0022-5347/91/1453-0654$03.00/0 Vol. 145, 654-656, March 1991
THE JOURNAL OF UROLOGY Copyright © 1991 by AMERICAN UROLOGICAL ASSOCIATION, IN C.
Printed in U.S.A.
THE EFFECT OF RADIATION THERAPY AND HYPERTHERMIA ON A HUMAN PROSTATIC CARCINOMA CELL LINE GROWN IN ATHYMIC NUDE MICE ISSAC KAVER, WARREN W. KOONTZ, JR., JOHN D. WILSON, JOHN M. GUICE JOY L. WARE*
AND
From the Division of Urology and the Departments of Surgery, Pathology and Radiology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia
ABSTRACT
The effect of radiation and/or hyperthermia on a human prostatic carcinoma xenograft in athymic nude mice was investigated. A human prostate carcinoma subline (1-LN-PC-3-lA) was inoculated subcutaneously in the thigh of male athymic nude mice. When tumors reached a size of approximately 200 mm. 3, they were treated with either radiation (X) or hyperthermia (H) alone, or in combination (X + H). In the combined treatment, hyperthermia was delivered immediately after radiation exposure. Comparison of the t ime required to reach twice the tumor volume observed at the time of treatment was used to define therapeutic impact on tumor growth. The combined treatment resulted in a median tumor volume doubling time of 35.5 days, compared to 18 days and 25.5 days, respectively, for hyperthermia or radiation alone. Analysis of tumor doubling time using a proportional hazards regression indicates that under the conditions of this experiment, the effect of radiation and hyperthermia for 1-LN-PC-3-lA tumors is additive. The impact of this treatment regimen in the management of prostatic cancer requires further investigation. KEY WORDS:
prostate carcinoma, xenograft, radiotherapy, hyperthermia
Prostatic cancer is the most common cancer in American men over the age of 50, and is the second leading cause of cancer deaths. In the United States in 1989, there were an estimated 28,500 deaths from the d1sease. 1 Despite the introduction of new operative techniques of nerve sparing radical prostatectomy and preservation of sexual function, the management of localized prostatic cancer remains controversial. 2• 3 External beam radiation therapy is an alternative treatment approach for localized prostatic cancer.4 Recently, interest in applying hyperthermia in conjunction with other modalities, such as radiation or chemotherapy, has increased. 5•6 The rationale for using hyperthermia is based on the observation t hat: 1) temperatures of 41 to 45C directly kills cells; b) acidic and nutritionally deprived cells are more heat sensitive; c) heat acts as a radiosensitizing agent, and d) the interaction of heat and radiation can be greater than additive.7 In the present study, we have investigated the effect of radiation and/or hyperthermia on a human prostatic carcinoma cell line grown in athymic nude mice. We report here our results and discuss the implications of hyperthermia as an adjuvant to radiation in the treatment of prostatic cancer. MATERIALS AND METHODS
Mice. Specific pathogen free male athymic nude mice were obtained from the Athymic Nude Mouse Core Facility of the Massey Cancer Center at the Medical College of Virginia. Mice were maintained in sterilized filter-topped cages, placed in a laminar flow cabinet, and provided sterile food and water. Mice were four to six weeks old at the time of tumor cell injection. Tumor cells. Tumor cell suspensions of a human prostate carcinoma subline (1-LN-PC-3-1A) 8 were prepared by harvesting the cells from subconfluent monolayer cultures with 0.25% trypsin and 0.02% EDT A. Nude mice were inoculated subcutaneously with 1 x 106 tumor cells in 0.1 ml. in the left posterior Accepted for publication October 30, 1990. * Requests for reprints: Department of Pathology, Medical College of Virginia, Box 662 MCV Station, Richmond VA 23298. Read at the Mid-Atlantic Section, American Urologic Association, 1989 Annual Meeting, September 17, 1989, Munich, Germany.
