Enhancement of iudr radiosensitization by low energy photons

Inr J. Radiatmn OncologyBiol Phys., Vol. 13, pp. 1071-1079

0360-3016/87 $3.00 + .OO Copyright 0 1987 Pergamon Journals Ltd.

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0 Original Contribution ENHANCEMENT OF IUdR RADIOSENSITIZATION BY LOW ENERGY PHOTONS RAVINDER NATH, PH.D.,*

PAUL BONGIORNI,

M.S.t

AND SARA ROCKWELL,

PH.D.*

Department of Therapeutic Radiology, Yale University School of Medicine, 333 Cedar St., New Haven, CT 065 10 The effect of the photon energy on the radiosensitization produced by iododeoxyuridine (IUdR) was examined using Chinese hamster cells in vitro. Radiosensitization by IUdR was considerably higher for 60 keV photons from 241Amsources than for the 860 keV photons (average energy) from ‘%a sources, under continuous low dose rate conditions applicable to intracavitary brachytherapy (a dose rate of 0.57 Gy/hr). Also, IUdR radiosensitization was higher for 250 kV X rays than for 4 MV X rays under the acute exposure conditions used in external beam radiation therapy (dose rates of 1 to 2 Gy/min). These data support the hypothesis that photons with energies just greater than 32.2 keV, the K-absorption edge of iodine, are more effective in causing cell damage than are photons of other energies, because their absorption results in the production of Auger electron cascades and therefore in the production of high linear energy transfer (LET) radiations. Iododeoxyuridine (IUdR), Americium-241, Brachytherapy, Intracavitary irradiation, Radiosensitizers. INTRODUCTION

tive radiosensitization of tumors in experimental animals and in human patients produced disappointing reA variety of problems were encountered, sults. ‘,2,‘2,‘4,28 including: (a) degradation of compounds by enzymatic cleavage, primarily in the liver, leading to dehalogenation and rapid excretion, (b) toxicity of the analogs, primarily to the bone marrow and skin, and (c) incorporation of the compounds into rapidly proliferating normal tissues, with the resulting radiosensitization of these tissues. A number of approaches have been used to overcome these difficulties. Dehalogenation can be reduced by intraarterial infusion, by shunting of the hepatic circulation, or by the use of inhibitors of dehalogenation.40 Normal tissue radiosensitization may be reduced by carefully restricting the procedure to use with neoplasms surrounded by normal tissues which are either non-proliferating or proliferating much less rapidly than the tumors. Examples of the latter approach are the treatment of brain tumors and osteogenic sarcomas.‘3~20~2’~27~32~36 The work presented in this paper was stimulated by the observation that IUdR radiosensitization can be enhanced substantially by the use of photon energies just above 33.2 keV, the K-absorption edge of iodine. The enhancement of IUdR sensitization appears to result

Iododeoxyuridine (IUdR) is an analog of thymidine, in which the methyl group in the 5-position of the pyrimidine ring has been replaced by iodine. The compound was first synthesized by Prusoff in 1959,34 for possible use as an antineoplastic agent. IUdR was found to produce only limited cytotoxicity when used alone, but to be a potent radiosensitizer when incorporated into DNA prior to irradiation.8,‘8,‘9 The mechanism for radiosensitization by IUdR is believed to be the release of halogen radical by free radical attack resulting in a free uracil radical which produces instability in the DNA backbone through abstraction of a hydrogen atom from the deoxyribose of DNA.35 Incorporation of IUdR into DNA takes place almost exclusively in actively proliferating cells. It was postulated that in the case of a growing neoplasm located in a region of non-proliferating normal tissue, IUdR incorporation, and thus radiosensitization, would be greater in the tumor than in the dose-limiting normal tissue. Radiosensitization by incorporated IUdR would then result in an enhancement of the therapeutic ratio and an improvement in the results ofradiation therapy.40 Initial attempts to use IUdR or BUdR to achieve selec-

Schulz for sharing some of his data on the relative biological effectiveness of 24’Amphotons before publication. The authors would also like to thank Dr. Ralph Fairchild and Brenda Laster of Brookhaven National Laboratory for neutron activation analysis of IUdR incorporation. We would also like to thank Deanna Jacobs for help in preparing this manuscript. Accepted for publication 4 February 1987.

