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0 Biology Original Contribution QUANTITATIVE COMPARISON OF RADIOLABELED ANTIBODY THERAPY AND EXTERNAL BEAM RADIOTHERAPY IN THE TREATMENT OF HUMAN GLIOMA XENOGRAFTS . JEFFERY A. WILLIAMS, Division
of Radiation
M.D.,
Oncology,
JAMES A. EDWARDS, B.S. AND LARRY E. DILLEHAY,
The Johns Hopkins
Oncology
Center,
600 N. Wolfe St., Baltimore,
PH.D.
MD 2 1205
Using “Yttrium radiolabeled antibodies, radioimmunotherapy was compared to fractionated external beam radiotherapy in the treatment of human glioma xenografts. Antibody treatments required administration of an approximately threefold greater total dose compared to external beam treatments to achieve the same tumor regrowth delay. Following multi-fraction external beam radiation treatments, tumor regrowth delay demonstrated a large fractionation effect (a/p = 2.3 Gy, 95% confidence limits 0.4-4.2 Gy), suggesting that much of the ineffectiveness of the antibody treatments could be caused by a large dose-rate effect in this system. Despite the large fractionation effect, the regrowth delay was small for a large single-fraction external beam irradiation, possibly because of tumor hypoxia. When compared to external beam radiation, radiolabeled antibody treatments resulted in a comparatively diminished tumor bed effect, suggesting radioimmunotherapy spares normal tissue surrounding the tumor. Radioimmunotherapy,
Glioma, Fractionated external beam, Xenograft, 9oY, Tumor bed effect.
INTRODUCTION Human malignant gliomas are rarely cured. Conventional postoperative external beam radiotherapy, however, extends median survival (28). When radiotherapy is conventionally fractionated, higher total doses yield further improved median, but not overall, survival (22). Hyperfractionated external beam radiotherapy improves neither median nor overall survival (5). More recently, radiolabeled antibodies have been used in the experimental treatment of human gliomas (4, 14, 15,30-35). P96.5, a murine IgG2a monoclonal antibody, specifies a neuroectodermally-derived cell surface antigen of molecular weight 97,000 (8) and targets experimental glioma in vivo (30-35). In nude mice bearing human glioma xenografts, intravenous administration of “Y-radiolabeled P96.5 causes striking accumulation of injected dose in tumor with resultant regression and growth delay (30-35). Such radiolabeled antibody therapy differs, however, from external beam radiotherapy in several important respects: 1. The dose rate to the tumor is both low and continuously decreasing after the initial tumor uptake ( 13, 16).
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requests
to: Larry E. Dillehay, Ph.D. work was supported by The Preuss Foundation for Brain Tumor Research, National Institute of Health grant PO1 CA4379 1, and Hybritech.
Acknowledgements-This
2. The total dose to the tumor is delivered over a comparatively short time (days rather than weeks) (19). 3. Both geometric and biological factors may render absorbed dose (Gy) non-uniform within the tumor. Absorbed radiation dose at the periphery of spherical tumors exhibiting uniform uptake of radiolabeled antibody may be less than the dose rate at the center (11). Differences in regional blood flow, antigen density or availability, or antibody specificity may cause nonuniform distribution of antibody and resultant inhomogeneity of tumor absorbed dose (6,9, 13). Despite these important differences, the comparative efficacies of external beam and radiolabeled antibody therapy have not been measured in the treatment of human gliomas. We therefore compared growth delay following varying regimens of 9oY radiolabeled antibody therapy or external beam radiotherapy in a human glioma xenograft-nude mouse model. METHODS
AND MATERIALS
Growth of human U-251 glioma xenografts in nude mice, labeling and biodistribution of ’’‘In and 9oY radiolabeled antibodies, dosimetry, and measurement of xe-
Accepted
for publication
3 February
1992.