thigh. Tumor cell growth was monitored and measured with calipers three times a week. When the tumors reached a volume of approximately 200 mm.3 , mice were randomized into one of four treatment groups: hypert hermia alone, radiation alone, radiation plus hyperthermia, and untreated tumor-bearing animals which served as controls. Hyperthermia treatment. Hyperthermia treatment was carried out by heating the tumor in a water bath. Prior to treatment, mice were anesthetized intraperitoneally with sodium pentobarbital (30 mg./ kg.). The mice were placed into a longitudinally halved 50 ml. plastic centrifuge tube, which was fixed in a horizontal position, and the tumor-bearing leg was brought out through an opening in the bottom of the tube. The size of the opening was large enough to avoid vascular occlusion and tissue hypoxia. The tumor was then treated by lowering the tube onto the surface of t he water, so that the entire leg was immersed. The bath water temperature was monitored with thermometers to maintain a constant temperature of 43C ± 0.2C. Treatment time was 45 minutes. Preliminary investigation using a thermocouple placed into the tumor showed that the tumor equilibrated with the bath water temperature (43C) within two minutes; upon removal from the bath, the tumor temperature fell below 37C within 30 seconds. In preliminary experiments, a thermocouple placed into the rectum of mice heated in this manner revealed no elevation of rectal temperature over t he course of the treatment. Radiation treatment. Local irradiation of the tumors was carried out using a Philips RT250 orthovoltage x-ray machine operated at 225 kV, 17 mA, with a 0.4 Thoreus filter (HVL = 2.2 mm./ cu) Except for the tumor-bearing leg, the animals were shielded with t hree mm. of lead during the exposures. The dose rate at the surface of the tumor was 166 cGy/ min. as measured with an ion chamber (Victoreen, model 154) and thermoluminescent dosimeters. All tumors received 750 cGy in a single exposure. Combined treatment. In t he combined treatment, the tumors were irradiated first, then exposed to water bath hyperthermia for 45 min. at 43C as described above. This treatment sequence was selected to mimic t he order in which t hese modalities are
654
the end of radiation sxposure ar2d ths ·""fi·''"'···u:; thermia treat.rnent varied betvveen five Tunior measurernents. A.fter sponse vvas assessed three tirnes each v1eek axes of the tumor rr1easurernents "Nere converted to tun1or volur.aes •;vhere 'l is the vohnr1e in :mn1c 3 and L formuia V = and width of the tun1or in rnmo, respec·· and VV are the
the data. The Statistical interest in the tumor volume as the time at the time of treatment. An ~~.n,·,mw, of
~~~··"L''"
variable of time defined
time v1as determined fron1 the tun1or 1neasuren1ents for individual tumor and entered into the
1-'he RESULTS
oc:rves for mice 1n each of the four The combined l, The 1r1ean tun1or volum.e at groups are illust:rated 1n tin1e of treatrr1ent was 216 1nm. SE = ± three H) treatment rnm. It can be seen had a marked while smaller
effects vi1sre obse:c'-.red in the groups ·""''°"'"nn radiation or alone. The tumors in mice which received radiation either failed to grow or regressed the observation interval. Although the graph 21 to 22 :measurement of tumor was taken for more than 40 in some of the cases combined treatment. For most of the control animals, urement of tumor was carried out for only three weeks. This was due to the development of bulky tumors in the untreated which resulted in animal morbidity. The effects of treatments on tumor volume doubling time are summarized in table 1. Exposure of these tumors to heat, radiation, or radiation followed heat, extended the tumor volume doubling time, relative to untreated controls. Although tumors exposed to radiation or to hyperthermia exhibited longer tumor volume doubling times, the greatest volume doutime occurred in mice which received radiation followed Mice which received combination treatment plus hyperthermia exhibited a tumor volume doubling time of 35 substantially longer than the tumors in the control mice The results of the proportional hazards regression analysis of tumor volume doubling times are summarized in table 2. The increased tumor volume doubling times of the groups treated with radiation alone or radiation followed hypersignificant, relative to controls (p = 0.0029 The increased tumor volume nnQP,·vp,n in mice treated with hyperthermia rucl1'uu;;11 the impact of se~ -·-··-·-· exposure cucuc,ua was greater that of either agent the interaction between radiation and heat under these conditions was additive since the interaction term in the proportional hazards model was not (p = DISCUSSION
Since the al. 10 of new operative for nerve and preservation of potency, much interest has directed to the performance of radical prostateotomy for localized prostatic cancer. Aln,.,,t,,·tP,r'rr,mv continues to offer the radical best chance of cure for clinical ,v,.a,w,cu cancer, more than 50 percent of the po!cH,rncr, advanced TABLE 1.