* Professor. t Associate in Research. Supported in part by USPHS grant number CA-39044, awarded by the National Cancer Institute. Reprint requests to: Ravinder Nath, Ph.D. Acknowledgements-The authors would like to express their thanks to Dr. J. J. Fischer for many discussions and suggestions on the work presented in this paper. We are grateful to Dr. R. J. 1071

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from the effects of the Auger electron cascade which follows the creation of a vacancy in the K-shell of iodine via photoelectric effect.‘,” Photons interact with matter, producing electron tracks which create ionization and excitation events over a distance considerably larger than cellular dimensions. These primary events produce radiolysis in the intracellular and extracellular materials and direct hits on sensitive targets in the cells, leading to cell lethality. When a photon with an energy just above the K-absorption edge, 33.2 keV for iodine, interacts by photoelectric effect, it is most likely to knock out a K-shell electron. The process of filling the K-shell vacancy leads to a cascade of Auger electrons and the release of fluorescent X rays. Filling the K-shell vacancy in iodine results in the emission of an average of approximately 9 Auger electrons, with a range of 1 to 18 electrons. The Auger cascade also leaves behind a highly charged iodine molecule.4 The low energy electrons produced by the Auger cascade have high specific ionizations and sub-cellular ranges. The resulting radiobiological effects are similar to those observed with high LET radiations: a reduced shoulder on the radiation cell survival curve and a reduced oxygen enhancement ratio.** Since the Auger electrons produce highly localized energy deposition (equivalent to high-LET charged particles such as alpha particles) in a small volume (about the size of a cell’s nucleus),4’ these effects lead to an increased radiochemical yield of halogens,7T’5increased DNA fragmentation and chromosome breaks in microorganisms,23*24increased cytotoxicity,3,16,33 and a reduced oxygen effect. ** The mechanisms involved in the biological effects of Auger cascades have been studied in detail, primarily in experiments attempting to explain the extreme radiotoxicity of ‘251-labelled IUdR relative to 3H- and ‘311-labelled IUdR.3,5,16,33More details about the exact nature of the mechanism of biological damage by Auger electrons have been obtained by double-labelling of IUdR with both 1251and 14Cto differentiate molecular fragmentation effects from Auger electron effects”; by comparing the effects of membrane-bound 1251versus DNA-bound ‘*‘I to elucidate the primary site of radiation damage by Auger electrons41; by comparing ‘251-albumin, “Fe-transferrin, ‘251-iodoantipyrine, and ‘*‘IUdR to differentiate between intracellular, extracellular, and intranuclear sites of radiation action$ and by studying the lengths of the DNA fragments produced by Auger electrons from 1251-labelled iododeoxycytidine.26 It appears from these studies that the range of Auger electrons from iodine bound to DNA extends up to 70 A”, but that more than 70% of the energy deposition occurs within 15 to 20 A” of the double helix.26 To investigate this phenomenon of enhancement of IUdR radiosensitization by photon-induced Auger elec* DON LINE, American Type Culture Collection CCL 1b, Rockville, MD.

July 1987,Volume13, Number7 trons, we performed a number of experiments to compare IUdR radiosensitization at photon energies just above 33.2 keV, the K-absorption edge of iodine, with that at energies considerably higher than 3 3.2 keV. These experiments were performed using Chinese hamster cells in vitro and cell survival was determined by a cloning assay. One set of experiments was performed with continuous low dose rate irradiations and the other with acute dose rates. Both studies modeled situations of potential interest in clinical radiotherapy. Our study supports the earlier observation of enhancement of IUdR radiosensitization by Fairchild et ~1.~~” with acute exposures of filtered, orthovoltage X rays and presents the first experimental observations of the same phenomenon under conditions of continuous low dose rate irradiation. METHODS

