T tion therapist is the reproductive death

Size: px

Start display at page:

Download "T tion therapist is the reproductive death"

Cleopatra Mills
5 years ago
Views:

1 SPATIAL DISTRIBUTION OF HUMAN CELL SURVIVAL IN A NEUTRON BEAM DESIGNED FOR THERAPY PAUL TODD, PHD,* CAKTEK B. SCHKOY, MS,t F. H. ATTIX, PHD,~ AND RICHAKD B. THEIJS, BSS The survival of cultured human (T-1 kidney) cells was determined at various locations in a tissue-equivalent liquid phantom irradiated with various doses of fast neutrons in the 10 x 10 cm collimated beam at the Naval Research Laboratory cyclotron. Conditions were: modal neutron energy = 17 MeV, dose rate = 60 rad/min, TSD = 125 cm, HVL = 11 cm tissue-equivalent fluid. When results were expressed in terms of cell inactivation as a function of dose, it could be seen that cell inactivation in all parts of the phantom was due to neutrons. For specified single doses at depth, cell isosurvival contours were plotted in two dimensions, and these contours were transformed to equivalent gamma-ray isodose contours by using the gamma-ray cell survival curve. The results of these experiments provide a preliminary indication that treatment planning with this neutron beam can be based on a space-independent, dosedependent RBE. Cancer 34:33-38, HE END-POINT OF INTEREST TO THE KADIA- T tion therapist is the reproductive death of the human cell. Improvements in radiation therapy consist of improved preferential inactivation of tumor cells. Fast neutrons offer the possibility of more effectively inactivating cells that are low in oxygen, but their scattering and attenuation in tissue are comparable to that of gamma rays from cobalt-60. The therapeutic neutron beam of the NRL cyclotron is produced by bombarding beryllium with 37.5 MeV deuterons, thereby pro- Presented at the Fifteenth Annual Meeting of the American Society of Therapeutic Radiologists, New Orleans, Louisiana, October 24-28, From the Naval Research Laboratory ant1 the Pennsylvania State University. Supported by the National Cancer Institute throiigh grants to Georgelown University and Medical College of Virginia. * Associate Professor of Biophysics, The Pennsylvania State University. t Rcscarch Assistant, The Pennsylvania State University. t Consultant, Naval Research Laboratory, Washirigton, D.C. $ Physicist, Cyclotron Laboratory, Naval Research Laboratory, Washington, D.C. Address for reprints: P. Todd, 618 Life Sciences Building, University Park, PA The authors gratefully acknowledge the encouragement and support of Dr. C. R. Rogers, Dr. R. 0. Rondelid, and Dr. J. R. Andrews, and the skilled assistance of the staff of the Naval Research Laboratory Cyclotron. Received for publication December 5, during a higher energy neutron beam than has been previously tested in detail for radiation therapy. The following important differences need to be considered: the chargedparticle spectrum will be richer in fast protons and heavy ions, the attenuation length is much,greater, collimation is more effective, and the prompt gamma ray spectrum is different.10 These physical features of the beam have corresponding biological consequences: the biological effectiveness of the spectrum of charged particles produced in tissue needs to be determined; the oxygen dependence of cell inactivation needs to be determined; the spatial variation of effectiveness and oxygen dependence needs to be determined; and the effectiveness of radiation scattered outside the treatment zone needs to be determined. It has been repeatedly pointed out that knowledge of spatial distribution of dose alone is not an adequate characterization of a particle beam for therapy.11 The spatial distribution of the pertinent biological effect must be known, in order to realistically evaluate effectiveness, safety, and treatment protocols. Earlier work indicates that neutron doseresponse curves for cultured mammalian cells do not change greatly with increasing neutron energy above about 5 MeV; that is, relative biological effectiveness (RBE) is nearly con-

34 CANCER July 1974 Vol. 34 stant.4 Similarly the oxygen enhancement ratio (OER) appears to be relatively independent of neutron energy above about 5 MeV.