Effect of different treatments on tumor volume-doubling time
No. of Mice
Treatment
18
Control
10 12 11
43C 45 min, 750 750 1
Median (Days)
Range (Days)
8.5 18.0
11-29
25.5
3-49 4-59 24-47
Volume doubling times for 1-LN tumors
various treatments. times for the indicated nurnbei- of animals are shown for each treat1nent group. Tumor volume at the time of treatment was al'JJ>c'AHHa,,e,y 200 mm 3. * This value excludes animals sacrificed before 2x treatment volume vvas reached (sacrificed on 35, 40 and 45 days post-treatment). \1 a:tuss for the median and range of '!Olume
TABLE 2.
Fm. 1. Growth curves of 1-LN tumors receiving following treatments: x-rays, 750 cGy (L); heat, 45 min. at 43C (~); 750 cGy followed by 45 min at 43C (li!lil); no treatment (@il). Growth of all tumors prior to selection for treatment is indicated by open circles. Tumor size is expressed as tumor volume on day of measurement relative to volume determined on day of treatment (day 0). Each symbol represents mean relative tumor volume of all tumors measured in that group on indicated day. Dotted lines indicate day of treatment (TX), relative tumor volume at time of treatment (V), and two times relative volume at time of treatment (2V).
Results of 1-LN tumor growth analysis for 1-LN tumors Treatment Contrast
p-Value
Heat vs. control X-ray vs. control X-ray+ heat vs. control X-ray+ heat vs. x-ray X-ray + heat vs. heat X -ray /heat interaction
0.0916
0.0029* 0.0003* 0.0930 0.0543 0.8978
* Significant difference, p c5 0.01.
Summary of the proportional hazards regression analysis of volume doubling times for 1-LN tumors receiving various treatments. The p-values for comparisons of tumor volume doubling times for animals in various treatment groups are indicated (a Bonferroni correction for multiple comparisons has been applied).
656
KAVER AND ASSOCIATES
disease at the time of diagnosis, or medical conditions which render them poor candidates for radical surgery. 11 Thus, external beam radiation therapy remains a good alternative treatment for localized prostatic cancer. The survival rate of patients with clinical stage C disease, treated by external beam radiation, has been reported to be 58 to 72% at five years, 36 to 47% at 10 years, and 22 to 27% at 15 years. 12 The question is whether hyperthermia in combination with irradiation will improve survival of patients with prostatic cancer confined to the gland. The study reported here was undertaken to investigate the effect of hyperthermia in conjunction with radiation on a human prostatic carcinoma growing in athymic nude mice. Although our results have shown that the effect of the combined treatment at these dose levels was only additive, the tumor doubling time in the combined treatment group was substantially increased over those obtained for hyperthermia and radiation alone. Our results are in agreement with those obtained by Gottlieb et al. 13 who used microwave heating and radiation in the Dunning rat prostate tumor model. They have also shown an additive effect in the interaction of radiation and hyperthermia. The rationale for the use of hyperthermia in combination with radiation is that hypoxic cells and nutritionally deprived cells which are radioresistant are often found to be heat sensitive. 7 Therefore, the physiological state of the cells in a solid tumor can be expected to influence the response of the tumor to these modalities. It should be stressed that prostatic carcinomas must be expected to consist of a heterogeneous population of cells with respect to these factors. Thus, it is reasonable to speculate that radiation in combination with heat may result in a better tumor response for localized prostatic cancer. There is still great controversy regarding the impact of hyperthermia on promotion of distant metastasis. 