AND MATERIALS

Cells Experiments were performed using Chinese hamster lung cells.* This system has been in place in our laboratory for several years, and the investigators have had considerable experience with the system.3”37,38 Chinese hamster cells are grown as monolayers in 75 cm2 Falcon tissue culture flasks, in a humidified 95% air-5% CO* atmosphere at 37°C. These cells are maintained in basal medium with Eagle’s salts, supplemented with fetal calf serum (15% V/V), antibiotics, MEM, vitamins, and Lglutamine. Under these conditions, the population doubling time during exponential growth is approximately 12 hr. Stock cultures are subcultured at 3-to-4 day intervals. Cytotoxicity studies Monolayers for experiments were prepared by plating cells, suspended from exponentially growing stock culture, into 60-mm Falcon tissue culture dishes. The cells were incubated for 18 hr prior to IUdR treatment or irradiation, to allow them to attach and to progress into logarithmic growth. Once the cells were in exponential growth, the growth medium was removed and replaced by growth medium containing 10e6 to 10m4M IUdR. The cells were then allowed to grow for 1 to 7 days. Replacement of medium with fresh medium containing IUdR twice a day was also performed to examine the toxicity, IUdR incorporation and radiosensitization as a function of IUdR concentration, duration of IUdR treatment, and number of IUdR treatments. Controls without IUdR treatment were handled similarly. The medium on the cultures was replaced by fresh medium containing IUdR just before the beginning of the irradiation, to ensure that IUdR was available in the medium throughout the irradiation, which lasted 1 to 48 hr.

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During irradiation, cells were maintained in a humidified 95% air-5% CO2 environment at 37°C. Unirradiated controls were maintained analogously. After irradiation, the cells were washed with Hanks’ balanced salt solution, trypsinized, and counted using a Coulter counter,? equipped with a Channelizer to allow assessment of and correction for any changes in cell size. Cells were plated for colony formation in at least four dishes per data point and were allowed to grow in a humidified 95% air-5% CO2 environment at 37°C for 10 days. The colonies were then stained and counted. Experiments were planned to obtain approximately 100 colonies in each dish, by adjusting the number of cells plated per dish appropriately. Controls receiving IUdR treatment but no irradiation were also examined to assess the toxicity of the drug alone.

Measurement of IUdR incorporation Average incorporation of IUdR into the DNA of the cells was measured using neutron activation. The neutron irradiations and assays were carried out at the Brookhaven National Laboratory by Dr. Ralph Fairchild and Brenda Laster, using their standard techniques.” In these studies 10’ cells were explanted from the tissue culture flasks and treated with 10m4M IUdR for 1 day. After IUdR treatment, the cells were irradiated in a thermal neutron beam and the neutron-induced lz81 activity was measured. The percent incorporation of IUdR (i.e., percent replacement of thymidine by IUdR) was calculated from the “*I activity, assuming that the mass of the DNA was 8.0 X 10-‘2 gm per cell, that the molecular weight of nucleoside monophosphate was 309, and that 29% of the bases in the DNA were thymidine.25

Irradiation techniques Cells in petri dishes were irradiated with specially-designed 24’Am and 226Ra source irradiators made of polystyrene with a central hole for placing the petri dishes on top of the sources, with or without some polystyrene spacers in between the sources and the tissue culture dish. The 24’Am irradiator contains a disc source of 24’Am with an activity of 9.7 Ci. The americium is in the form of americium oxide, which is bonded to an aluminum substrate and then double encapsulated in 1 mm of titanium. The 24’Am source in this irradiator was purchased for feasibility studies of 24’Am application for intracavitary irradiation of uterine cancers29,30and for the study of the relative biological effectiveness (RBE) of 24’Am gamma rays relative to “‘Ra gammas (Schulz, R.J., Bongiorni, P., unpublished data, 1986). The 226Rairradiator consists of 137 mg of 226Raspread around in a circular pattern to provide a uniform dose t Coulter Corp., Model ZBI, Hialeah, FL.

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distribution at the petri dish. Different dose rates over the range of 10 to 100 cGy/h were obtained by varying the thickness of the polystyrene spacers. The 24’Am irradiator was placed in a 37°C water-jacketed incubator and surrounded in lead foil of 1 mm thickness to shield the gamma rays. Adequate shielding for the 226Ra irradiator within a commercial incubator is not easily accomplished because of the thickness and weight of lead necessary. Shielding the whole incubator would have required a tremendous amount of lead. Therefore, our radium irradiator was placed in a specially-designed, small incubator which is surrounded by lead and kept in a well-shielded, restricted area. External beam orthovoltage irradiations were performed using a 250 kV X ray machine* with a source to surface distance of 60 cm. The dose rate was 204 cGy/min and the HVL of the X ray was 0.3 mm of Cu. The megavoltage X ray irradiation was performed using a clinical radiotherapy machine9 (4 MV X rays, SSD = 100 cm, dose rate = 150 cGy/min). A lucite sheet with a thickness of 1.25 cm was placed on top of the petri dish to establish charged particle equilibrium for 4 MV X rays.