2 34 CANCER July 1974 Vol. 34 stant.4 Similarly the oxygen enhancement ratio (OER) appears to be relatively independent of neutron energy above about 5 MeV. Plausible explanations have been offered for this.2 As neutrons are attenuated by passage through tissue or tissue-equivalent material, the charged-particle spectrum changes slightly as a function of depth. There exist conflicting reports whether RBE and OER change with depth correspondingly.3~7 It is evident that the spatial distribution of biological effect (human cell lathality) and its oxygen dependence in and around this new therapeutic neutron beam need to be determined. The objectives of this research therefore were: 1. A biological evaluation of the effectiveness of beam collimation. 2. Biological confirmation of the depthdose profile. 3. Testing of spatial dependence of biological effectiveness. 4. Biological determination of isodose contours in gamma-ray equivalent doses. Corresponding physical measurements accompanied all of the biological determinations, so that correlations between dose and effect were possible at all points studied in a tissueequivalent liquid phantom. The biological endpoint studied was the reproductive lethality of human kidney T-1 cells forming colonies in culture. TARGET 7 IONIZATION CHAMBERS 125 CM * SAMPLE / HOLDER TISSUE BENELEX EQUIVALENT LIQUID COLLIMATOR PHANTOM FIG. 1. Sketch, not to scale, of the exposure arrangement for cultured human cells at the NRL cyclotron. Positions 1, 2, and 3 in the tissue equivalent liquid phantom are defined in Table 1. Depth = 0 is defined at the front exterior face of the phantom, 125 cm from the surface of the beryllium target. tions of cell exposure using tissue-equivalent ionization chambers and thermoluminescence dosimeters, so that total dose and neutron dose are known at all locations under the exposure conditions. The neutron dose rate was rads/min at the face of the phantom, where 5.1% of the total dose was due to gamma rays. Table 1 lists neutron and gamma ray doses at the three principal points of interest in the phantom indicated in Fig. 1. Figure 2 describes the arrangement for irradiating human cells in submersible coverslip holders. In these experiments, the beam was collimated to 10 x 10 cm at a point 125 cm from the Be target. Coverslip holders or tissue-culture bottles were immersed in tis- MATERIALS AND METHODS Neutron Dosimetry and Exposure Dosimetric methods and beam production have been summarized elsewhere.10 Measurements have been made throughout the exposure phantom and under the exact condi-., TABLE 1. Neutron, Gamma-Ray, and Total Dosei Delivered at the Center of a 10 x 10 cm Beam at Positions 1, 2, and 3 Defined in Fig. 1. Based on 100 rads (Total) at the Ionization Chambers Position Depth in phantom, cm Total dose, rads Neutron dose Gamma dose Percent gamma dose FIG. 2. Photograph of Lexan polycarbonate coverslip holders. Upper left, 10-cm holder with slotted inner wall removed to illustrate single-block construction. Upper right, 3-cm holder with slotted inner walls inserted, showing slots into which coverslip cultures arc inserted. Lower part of photo shows covers with captured silicone 0 rings and nylon screws. No glass or metal is used in the exposures.

No. 1 HUMAN CELL SURVIVAL IN A NEUTRON BEAM - sue-equivalent fluid contained in a I x 1 x I ft cubic container made of lucite. Cells Human kidney T-1 cells were used as previously described.

