevious study we reported a significant increase in tumour incidence after localizedhyperthermia 90 days after irradiation of the rat cervical region (Sminia et al. 1990) .

The present study is a retrospective analysis of data on tumour inductionobtained from an earlier investigation on the effects of local hyperthermia applied5-10 min after X-irradiation of the cervical spinal cord (Sminia et al. 1991), andrecent data with an interval of 7 h between heat and X-rays .

2 . Materials and methods2 .1 . Animals

Female Wistar rats, aged 12-13 weeks were used in all experiments . For the5-10 min interval experiments, animals (WU/CPB rats) were obtained from HarlanCPB, Zeist, The Netherlands, whereas for the 7 h interval experiments, animalsfrom the same rat strain were obtained from Iffa Credo, Someren, The Nether-lands. Animals were anaesthetized with sodium pentobarbital (Nembutal®50 mg/kg body weight i .p .) about 15 min before hyperthermia . The analgesicpentazocine (1 .8 mg/animal i.p .) was administered about 10 min thereafter . Whenanimals were irradiated, light anaesthesia was obtained by about half of the sodiumpentobarbital dose and no pentazocine was administered. After treatment, animalsrecovered from anaesthesia in an infant incubator (Air-Shields Europe, Shannon,Ireland) at a temperature of 32 °C . Treatment groups consisted of seven to 17animals. The total number of animals included in the present study was 490. Theexperimental protocol was approved by the animal welfare commission of theUniversity of Amsterdam (DEC/TB2 .1) . The animal experiments were performedaccording to the regulations of the veterinarian inspection .

2.2. IrradiationThe cervical region of the animals was irradiated using a Siemens Stabilipan

X-ray generator operated at 250 kV and 15 mA, filtered with 1 mm Cu . Theanaesthetized animal was placed in supine position on a 10 mm thick lead shield .Local irradiation of the spinal cord, cervical 5-thoracic 2, was via a rectangular fieldof 15 mm x 20 mm. Irradiation was performed in air at a dose-rate of 326 cGy/minwith a focus-skin distance of 24 .2 cm. The dose measured at the ventral side of theanimal was about 80% of the dose measured at the position of the spinal cord . Thetemperature in the irradiation room was kept at 26°C to avoid a drop in bodytemperature of the anaesthetized animals during irradiation .

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2.3. HyperthermiaThe rat cervical vertebral column, including the spinal cord region cervical

5-thoracic 2, and immediate adjacent tissues were heated using a 434 MHzmicrowave applicator. Surrounding tissues were only slightly heated . Technicalaspects of this non-invasive heating system and details on thermometry have beendescribed previously (Sminia et al. 1987, 1988). In all experiments the temperaturewas regulated using a reference thermocouple probe which was placed against oneof the cervical vertebra 6, 7 or thoracic 1 . Animals were heated for 30 min at areference temperature of 42 °C, 43°C and 44°C. If the cervical vertebral column washeated at a reference temperature of 43 .0±0.1°C (mean ± standard deviation) for30 min, the mean average temperature inside the vertebral canal was 42 .1 ±0 .4 °C .The temperature measured in the intervertebral discs was as high as the referencetemperature+ 0-1'C . The mean temperature in the oesophagus, which is locatedjust below the heated vertebral column, was 40 . 3 ± 1 .0 °C (n = 8) . Lateral to thevertebral column at 5 mm and 10 mm, mean temperatures were respectively41 .8±0 .6°C and 40 .4±0 .7° C (n=4) . The mean rectal temperature was 38 . 2±0.4°C(n = 8) . In sham-treated animals only the reference thermocouple probe was placedin position for 30 min .