Dosimetry The dose to cells in the monolayers was measured by using a FeS04 Fricke dosimeter with standard formulation (1 .O mM ferrous sulfate, 1.O mM sodium chloride, 0.8 N sulfuric acid). Ferric ion yields, G-values, were taken to be 14.8 for 241Am and 15.5 for 226Ra. Optical absorbance measurements were taken at 224 nm and 304 nm. It has been our experience that when the ratio of optical absorbancies is within 2.06 to 2.10, then the Fricke dosimeter is functioning well; 5 ml of dosimeter solution was placed in 60 mm Falcon petri dishes, and the dishes were then placed in the identical location used for petri dishes containing experimental monolayer cells. Falcon dishes were used for Fricke dosimetry because the inner surface of the dish has a negligible effect on the optical absorbance of the unirradiated dosimeter solution. The dose to the monolayer of cells adhering to the petri dish was calculated from the dose to the FeS04 solution by multiplying by the ratio of mass energy absorption coefficients of muscle and FeS04 solution and applying a correction factor for the distance of the cells from the interface. Details of the dosimetry have been described elsewhere (Schulz, R.J., Bongiomi, P., unpublished data, 1986). RESULTS

Before starting with the determination of IUdR radiosensitization, we examined the toxicity of IUdR alone in our tissue culture system as a function of incubation time # Picker Vanguard Therapy Unit, Highland Heights, OH. QVarian Clinac 4 with U-filter, Palo Alto, CA.

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and IUdR concentration. Figure la shows the relative survivals of cells treated with IUdR in concentrations of 10W3,10A4,or 10W5M for up to 15 days. The relative survival is the ratio of cell survival (colony-forming ability) with IUdR treatment to that without IUdR. The IUdR toxicity is quite small at 1O-’M for up to 10 days of incubation and at 10m4M up to an incubation time of 3-4 days. In contrast, 10m3M is extremely toxic even with a 1 day incubation time. Figure lb shows the results of another set of toxicity studies in which relative survivals were measured for concentrations from lo-” to lop2 M and incubation times of 2 and 3 days. IUdR toxicity was quite small in the case of 2 day incubation time for concentrations up to lop4 M and in the case of 3 day incubation time for concentrations up to 10e5 M. IUdR incorporation in the DNA using a single dose of lo-’ M IUdR was found to be 14.5, 14.4, and 18.1% for incubation times of 1, 2, and 3 days, respectively. The lack of a significant increase in IUdR incorporation with larger incubation times of 2 and 3 days may indicate depletion of IUdR available after a single dose of IUdR. To increase the IUdR incorporation, in later experiments, a higher concentration of 10d4 M and reinoculation of