3 No. 1 HUMAN CELL SURVIVAL IN A NEUTRON BEAM - sue-equivalent fluid contained in a I x 1 x I ft cubic container made of lucite. Cells Human kidney T-1 cells were used as previously described.'* Medium was Eagle's MEM supplemented with 10% fetal calf serum.e+ Preparation of Cells for Irradiation Cells were trypsinized from a logphase (1 day old) monolayer, counted, and seeded in predetermined numbers on 20 x 22 mm plastic (LUX, Microbiological Associates, Inc.) coverslips in 0.5 ml. of complete medium in 35 mm plastic (Falcon Plastics, B-D Corp., Los Angeles, Calif.) petri dishes. These cells were incubated for 7.0 hours. The coverslips were then transferred to slotted rectangular polycarbonate containers, which spaced the coverslips 0.25 cm apart along the length of the container with the coverslips in vertical orientation, as illustrated in Fig. 1. The containers, depicted in Fig. 2, were fabricated in the shop of the Naval Research Laboratory Cyclotron Laboratory, Washington, D.C. After coverslips were inserted (3 groups of 5 per 10-cm container, and 8 per 3-cm container), the containers were completely filled with medium, sealed, and transported for about 5.0 hours before exposure to neutrons. At this time there were approximately 1.3 test cells/colony. Containers were placed in the collimated beam so that coverslips were distributed, perpendicular to the beam, from the point marked 1 (front, 2.3 cm) to the point marked 3 (back, 12.3 cm) in Fig. I. Five hours after irradiation, coverslips were transferred to 35-mm petri dishes containing 3 ml each of complete medium. These cultures were fed every 4 days. Colony formation was completed at 9-10 days, at which time the coverslips were stained with methylene blue and the colonies were counted under a dissecting microscope. Colony counts on groups of 5 or more adjacent coverslips were averaged to calculate local survivals. RESULTS Spatial Distribution of Dose The central axis depth-dose profile was determined within the tissue equivalent liquid phantom with a l-cm3 tissue-equivalent ioni- * Chemicals and solutions, including serum. ohtained from Grand Island Biological Co., Grand Island, N.Y. 40r Todd et al. 35 I 20 0 W IA DEPTH, CB FIG. 3. Depth-dose profile along the central axis of the beam in the phantom. Empty circles give measurements made with 1 cm3 tissue-equivalent ionization chamber, and filled circles give measurements with thermoluminescence dosimeters. zation chamber. The results, shown in Fig. 3, indicate that the dose at 10 cm depth is greater than half the dose at entrance, so that the depth-dose profile is superior to that of gamma rays of Wo. Thermoluminescence dosimetry was performed at cell sample locations 1, 2, and 3 (Fig. 1) and found to be in agreement with ionization-chamber measurements, as also indicated on Fig. 3. These data are presented in detail in Table 1. The uniformity of the dose across the beam was examined, because therapy places stringent requirements on transverse uniformity and because cell irradiations were performed to evaiuate the effectiveness of collimation. The results, shown in Fig. 4, indicate a small 4 W w I n DISTANCE FROM CENTERLINE, CM FK. 4. Transverse dosimetric scan at the neutron beam in the phantom at depths of 0, 5, and 10 cm. The 5 cm curve is emphasized, due to its relationship to Figq. 7 and 8 experiments.

36 CANCER July 1974 VOl. 34 DEPTH IN PHANTOM, CM FIG. 5. Cell survival as a function of depth in the phantom for four neutron doses, 69, 138, 183, and 229 rads delivered at 12.3 cm depth in phantom.