14 In our experiments, evidence of lymphatic metastasis was observed in some cases of the combined treatment group. This may be related to the increased tumor doubling time in this group rather than a true treatment-induced increase in the metastatic potential of the cells. Previous investigations indicated that up to 88% of mice bearing 1-LN tumors subcutaneously on the dorsal surface also develop one or more lymphatic metastases if followed for 36 days. 15 The relation, if any, between hyperthermia and radiation, and subsequent metastasis in this system, is currently under investigation. In conclusion, our current findings have shown an additive effect of combining radiation and hyperthermia in the treatment of human prostatic tumors grown in nude mice. Hyperthermia, in combination with radiation, has been clinically shown to increase the efficacy of radiation therapy in a variety or tumor types. 16 Today, with the introduction of new technologies in heat delivery and temperature monitoring, hyperthermia can be delivered safely to the prostate gland. 17 With this in mind, it is clinically possible that applying hyperthermia in conjunction with curative external beam radiation for local prostatic cancer might yield a better local control of the disease. This has yet to be fully evaluated by a randomized study, with survival being the major endpoint. Our previous findings in vitro, 18• 19 and the present results in vivo, coupled with other studies, 20 may provide important information for designing appropriate clinical trials with radiation in combination with
hyperthermia in patients with locally advanced prostatic cancer. Acknowledgments. We gratefully acknowledge Susan Quinn and Martha Meade for their conscientious care of the athymic nude mice used in these experiments, We thank Norman Palmer, Animal Care Supervisor, for his technical advice and support of the Nude Mouse Core Facility. We thank Dr. Vernon Chinchilli, Department of Biostatistics, for biostatistical analysis of the data in this report. REFERENCES
1. Silverberg, E. and Lubera, J.: Cancer statistics, 1989. CA, 39: 3, 1989, 2. Paulson, D. F,: Radical surgery versus radiotherapy for adenocarcinoma of the prostate. J. UroL, 128: 502, 1982. 3. Pilepich, M. V,, Krall, J.M., Bagshaw, M.A., Emami, B. N,, Asbell, S. 0., Bard, R. H. and Hanks, G. E.: Radical prostatectomy or radiotherapy in carcinoma of prostate. The dilemma continues. Urology, 30: 18, 1987. 4. Bagshaw, M.A., Gordon, R.R., Pistenma, D. A., Castellino, R. A. and Meares, E. M. Jr.: External beam radiation therapy of primary carcinoma of the prostate. Cancer, 36: 723, 1975. 5. Kim, J. H., Hahn, E.W. and Antich, P. P.: Radiofrequency hyperthermia for clinical cancer therapy. Nat. Cancer. Inst. Monogr., 61: 339, 1982. 6. Yerushalmi, A., Sarvadio, C., Leib, Z., Fishelovitz, Y., Rakowsky, A. and Stein, J. A.: Local hyperthermia for treatment of carcinoma of the prostate; a preliminary report. Prostate, 6: 623, 1984. 7. Suit, H. D. and Gerweck, L. E.: Potential for hyperthermia and radiation therapy. Cancer Res., 39: 2290, 1979. 8. Ware, J. L., Paulson, D. F., Mickey, G. H. and Webb, K. S.: Spontaneous metastasis of cells of the human prostate carcinoma cell line PC-3 in athymic nude mice. J. Urol., 128: 1064, 1982. 9. SAS Institute, Inc. (1985) SUGI Supplemental Library Users' Guide. Version 5 Edition. Cary, NC: SAS Institute, Inc. 10. Walsh, P. C., Lepor, H. and Eggleston, J.C.: Radical prostatectomy with preservation of sexual function: anatomical and pathological considerations. Prostate, 4: 473, 1983. 11. Murphy, G. P., Natarajan, N. and Pontes, J. E.: The National Survey of prostate cancer in the United States by the American College of Surgeons. J. UroL, 127: 928, 1981. 12. Hanks, G. E.: Treatment of locally advanced prostate cancer with radiation therapy. Urology, 33: 37, 1989. 