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IUdR every 12 hr was employed. For 10e4 M concentration with the addition of IUdR at the beginning and again after 12 hr, the IUdR incorporation was found to be 39.2% for an incubation time of 1 day. Figures 2a and b show survival curves for cells treated with 10m4M IUdR continuously during 1 day of growth, then irradiated with 24’Am or 22aRa sources at dose rates of 55.5 or 58.7 cGy/hr, respectively. The survival curves for cells treated under identical conditions but without IUdR were also measured and are shown in Figures 2a and b; these indicate a relative biological effectiveness (RBE) of 1.3 for 24’Am photons relative to 226Ra photons, at a dose rate of about 57 cGy/hr in our in vitro system (Schulz, R.J., Bongiorni, P., unpublished data, 1986). Using the survival data shown in Figures 2a and b, sensitization factors, defined as the ratios of the doses required to obtain the same cell survival without and with IUdR, were calculated at different survival levels, and are shown in Figure 2c. Relative survivals, defined as the ratios of survivals with and without IUdR, were also calculated at different radiation doses and are shown in Figure 2d. It is clear from Figure 2c that IUdR radiosensitization is larger by a factor of about 1.5 for 24’Am photons than for 226Ra photons. The relative survival with IUdR for 241Amphotons is a factor of about 10 less than that for 226Raphotons, as shown in Figure 2d. Similar studies with lO-5 M IUdR showed less sensitization, as expected, and confirmed the pattern of enhancement with 24’Am and 226Ra(data not shown). To untangle some of the factors involved in IUdR radiosensitization with continuous low rate irradiations, such as IUdR uptake, cell kinetic parameters, repair, and the high LET of the Auger electrons, we initiated a series of experiments comparing IUdR radiosensitization after acute exposures of cells to 250 kV and 4 MV X rays. The HVL of the 250 kV X ray beam employed is 0.3 mm of Cu; this HVL is similar to that for 24’Am photons. However, the energy spectrum for 250 kV X ray is broad while that for 24’Am photons is monoenergetic. Figures 3a and b show survival curves for cells treated with 1Oe5 M IUdR for 3 days and then irradiated. Survival curves for cells irradiated without IUdR treatment were also measured and are shown in Figures 3a and b; these data indicate an RBE of 1.15 for 250 kV X rays relative to 4 MV X rays. IUdR sensitization factors as a function of survival and relative survival with IUdR as a function of dose (shown in Figures 3c and d), again illustrate the enhancement of IUdR-induced radiosensitization by the use of a photon spectrum closer to the K-absorption edge of iodine. The enhancement of radiosensitization with 250 kV X rays, relative to that for 4 MV X rays, is a factor of about 1.5. DISCUSSION Exact mechanisms for the observed enhancement of IUdR radiosensitization by lower energy photons ob-

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served in our experiments are not understood at this stage and can only be speculated upon. Our results are certainly consistent with the hypothesis proposed by Fairchild et al.,” that photons with energies just greater than 33.2 keV, the K-absorption edge of iodine, produce

more Auger electron cascades than do higher energy photons and that these Auger electron cascades, which have a high LET and a subcellular range, cause greater damage to the IUdR-substituted DNA than do the low LET photons. Based upon this hypothesis, an extensive

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theoretical analysis of dose enhancement by halogenated thymidine analogs has been carried out by Fairchild et uL~*‘~*” This analysis takes into account dimensions of the cell nuclei, photoelectric cross sections, Auger electron energies, fluorescence yields, average numbers of Auger electrons produced, and HVL’s. This analysis

shows that the most important parameter determining the photon-induced Auger effect is the ratio of the cross sections for photon absorption by the halogen in the thymidine analog in substituted DNA and this cross section for normal DNA. They calculated this cross section ratio, C, as a function of photon energy and found that the

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ratio for iodine is significantly higher than that for bromine. In fact, A&tine has an even higher cross section ratio, and would be even better for inducing Auger cascade; however, no stable isotope of astatine is known to exist. It is concluded by Fairchild et al. that “of the several halogens then, iodine appears to be the only viable choice, if it is hoped to take advantage of stimulated Auger cascades.” Using their model, Fairchild et al.” have calculated therapeutic gain factors (defined as ratios of dose to tumor containing IUdR and dose to normal tissue without IUdR) for acute, fractionated, and protracted irradiations. They report therapeutic gain factors of 2.2, 5.4, and 11 for acute irradiation using 40 keV photons and percent IUdR replacements of 5, 25, and 50%, respectively. For protracted irradiation using 40 keV photons, the reported values for therapeutic gain factors are 17, 44, and 84 for percent IUdR replacement of 5, 25, and 50% (see Table 1 of reference 11). In our experiments, we have compared the IUdR radiosensitization factors for 24’Am photons with that for 226Ra photons. Note that the enhancement factors calculated in this manu-