4 36 CANCER July 1974 VOl. 34 DEPTH IN PHANTOM, CM FIG. 5. Cell survival as a function of depth in the phantom for four neutron doses, 69, 138, 183, and 229 rads delivered at 12.3 cm depth in phantom. amount of beam divergence over the first 10 cm of depth in the phantom. However, at 0, 5, and 10 cm depth, all transverse dose profiles fell to 5OY0 of the center-line dose at a distance of 5.7 cm from the center line. At a depth of 5 cm, near where cell survival experiments were done, the dose outside the beam (8-10 cm from the center line) was 10% of the dose at the center line. Cell Survival as a Function of Depth Cell survival approximates a logarithmic transformation of the dose. At high doses one expects logarithmic dependence of survival upon depth, when the dose decreases nearly linearly, as in Fig. 3, over the range of interest. Measurements of cell survival as a function of depth, as shown in Fig. 5, are con- FIG. 7. Photograph of tissue-culture bottles containing methylene-blue stained colonies of T-1 cells. Colonies are individually countable at a magnification of 3X. The upper bottle was not irradiated. The lower bottle was irradiated with its right (top) end in the neutron beam and its left (bottom) end outside the beam. sistent with this expectation. Survival points at all depths fell within two standard deviations of a single survival curve, shown in Fig. 6, intersecting 10% survival at 280 rads, having extrapolation number of 3.0 and Do of 86 rads. The RBE at any point in the beam lies between 1.8 at high and 3.7 at low doses. Biological Evaluation of Beam Collimation To evaluate cell survival as a function of distance from the center line, bottle cultures of cells (30-ml plastic Falcon Tissue Culture bottles) were suspended in the phantom so that the lower halves of the bottles were out- ; GOO REUTRON DOSE, PADS FIG. 6. Cell survival as a function of dose. using data of Fig. 5. Different plotting symbols cokespogd to different depths: squares--2.1 cm, dots-7.2 cm., and circles cm. Upper curve gives gamma-ray survival DISTANCE FROM CENTERLINE, CM FIG. 8. Cell survival as a function of distance from the beam centerline, using colony counts from bottles such as those in Fig. 7 (empty circles). Filled circles give total dose, which was 230 rads at the center of :he beam.

No. 1 HUMAN CELL SURVIVAL IN A NEUTRON BEAM * Todd et al. 37 side the neutron beam and the upper halves (end with screw-cap) were in the beam. The number of colonies that grew in 0.

5 No. 1 HUMAN CELL SURVIVAL IN A NEUTRON BEAM * Todd et al. 37 side the neutron beam and the upper halves (end with screw-cap) were in the beam. The number of colonies that grew in 0.25-cm intervals was compared with identical intervals of unirradiated bottles, and survival was calculated as a function of position along the long axis of the bottles. A bottle irradiated this way is compared with an unirradiated bottle of cells after colony formation has taken place (6-8 days) in Fig. 7. The technique of scoring survival should be clear from this photograph. Results of quantitative counting of colonies in duplicate bottles as a function of distance from the beam center line are presented in Fig. 8. A dose of 235 rads led to 30% survival across the full breadth of the beam, 80% survival 1 cm outside the beam, and 90% (or greater) survival 2 cm outside the beam. The experiment described by Fig. 8 was repeated at several doses at 6.5 cm depth in the phantom, and survival was scored inside and outside the beam only. The data were used to construct dose-survival curves inside or outside the beam, and fall within one standard deviation of the same survival curve (Fig. 9), indicating that cell lethality outside * 01 - I 1 I DOSE, RADS Frc.. 9. Dose-survival curves for cells irradiated at a depth of 6.5 cm in the phantom, in and out of the beam and at the edge of the beam. Empty circle points are for cells in the beam. The lower curve is based on actual dose. Thc upper curve is for cells outside the beam, using the abscissa to denote the corresponding dose delivered at the beam ccnterline. 10 I I [ l I I ' 1 I I UEPTH IN PHANTOM, GI1 Frc. 10. Isosurvival plots for human cells in tissucequivalent phantom. Contours are given for 5, 10, 20, 50, 80, and 100% survival. Each survival value is followed by the dose (in parentheses) of gamma radiation that leads to the same survival. the beam is due to scattered neutrons and not to gamma rays. Isosurvival Profiles A constant neutron dose of 183 rads was delivered at 12.3 cm depth in the phantom. Bottle cultures were placed across the beam at various depths to determine the transverse biological effect profiles. Distances from the center line at which 80, 50, 20, 10, and 5% cell survivals occurred were determined. These locations are plotted as points in Fig. 10. The points are connected to give approximate isosurvival profiles, and the gamma ray dose that leads to the same amount of cell inactivation is given in parentheses on each contour. It should now be possible to construct such diagrams directly from isodose profiles and the neutron and gamma-ray survival curves and use them in treatment planning. RBE 11 I I I I I l i l l I I I I I Ill NEUTRON DOSE, RADS Frc. 11. Logarithmic plot of neutron RBE vs. ncutron dose for human cell inactivation. The low-do% flat line is RBE T 3.7 at all doses below 50 neutron rads or about 180 gamma-ray rads. The logarithmic slope of the slanted line is -I/. The high-tlosc flat line is RBE = 1.8 at all doses above 600 neutron ratls.