13. Gottlieb, C. F., Seibert, G. B. and Block, N. L.: Interaction of irradiation and microwave-induced hyperthermia in the Dunning R3327G prostatic adenocarcinoma model. Radiology, 169: 243, 1988. 14. Hill, S. A. and Denkekamp, J.: Does local tumour heating in mice influence metastatic spread? Br. J. Radio!., 55: 444, 1982. 15. Ware, J. L. and DeLong, E. R.: Influence of tumour size on human prostate tumour metastasis in athymic nude mice. Br. J. Cancer, 51: 419, 1985. 16. Storm, F. K., Seaton, E. F., Baker, H. W., Roe, D., Morton, D. L.: Tumor stabilization after hyperthermia: an important criterion of response to thermal therapy. J. Surg. Oncol., 34: 143, 1987. 17. Servadio, C., Leib, Z. and Lev, A.: Diseases of the prostate treated by local microwave hyperthermia. Urology, 30: 97, 1987. 18. Raver, I., Ware, J. L. and Koontz, W. W., Jr.: The effect of hyperthermia on human prostatic carcinoma cell lines: evaluation in vitro. J. UroL, 141: 1025, 1989. 19. Raver, I., Ware, J. L., Wilson, J. D. and Koontz, W.W., Jr.: The effect of radiation combined with hyperthermia on human prostatic carcinoma cell lines in culture. Urology (in press). 20. Yerushalmi, A., Shpirer, ZS., Hod, I., Gottesfeld, F. and Baos, D. D.: Normal tissue response to localized deep microwave hyperthermia in the rabbit's prostate: a preclinical study. Int. J. Rad. Oncol. Ciol. Phys., 9: 77, 1983.
THE JOURNAL OF UROLOGY Copyright © 1991 by AMERICAN UROLOGICAL ASSOCIATION, IN C.
Printed in U.S.A.
THE EFFECT OF RADIATION THERAPY AND HYPERTHERMIA ON A HUMAN PROSTATIC CARCINOMA CELL LINE GROWN IN ATHYMIC NUDE MICE ISSAC KAVER, WARREN W. KOONTZ, JR., JOHN D. WILSON, JOHN M. GUICE JOY L. WARE*
AND
From the Division of Urology and the Departments of Surgery, Pathology and Radiology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia
ABSTRACT
The effect of radiation and/or hyperthermia on a human prostatic carcinoma xenograft in athymic nude mice was investigated. A human prostate carcinoma subline (1-LN-PC-3-lA) was inoculated subcutaneously in the thigh of male athymic nude mice. When tumors reached a size of approximately 200 mm. 3, they were treated with either radiation (X) or hyperthermia (H) alone, or in combination (X + H). In the combined treatment, hyperthermia was delivered immediately after radiation exposure. Comparison of the t ime required to reach twice the tumor volume observed at the time of treatment was used to define therapeutic impact on tumor growth. The combined treatment resulted in a median tumor volume doubling time of 35.5 days, compared to 18 days and 25.5 days, respectively, for hyperthermia or radiation alone. Analysis of tumor doubling time using a proportional hazards regression indicates that under the conditions of this experiment, the effect of radiation and hyperthermia for 1-LN-PC-3-lA tumors is additive. The impact of this treatment regimen in the management of prostatic cancer requires further investigation. KEY WORDS:
prostate carcinoma, xenograft, radiotherapy, hyperthermia
Prostatic cancer is the most common cancer in American men over the age of 50, and is the second leading cause of cancer deaths. In the United States in 1989, there were an estimated 28,500 deaths from the d1sease. 1 Despite the introduction of new operative techniques of nerve sparing radical prostatectomy and preservation of sexual function, the management of localized prostatic cancer remains controversial. 2• 3 External beam radiation therapy is an alternative treatment approach for localized prostatic cancer.4 Recently, interest in applying hyperthermia in conjunction with other modalities, such as radiation or chemotherapy, has increased. 5•6 The rationale for using hyperthermia is based on the observation t hat: 1) temperatures of 41 to 45C directly kills cells; b) acidic and nutritionally deprived cells are more heat sensitive; c) heat acts as a radiosensitizing agent, and d) the interaction of heat and radiation can be greater than additive.7 In the present study, we have investigated the effect of radiation and/or hyperthermia on a human prostatic carcinoma cell line grown in athymic nude mice. We report here our results and discuss the implications of hyperthermia as an adjuvant to radiation in the treatment of prostatic cancer. MATERIALS AND METHODS