2.4. Follow-up and statistical analysis of the dataThe follow-up period was 21 months for the animals obtained from Harlan CPB

which were used in the 5-10-min interval experiments . Animals obtained from IffaCredo showed a higher late incidence of X-ray-induced tumours (see e .g. Figure 1)so that, for these animals, the follow-up had to be restricted to 18 months . Duringfollow-up, animals were examined frequently for general condition as well as theincidence of palpable tumours . The tumour incidence results were analysed usingthe methodology of survival analysis correcting for loss of animals from theexperiment. Animals could be lost due to intercurrent disease or due to severeneurological complications occurring as a result of radiation or heat damage to thespinal cord . This was most pronounced at irradiation doses exceeding 24 Gy with orwithout hyperthermia. The results of these observations are published separately(Sminia et al . 1991). The time interval from the day of treatment to the day ofobservation of a palpable tumour was evaluated using the statistical package ofsocial sciences (SPSS-X User's Guide, 3rd edn, 1988. SPSS Europe BV, Gorin-chem, The Netherlands) . This procedure calculates survival functions whichcharacterize the distribution of survival times in a population using the actuarialmethod developed by Berkson and Gage (1950). The probability of tumour-freesurvival was calculated for the different treatment groups using a non-parametrictechnique developed by Lee and Desu (1972) . Significant differences in the latentperiod between the various treatment groups was calculated using Student's t-test .

2.5. HistologyAll palpable tumours larger than 0.5-1 cm in diameter were excised and

included in the study . Standard histological techniques were used to prepare tissuesections .

3 . Results3.1 . Tumour incidence

Figures 1(a) and 1(b) are plots of the cumulative proportion of animals survivingtumour-free after 16 Gy alone or 16 Gy followed by 30 min heating at 42 °C, 43 ° C or

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(a)

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WEEKS AFTER TREATMENT

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Figure 1 . Percentage of animals surviving without evidence of a tumour inside the treatedvolume as a function of the follow-up period . Animals were treated with 16 Gy alone(0) or followed by hyperthermia for 30 min at 42°C (0), 43 °C (A) and 44°C (V)after 5-10 min (a) or 7 h (b) .

44°C, given at 5-10 min and 7 h after irradiation . These figures show that 30 minheat treatment at 42°C or 43°C did not affect the incidence of tumours, but that30 min 44°C significantly increased tumour incidence. Figure 2(a) shows that afterhigher irradiation doses, i .e. 20 Gy, only a mild heat dose of 30 min 43 °C appliedshortly after irradiation is required to observe a significant increase in radiationcarcinogenesis . Increasing the temperature to 44°C for 30 min (given at 5-10 minafter 20 Gy) did not result in a further increase in tumour incidence. Increasing thetime interval between 20 Gy and 30 min heating at 44 °C to 7 h did, however, resultin a significant increase of radiation carcinogenesis (Figure 2b) . With a furtherincrease in irradiation dose to 24 Gy, the augmenting effect of hyperthermia on the

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WEEKS AFTER TREATMENT

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Figure 2 . Percentage of animals surviving without evidence of a tumour inside the treatedvolume as a function of the follow-up period . Animals were treated with 20 Gy alone(0) or followed by hyperthermia for 30 min at 42°C (0), 43 °C (A) and 44°C (V)after 5-10 min (a) or 7 h (b) .

radiation carcinogenesis was observed only with 30 min at 44 °C applied 7 h afterX-rays (Figure 3b) . Animals with a long follow-up after 24 Gy followed by 30 minat 43 °C were, however, not available .

No significant differences in the percentage of animals with a tumour outside thetreated volume were observed between most treatment groups, except in groupstreated with X-rays followed by heat after 7 h (p=0.0023 vs. heat alone) .

Figure 4 shows the percentage of animals (pooled data) surviving tumour-free asa function of follow-up time . Hyperthermia for 30 min at 42-44°C was notcarcinogenic, but if applied 5-10 min after X-ray irradiation with 16-28 Gy itresulted in a significant increase in tumour incidence relative to irradiation alone .

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0 26 52

WEEKS AFTER TREATMENT

Figure 3 . Percentage of animals surviving without evidence of a tumour inside the treatedvolume as a function of the follow-up period . Animals were treated with 24 Gy alone(0) or followed by hyperthermia for 30 min at 42°C (0), 43'C (A) and 44°C (V)after 5-10 min (a) or 7 h (b) .