6 38 CANCER Jzily 1974 VOl. 34 DISCUSSION Therapeutic Adequancy of the NRL Beam It appears that the NRL fast neutron beam has sufficient uniformity of dose, effectiveness, and collimation to be used with confidence in radiation therapy. The depth-dose profiie and collimation are at least as good as in the case of gamma rays of WCo. The cell data should be ultimately utilizable in the daily dose calculation. Dose Dependence of RBE From the survival curves of Fig. 6 it can be determined that the RBE (ratio of gamma-ray to neutron dose that produces the same survival) depends upon dose. Below about 50 neutron rads, the RBE is constant (dose-independent) with a value of about 3.7. Above about 500 neutron rads, the RBE becomes asymptotic to the ratio of D,s of the gammaray and neutron survival curves-a value of about 1.8. At neutron doses between these the RBE depends upon dose according to D-n,9 as shown in Fig. 11. Therapeutic Dose Calculation This result has important applications. When the dose to the patient is calculated in neutron rads, the gamma-ray contamination can be neglected? or it can be added arithmetically to the neutron dose.8 For treatment planning based upon neutron and gamma-ray doses, the correct neutron-equivalent dose is given by D D = D, +R+ But D, will usually be rads, so the proper RBE to be used in the above calculation is 3.7, the limiting value determined by the initial (low-dose) slopes of the survival curves. REFERENCES 1. Barendsen, G. W.: Responses of cultured cells, turnours, and normal tissues to radiations of different linear energy transfer. Curr. Top. Radiat. Res. 4: , Barendsen, G. W., and Broerse, J. J.: Dependence of the oxygen effect on the energy of fast neutrons. Nature (Lond.) 212: , Berry, R. J.: OER and cell cycle. In Conference on Particle Accelerators in Radiation Therapy. Los Alamos Scientific Laboratory Report LA-5180-C, 1973: pp Broerse, J. J., Barendsen, G. W., and van Kersen, G. R.: Survival of cultured human cells after irradiation with fast neutrons of different energies in hypoxic and oxygenated conditions. Znt. J. Radiat. Biol. 13: , Catterall, M., Rogers, C. R., Thomlinson, H. R., and Field, S. B.: An investigation into the clinical effects of fast neutrons. Br. J. Radiol. 44: , Eagle, H.: Amino acid metabolism in mammalian cell cultures. Science 130:432437, Evans, R. G., Pinkerton, A,, Djordjevic, R. Mamacos, J.. and Laughlin, J. S.: Changes in biological effectiveness of a fast neutron beam at different depths in tissue equivalent material. Radiat. Res. 45: , Hussey, D.: Personal communication-the full neutron plus gamma-ray dose is used in treatment planning at Texas A & M-M. D. Anderson Hospital clinical trial Kellerer, A. M., and Rossi, H. H.: RBE and the primary mechanism of radiation action. Radint. Res. 47:15-34, Theus, R. B.: Neutron delivery. In Conference on Particle Accelerators in Radiation Therapy. Los Alamos Scientific Laboratory Report LA-5180-C, 1973: pp Todd, P.: CeIlular aspects of dose fractionation and recovery in particle radiation treatments. In Conference on Particle Accelerators in Radiation Therapy. Los Alamos Scientific Laboratory Report LA-5180-C, 1973; pp Todd, P.: Heavy ion irradiation of cultured human cells. Radiation Res. [Suppl.] 7: , 1967.

LET, RBE and Damage to DNA

LET, RBE and Damage to DNA Linear Energy Transfer (LET) When is stopping power not equal to LET? Stopping power (-de/dx) gives the energy lost by a charged particle in a medium. LET gives the energy absorbed