Mice. Specific pathogen free male athymic nude mice were obtained from the Athymic Nude Mouse Core Facility of the Massey Cancer Center at the Medical College of Virginia. Mice were maintained in sterilized filter-topped cages, placed in a laminar flow cabinet, and provided sterile food and water. Mice were four to six weeks old at the time of tumor cell injection. Tumor cells. Tumor cell suspensions of a human prostate carcinoma subline (1-LN-PC-3-1A) 8 were prepared by harvesting the cells from subconfluent monolayer cultures with 0.25% trypsin and 0.02% EDT A. Nude mice were inoculated subcutaneously with 1 x 106 tumor cells in 0.1 ml. in the left posterior Accepted for publication October 30, 1990. * Requests for reprints: Department of Pathology, Medical College of Virginia, Box 662 MCV Station, Richmond VA 23298. Read at the Mid-Atlantic Section, American Urologic Association, 1989 Annual Meeting, September 17, 1989, Munich, Germany.
thigh. Tumor cell growth was monitored and measured with calipers three times a week. When the tumors reached a volume of approximately 200 mm.3 , mice were randomized into one of four treatment groups: hypert hermia alone, radiation alone, radiation plus hyperthermia, and untreated tumor-bearing animals which served as controls. Hyperthermia treatment. Hyperthermia treatment was carried out by heating the tumor in a water bath. Prior to treatment, mice were anesthetized intraperitoneally with sodium pentobarbital (30 mg./ kg.). The mice were placed into a longitudinally halved 50 ml. plastic centrifuge tube, which was fixed in a horizontal position, and the tumor-bearing leg was brought out through an opening in the bottom of the tube. The size of the opening was large enough to avoid vascular occlusion and tissue hypoxia. The tumor was then treated by lowering the tube onto the surface of t he water, so that the entire leg was immersed. The bath water temperature was monitored with thermometers to maintain a constant temperature of 43C ± 0.2C. Treatment time was 45 minutes. Preliminary investigation using a thermocouple placed into the tumor showed that the tumor equilibrated with the bath water temperature (43C) within two minutes; upon removal from the bath, the tumor temperature fell below 37C within 30 seconds. In preliminary experiments, a thermocouple placed into the rectum of mice heated in this manner revealed no elevation of rectal temperature over t he course of the treatment. Radiation treatment. Local irradiation of the tumors was carried out using a Philips RT250 orthovoltage x-ray machine operated at 225 kV, 17 mA, with a 0.4 Thoreus filter (HVL = 2.2 mm./ cu) Except for the tumor-bearing leg, the animals were shielded with t hree mm. of lead during the exposures. The dose rate at the surface of the tumor was 166 cGy/ min. as measured with an ion chamber (Victoreen, model 154) and thermoluminescent dosimeters. All tumors received 750 cGy in a single exposure. Combined treatment. In t he combined treatment, the tumors were irradiated first, then exposed to water bath hyperthermia for 45 min. at 43C as described above. This treatment sequence was selected to mimic t he order in which t hese modalities are
654
the end of radiation sxposure ar2d ths ·""fi·''"'···u:; thermia treat.rnent varied betvveen five Tunior measurernents. A.fter sponse vvas assessed three tirnes each v1eek axes of the tumor rr1easurernents "Nere converted to tun1or volur.aes •;vhere 'l is the vohnr1e in :mn1c 3 and L formuia V = and width of the tun1or in rnmo, respec·· and VV are the