With an interval of 7 h between X-rays and heat, radiation carcinogenesis was alsosignificantly increased (Figure 5) .

3 .2 . Latent periodThe latent period for tumours inside the volume irradiated with 15-28 Gy of

X-rays was 472+19 days (mean +SEM, n=24) . Hyperthermia for 30 min at42-44°C applied 5-10 min and 7 h after X-rays led to a slight but significantshortening of the latent period to 404 ± 34 days (p < 0 .05; n = 22) and 348 ± 6 days(p < 0 .001 ; n = 33), respectively . Latency for the various subgroups did not differ

78 104

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HEAT (H) 5-10 MINUTES AFTER X-RAYS (X)

0

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WEEKS AFTER TREATMENTFigure 4 . Tumour-free survival in animals that were either sham-treated (n=17),

X-irradiated (X) at a dose of 15-32 Gy (n=124), heated (H) for 30 min at 42-44°C(n=38) or received the combined treatment of X-rays followed by heat 5-10 minlater (X+H; n=120) . p-values: sham vs X=0.0601; sham vs X+H=0.0053 ;H vs X+H=0.0000; X vs X+H=0 .0015 .

significantly. For tumours located outside the treated volume it was 465 ± 15 days(n = 88) .

3 .3 . Pathological findingsIn the dissected tumours, two types of morphology dominated . One type was a

solid tumour with a smooth plane of transection . These tumours were locateddorsolateral in the rat cervical region and had a volume doubling time of 4 . 5 + 1 .6days (n = 3) . On histological examination the tumours consisted of sheets of cellswith irregular-shaped nuclei . Some cells had an extensive fibrillar eosinophiliccytoplasm. A considerable number of cells was in mitosis . These tumours wereclassified as rhabdomyosarcomas . Similar, less well-differentiated tumours wereclassified as sarcomas . The other tumour type, often observed outside the treatedvolume, was a soft tumour with a lobular appearance that proved to be benign ormalignant mammary tumours: adenomas, cystadenomas, adenofibromas andadenocarcinomas . Other diagnoses made were epidermic cysts and squamouscarcinomas. No neuronal tumours were found . Table 1 shows that after X-raysalone 38% of the tumours (6/16) were sarcomas. After X-rays followed by heat5-10 min or 7 h later, 86% of the tumours (38/44) were sarcomas. From thesetumours, 79% were of the rhabdomyosarcoma type (30/38), 16% not clearlydifferentiated sarcomas (6/38) and 5% osteosarcomas (2/38) .

4. DiscussionThe observation that hyperthermia alone did not increase the incidence of

spontaneous tumours in the treated volume confirms data reported by Urano (1981)

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HEAT (H) 7 HOURS AFTER X-RAYS (X)

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Figure 5 . Tumour-free survival in animals that were either sham-treated (n=17),X-irradiated (X) at a dose of 16-28 Gy (n=41), heated (H) for 30 min at 42-44 °C(n=28) or received the combined treatment of X-rays followed by heat 7h later(X+H; n=122) . p-values: H vs X+H=0 .0001 ; X vs X+H=0 .0016 .

for hyperthermia applied to the mouse foot . Also the data from Baker et al. (1988)and data from our previous study (Sminia et al . 1990) agree that hyperthermia aloneis not carcinogenic .

Dose-response curves for cancer induction in experimental animals by X-raysalone tend to a plateau or to a peak in incidence (see e .g. Coggle 1983, Barendsen1986, Broerse 1989). Such curves may be explained by assuming a competitionbetween cell transformation and cell sterilization . Depending on the radiation dose,a normal cell will be transformed to a cancer cell or, at higher dose, may be killed orlose its ability to proliferate . The present study shows a significant increase in

Table 1 . Number and percentage of animals with a sarcoma found in the cervical region ofthe rat

Animals were either sham-treated, X-irradiated (X), heated (H) or received the combina-tion treatment of irradiation followed by hyperthermia after 5-10 min or after 7 h (X + H) asdescribed in § 2 (99% confidence limits in parentheses) .