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script reflect only the enhanced sensitization resulting from the differences in the photon energies. The enhancement of radiosensitization for 24’Am relative to 226Ra observed in our work is in the range of 1.3 to 1.5; this value is consistent with estimates from the theoretical model of Fairchild et al.” and with Fairchild’s data comparing the survival curves for V79 cells irradiated with 13’Cs and with 70 kV X rays, with and without IUdR (Fig. 6, reference 10). Assuming that tissue damage from 226Ra results primarily from Compton scattering, that is, no Auger electron effects, we have estimated the calculated ratio of sensitization factors for 241Am and 226Rato be 1.43 and 2.72 for percent IUdR replacement of 5 and 25%, respectively. These calculated values of enhancement of IUdR radiosensitization by low energy photons are in reasonable, qualitative agreement with our measured data. Fairchild et al.I1 have calculated therapeutic gain factors that might be obtained using photon activation therapy, rather than photons alone, throughout a 30 fraction, 4-6 week course of radiotherapy. These should probably be considered as very optimistic estimates, as their calculation rests on a number of very tenuous assumptions, including: (a) the assumption that IUdR is incorporated uniformly throughout the tumor and that no IUdR is incorporated by normal tissues, (b) an assumption of a therapeutic gain of 2 resulting from an increased killing of hypoxic tumor cells due to the lowered OER of the Auger electrons, (c) an assumption that the normal tissue will repair radiation damage (resulting only from photons) whereas no repair will occur in tumor cells receiving photon-induced Auger electron therapy. However, while the values of therapeutic gain factors quoted by Fairchild may prove to be overly optimistic, there is reason to expect that the use of photon energies producing Auger electrons should improve the therapeutic ratios attainable with IUdR in the clinic. As the combination of halogenated pyrimidines with conventional radiotherapy has been shown to have clinical merit under certain circumstances,20q2’ this potential approach to improving the clinical use of IUdR appears to us to merit further consideration. Note that the ratio C is essentially constant in the photon energy range of 33 to 60 keV, indicating that within this range the photon energy is not critical. We have chosen to use 60 keV photons from 24’Am because of our ongoing research developing 241Am sources for use in clinical brachytherapy. 29*30 However, if the concept of increasing the radiosensitization by IUdR by using photons of appropriate energy is valid, other radioactive sources in this energy range would also be applicable. For example, ‘45Sm, with photon energies of 30 to 61 keV, which is currently being developed and tested at Brookhaven as a clinical brachytherapy source, could also be used in this application.” In this paper, we present experimental evidence indicating the validity of the hypothesis, proposed by Fair-

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child et al .?9,‘o,” that photon-induced Auger electron cascades can lead to an enhancement of IUdR radiosensitization. We observed that the radiosensitization obtained with continuous low dose rate irradiations with 241Am photons is greater than that with 226Raphotons by a factor of 1.5 to 2.0. We also observed a similar effect for acute exposures of 250 kV X rays relative to 4 MV X rays. To further test this hypothesis, similar experiments with photon beams with energies lower than 33.2 keV must be performed. An elegant experiment to confirm this mechanism would use the monochromatic photons from a synchrotron light source with photon energies just below and above the K-absorption edge of iodine. Such an experiment is being conducted by Fairchild et al. at Brookhaven National Laboratory and preliminary results confirm the role of photon-induced Auger electron cascades. Similar experiments for the determination of BUdR sensitization have been performed using photons with energies just below and above the K-absorption edge of bromine, from a monochromatic synchrotron radiation source at the Photon Factory, National Laboratory for High Energy Physics in Japan.39

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To exploit the Auger cascade effect, radiation therapy must be carried out with photon sources of relatively low energy. Orthovoltage X ray generators are, of course, readily available and could be used with minimal filtration to produce photon beams with sufficiently low energies to stimulate Auger cascades. Such beams would offer considerable disadvantages with regard to dose distribution in comparison with the more widely used modern megavoltage sources. Nevertheless, these orthovoltage sources could be used for the treatment of certain superficial tumors, intraoperative treatment, intra- or transcavitary treatment, etc. A number of isotopes which emit low energy photons suitable for inducing Auger cascades are presently being developed for use in brachytherapy; these include 24’Am (60 keV photons, 453 year half-life) which is actively under study in this laboratory29g30 and ‘45Sm(30-6 1 keV photons, 340 day half-life) which is being developed at Brookhaven National Laboratory. ’’ The phenomenon described in this report might therefore be exploited in several clinical situations, and might offer a new mechanism for improving the therapeutic ratio obtained with halogenated pyrimidines. More laboratory work to predict the practicality and clinical potential of this approach appears warranted.

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