the data. The Statistical interest in the tumor volume as the time at the time of treatment. An ~~.n,·,mw, of
~~~··"L''"
variable of time defined
time v1as determined fron1 the tun1or 1neasuren1ents for individual tumor and entered into the
1-'he RESULTS
oc:rves for mice 1n each of the four The combined l, The 1r1ean tun1or volum.e at groups are illust:rated 1n tin1e of treatrr1ent was 216 1nm. SE = ± three H) treatment rnm. It can be seen had a marked while smaller
effects vi1sre obse:c'-.red in the groups ·""''°"'"nn radiation or alone. The tumors in mice which received radiation either failed to grow or regressed the observation interval. Although the graph 21 to 22 :measurement of tumor was taken for more than 40 in some of the cases combined treatment. For most of the control animals, urement of tumor was carried out for only three weeks. This was due to the development of bulky tumors in the untreated which resulted in animal morbidity. The effects of treatments on tumor volume doubling time are summarized in table 1. Exposure of these tumors to heat, radiation, or radiation followed heat, extended the tumor volume doubling time, relative to untreated controls. Although tumors exposed to radiation or to hyperthermia exhibited longer tumor volume doubling times, the greatest volume doutime occurred in mice which received radiation followed Mice which received combination treatment plus hyperthermia exhibited a tumor volume doubling time of 35 substantially longer than the tumors in the control mice The results of the proportional hazards regression analysis of tumor volume doubling times are summarized in table 2. The increased tumor volume doubling times of the groups treated with radiation alone or radiation followed hypersignificant, relative to controls (p = 0.0029 The increased tumor volume nnQP,·vp,n in mice treated with hyperthermia rucl1'uu;;11 the impact of se~ -·-··-·-· exposure cucuc,ua was greater that of either agent the interaction between radiation and heat under these conditions was additive since the interaction term in the proportional hazards model was not (p = DISCUSSION
Since the al. 10 of new operative for nerve and preservation of potency, much interest has directed to the performance of radical prostateotomy for localized prostatic cancer. Aln,.,,t,,·tP,r'rr,mv continues to offer the radical best chance of cure for clinical ,v,.a,w,cu cancer, more than 50 percent of the po!cH,rncr, advanced TABLE 1.
Effect of different treatments on tumor volume-doubling time
No. of Mice
Treatment
18
Control
10 12 11
43C 45 min, 750 750 1
Median (Days)
Range (Days)
8.5 18.0
11-29
25.5
3-49 4-59 24-47
Volume doubling times for 1-LN tumors
various treatments. times for the indicated nurnbei- of animals are shown for each treat1nent group. Tumor volume at the time of treatment was al'JJ>c'AHHa,,e,y 200 mm 3. * This value excludes animals sacrificed before 2x treatment volume vvas reached (sacrificed on 35, 40 and 45 days post-treatment). \1 a:tuss for the median and range of '!Olume
TABLE 2.
Fm. 1. Growth curves of 1-LN tumors receiving following treatments: x-rays, 750 cGy (L); heat, 45 min. at 43C (~); 750 cGy followed by 45 min at 43C (li!lil); no treatment (@il). Growth of all tumors prior to selection for treatment is indicated by open circles. Tumor size is expressed as tumor volume on day of measurement relative to volume determined on day of treatment (day 0). Each symbol represents mean relative tumor volume of all tumors measured in that group on indicated day. Dotted lines indicate day of treatment (TX), relative tumor volume at time of treatment (V), and two times relative volume at time of treatment (2V).