Treatment Sham H X X+H

Initial number of treated animals 17 66 165 242Tumour inside treated volume 2 2 24 55Pathological examination 1 2 16 44Number of sarcomas 0 0 6 38Percentage sarcomas 0 0 38(6-69) 86(73-100)

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HEAT 5-10 M/IUTES AFTER X-RAYS

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42'C ViivA 43'C 44 -C

20DOSE (Gy)

HEAT 7 HOURS AFTER X-RAYS42'C

43 'C • . :kti:? 44'C

20DOSE (ay)

24

24

Figure 6 . Percentage of animals that developed a tumour inside the treated volume within aperiod of 15 months. Animals were X-irradiated with 16, 20 and 24 Gy and heated5-10 min (a) or 7 h (b) later at 42 ° C, 43 °C and 44°C for 30 min. Significant differencesbetween the various treatment groups (n = 7-17 at the start of the experiment) werecalculated over the entire survival curves using the actuarial statistical methodp-values for significance are given in Figures 1-3 ; *, data not available .

radiation-induced carcinogenesis by hyperthermia. There was a trend for a maxi-mum increase in radiation carcinogenesis at a relatively low irradiation dose andhigh heat dose (16 Gy + 30 min 44°C), either applied 5-10 min (Figure 6a) or 7 hlater (Figure 6b) . This is in spite of the fact that the number of animals that could beincluded in the analysis was limited and the heat distribution was not uniform

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Table 2 . Thermal enhancement ratios (TER) for X-rays combined with hyperthermia onthe rat cervical spinal cord (C5-T2)

The intervals between X-rays and heat were 5-10 min and 7h. TERs + SE werecalculated from the ED 50 values (radiation dose with or without hyperthermia resulting in50% of the animals with neurological complications) obtained from dose-response curves foreither 'early delayed' or 'late delayed' foreleg paralysis and 'late' neurological symptoms (the5-10 min interval data are taken from Sminia et al . 1991) .

t The majority of animals from these groups was lost due to tumour development insidethe treated volume .

throughout the irradiated volume . Our observations suggest an optimum heat andirradiation dose for tumour induction, indicating that cell killing by heat plays arole in the balance between X-ray-induced cell transformation and cell sterilization .

Only a small number of in vivo studies on cancer induction after combinedtreatment of hyperthermia and radiation are available . In the experiments of Bakeret al . (1988), heat was applied immediately after irradiation of mouse skin . They didnot observe any effect of hyperthermia on radiation carcinogenesis . Urano et al .(1989), however, showed a significant increase in the number of tumours inside thetreated volume if heat was applied 20 min prior to or after irradiation of the mouseskin. No enhancement was observed if the interval between the heat and X-rays was2 days . Previous observations from our laboratory showed an increase in tumourinduction by heat applied 90 days after X-rays (Sminia et al . 1990). Interestingly,the increase in radiation carcinogenesis by hyperthermia in the present study isagain of the same extent, indicating that the process is independent of the timeinterval between X-rays and heat . This observation is in agreement with thegenerally accepted opinion that the process of carcinogenesis occurs in separatephases in which the time interval is of minor importance (see e .g. Hecker 1976) .The first phase, initiation, is a rapid and irreversible process. The second phase,promotion, can start a long time after the initiation phase by application of a varietyof chemical, physical and other agents . In our experiments, X-irradiation is theinitiator and hyperthermia seems to exert tumour-promoting activity .