Results of 1-LN tumor growth analysis for 1-LN tumors Treatment Contrast
p-Value
Heat vs. control X-ray vs. control X-ray+ heat vs. control X-ray+ heat vs. x-ray X-ray + heat vs. heat X -ray /heat interaction
0.0916
0.0029* 0.0003* 0.0930 0.0543 0.8978
* Significant difference, p c5 0.01.
Summary of the proportional hazards regression analysis of volume doubling times for 1-LN tumors receiving various treatments. The p-values for comparisons of tumor volume doubling times for animals in various treatment groups are indicated (a Bonferroni correction for multiple comparisons has been applied).
656
KAVER AND ASSOCIATES
disease at the time of diagnosis, or medical conditions which render them poor candidates for radical surgery. 11 Thus, external beam radiation therapy remains a good alternative treatment for localized prostatic cancer. The survival rate of patients with clinical stage C disease, treated by external beam radiation, has been reported to be 58 to 72% at five years, 36 to 47% at 10 years, and 22 to 27% at 15 years. 12 The question is whether hyperthermia in combination with irradiation will improve survival of patients with prostatic cancer confined to the gland. The study reported here was undertaken to investigate the effect of hyperthermia in conjunction with radiation on a human prostatic carcinoma growing in athymic nude mice. Although our results have shown that the effect of the combined treatment at these dose levels was only additive, the tumor doubling time in the combined treatment group was substantially increased over those obtained for hyperthermia and radiation alone. Our results are in agreement with those obtained by Gottlieb et al. 13 who used microwave heating and radiation in the Dunning rat prostate tumor model. They have also shown an additive effect in the interaction of radiation and hyperthermia. The rationale for the use of hyperthermia in combination with radiation is that hypoxic cells and nutritionally deprived cells which are radioresistant are often found to be heat sensitive. 7 Therefore, the physiological state of the cells in a solid tumor can be expected to influence the response of the tumor to these modalities. It should be stressed that prostatic carcinomas must be expected to consist of a heterogeneous population of cells with respect to these factors. Thus, it is reasonable to speculate that radiation in combination with heat may result in a better tumor response for localized prostatic cancer. There is still great controversy regarding the impact of hyperthermia on promotion of distant metastasis. 14 In our experiments, evidence of lymphatic metastasis was observed in some cases of the combined treatment group. This may be related to the increased tumor doubling time in this group rather than a true treatment-induced increase in the metastatic potential of the cells. Previous investigations indicated that up to 88% of mice bearing 1-LN tumors subcutaneously on the dorsal surface also develop one or more lymphatic metastases if followed for 36 days. 15 The relation, if any, between hyperthermia and radiation, and subsequent metastasis in this system, is currently under investigation. In conclusion, our current findings have shown an additive effect of combining radiation and hyperthermia in the treatment of human prostatic tumors grown in nude mice. Hyperthermia, in combination with radiation, has been clinically shown to increase the efficacy of radiation therapy in a variety or tumor types. 16 Today, with the introduction of new technologies in heat delivery and temperature monitoring, hyperthermia can be delivered safely to the prostate gland. 17 With this in mind, it is clinically possible that applying hyperthermia in conjunction with curative external beam radiation for local prostatic cancer might yield a better local control of the disease. This has yet to be fully evaluated by a randomized study, with survival being the major endpoint. Our previous findings in vitro, 18• 19 and the present results in vivo, coupled with other studies, 20 may provide important information for designing appropriate clinical trials with radiation in combination with
hyperthermia in patients with locally advanced prostatic cancer. Acknowledgments. We gratefully acknowledge Susan Quinn and Martha Meade for their conscientious care of the athymic nude mice used in these experiments, We thank Norman Palmer, Animal Care Supervisor, for his technical advice and support of the Nude Mouse Core Facility. We thank Dr. Vernon Chinchilli, Department of Biostatistics, for biostatistical analysis of the data in this report. REFERENCES
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