With respect to the time factor, there is a remarkable difference between thermalenhancement of radiation-induced neurological complications and the increase byhyperthermia of radiation carcinogenesis . Enhancement of the radiation response ofthe spinal cord was evident with short intervals between X-rays and heat (Sminiaet al. 1991), but had almost completely disappeared with an interval of 7h (seeTable 2) . These observations fit well with the fact that repair of sublethal radiation

Treatment Interval'Early

paralysis''Late

paralysis'

'Lateneurologicalsymptoms'

X-rays +30 min 41 . 1 °C 5-10min 1 .07±0 .08 1 .25±0 . 10 1 .27±0 . 127h 1 .01+0 .07 1 . 14+0 .08 1 .03+0 .08

X-rays+ 30min42 . 1 °C 5-10min 1 . 17±0 .08 1 .31±0 .07 1 .19±0 .077h 1 .02+0 .07 0 .99+0. 13 1 .00+0 .08

X-rays+ 30 min 42 . 9 °C 5-10min 1 . 12±0 .04 1 .16±0.07t 1 .11±0 .07t7 h 1-01+0-02 1 .17+0. 171 1 .14±0 . 13t

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damage of the spinal cord is almost completed within 7 h (Ang et al. 1987) . Ourresults emphasize the point that hyperthermia increases radiation carcinogenesisindependent of the time interval between the two modalities . Such a phenomenon iscompletely different from the radiosensitizing effect of hyperthermia in theradiation response of normal or malignant tissues, which is very much time-dependent (e.g . Table 2, Wondergem and Haveman 1985, Haveman 1986) .

The percentage of tumours located outside the treated volume was not signifi-cantly different between all treatment groups (pooled data), except one . In thisgroup a higher tumour incidence was found after X-rays and heat relative to heatalone. This observation may be explained by scattering of X-rays . We estimate thatthe scattered dose outside the irradiated volume was 1-5% of the dose applied tothe cervical region . No effect was found on tumour induction outside the heatedvolume relative to sham-treated animals . Baker et al. (1988), however, reported areduction of tumour incidence outside the treated volume after fractionatedhyperthermia of the mouse leg . A possible explanation for their observation mightbe that, in their experiments, localized heating of the mouse leg was accompaniedby systemic heating .

The latency for expression of X-ray-induced tumours was significantly short-ened by hyperthermia. Urano et al . (1989) also reported a significant reduction (by16%) of the median radiation-carcinogenesis latency by heat. Considering also thedata on latency from our previous study (Sminia et al . 1990), we conclude thathyperthermia reduces the latency of tumour induction by X-rays by 10-20% .

The macroscopical and histological features of the rhabdomyosarcomas that weobserved fit well with the description of these tumours given by Carter (1973) .These types of tumour rarely develop spontaneously, but when induced theyusually grow rapidly. In our experiments, deep hyperthermia combined with X-rays led to induction of rhabdomyosarcomas, neoplasms originating from musclecells. In the study of Urano et al . (1989), more than 60% of the tumours induced bytreatment with superficial hyperthermia and X-rays were classified as fibrosarco-mas (connective tissue neoplasms). It is well documented that radiation alone caninduce secondary neoplasms within the irradiated fields in long-term survivors,which are often of the sarcoma type (Coleman and Tucker 1989, Upton 1989) .Raabe et al. (1980) did, however, show that the frequency of radiation-inducedsarcomas in humans is much lower than in dogs and mice . Since, at present,hyperthermia is being used mainly in patients with short life expectancy, theoncogenic risk is of little relevance . However, if hyperthermia is to be used incombination with radiation with a curative intent in patients with a betterprognosis, the possible oncogenic effect would become important . The presentobservations stress the point that `the incidence of secondary neoplasias in long-term survivors, in and outside the heated volume, must be included in reportingresults' (International Consensus Meeting on Hyperthermia 1990) .

AcknowledgementsThe authors wish to thank Dr F . A. Stewart for assistance with the English . Mrs

A. van der Graaff is acknowledged for editing the manuscript and Mr E . Heeren forhis proficient preparation of histological sections . This study was supported by agrant from the Interuniversitair Instituut voor Radiopathologie en Stralen-bescherming .

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