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I. J. Radiation Oncology ??Biology 0 Physics
nograft growth delay following 9oY radiolabeled antibody treatments were performed as previously described (34, 35). Treatments were performed when tumors grew to 0.3-0.4 grams. External beam phantom dosimetry Tumor-bearing nude mice phantoms were constructed of polystyrene and mounted a fixed distance from the collimated 13’Cssource of a laboratory irradiator.* Using surface-mounted thermoluminescent dosimeters, the tumor phantom absorbed dose (Gy) per unit time of irradiation was measured. Human glioma xenograji therapy ‘oY-radiolabeled antibody therapy. Groups of at least nine tumor-bearing animals received intravenous injections of either 2% bovine serum albumin in phosphatebuffered saline (PBS), unlabeled P96.5, 100 uCi ‘oY-radiolabeled P96.5, or 200 uCi “Y-radiolabeled QC1054 (a monoclonal IgG anti-ferritin) as previously described (34, 35). External beam radiotherapy. Pairs of tumor-bearing nude mice were restrained in ventilated 50 ml conical centrifuge tubes and mounted in the 13’Csirradiator with tumor-bearing flanks facing the collimated source in a geometry identical to that used with the phantoms. The collimator shielded the animals anteriorly and posteriorly with 0.5 cm margins from the edges of the tumors. No collimation was used dorsally or ventrally, allowing irradiation of the inferior abdomen, rectum, and flank muscle and skin. Groups of at least four tumor-bearing animals received either no irradiation (control) or daily fractionated external beam irradiations. Tumor volumetrics Measurements of tumor length (L), width (W), and height (H) were made every 3 days and the product L X W X H, proportionate to tumor volume, was calculated. The logarithm of the ratio of tumor volume (V) to the initial tumor volume at the time of treatment (VCJ was plotted versus time for each animal as was the average of Log(V/V,,) for each treatment group. Tumor regression was defined as any decrease in an individual tumor volume below its initial value (V/V, < 1). The nadir in tumor volume for each treatment was defined as the lowest value of the average Log(V/VO). The delay in regrowth to the original volume (V/V, = 1) for each treatment was estimated from the plot of average Log(V/VO) versus time. The tumor bed effect (1) was quantitated by estimating the volume doubling time for the control tumors at the time of treatment and for the treated tumors at the time they regrew to V = VO.The three parameters of the Gompertz equation (23)
* Mark I, Model 68, J. L. Shepherd and Associates, San Fernando, CA.
Volume 24. Number I. 1992
Log V/V, = P,( 1 - eP(P2(t-P3))),
(Eq. 1)
were found which gave the best least-squares fit to the data for each tumor of Log(V/V,) versus time (t) for growth between V/V, = 1 and V/V, = 4. The tumor volume doubling time at V/V, = 1 was then calculated to be 0.3/(P1 *Pz). Some tumors could not be analyzed for the reasons indicated in Table I. RESULTS
In vivo thermoluminescent dosimetry Direct measurement of absorbed radiation dose in tumor by microthermoluminescent dosimetry revealed average absorbed doses (Gy) of 37.7 + 4.5 (s.e.m.) and 20.4 + 1.3 following administration of 100 uCi of T-radiolabeled P96.5 and QCIO54, respectively. These values were corroborated by the MIRD formalism of absorbed dose calculation (35). Human glioma xenograft therapy Radiolabeled antibody. Following intracardiac administration of buffer or unlabeled P96.5 (Controls) no tumors regressed, and the average time to grow to four times the initial volume was 6.1 -t 0.9 days. Following administration of 100 uCi “Y-radiolabeled P96.5, 9 of 10 tumors regressed, the average of Log V/V, reached a minimum at 57% of the initial treatment volume and subsequently reached the initial treatment volume 36 days after injection (Fig. 1 and Table 1). Following administration of 200 uCi “Y-radiolabeled QCI054, 7 of 9 tumors regressed, the average of Log V/V0 reached a minimum at 63% of the initial treatment volume and subsequently reached the initial treatment volume 28 days after injection (Fig. I and Table 1). Fractionated external beam radiotherapy. Figure 2 illustrates the growth of tumors following two of the external beam treatments. The treatment with a larger dose per fraction, three 4.5 Gy fractions, produced a larger regrowth delay than that with ten 1.5 Gy fractions, even though the total dose was smaller. The regrowth delay (time for the average of Log V/V, to return to 0) following each treatment is listed in Table 1. The time for regrowth to V/V, = 1 was also estimated for each tumor and the mean and standard error for each treatment (including antibody treatments) plotted versus the total dose in Figure 3. For treatments with the same dose per fraction, the regrowth delay increased with total dose. It can also be seen in Figure 3 that regrowth delays increased with increasing dose per fraction, except for the treatment with a single 10 Gy fraction. Comparison of radiolabeled antibody to external beam. In Figure 3 it can be seen that much larger doses were required to produce the same regrowth delay with anti-
113
Comparison of radiolabeled antibody and external beam therapies 0 J. A. WILLIAMS et a/.
Fig. 1. Effect of two radiolabeled antibody treatments on tumor growth. Each symbol is the average of Log V/V, for the tumors
Fig. 2. Effect of two fractionated external beam treatments on tumor growth. Each symbol is the average of Log V/V0 for the
in the group. Plus or minus the standard error is indicated by the vertical line whenever larger than the symbol.
tumors in the group. Plus or minus the standard error is indicated by the vertical line whenever larger than the symbol.
bodies as with the external beam treatments. We wanted to determine whether this ineffectiveness could be caused by a large fractionation/dose-rate effect, that is, that the antibodies effectively deliver irradiation in a large number of small fractions, which might be ineffective in this tumor. We first determined whether the response of the tumors to external beam could be described by the FDF formu-
lation of the linear-quadratic tumor-cell-kill model (26) of independently-acting fractions (complete repair of sublethal damage between fractions). In this model, for treatments that deliver a total dose D in fractions of size d, the response depends on the total effect, TE, defined by: TE = D*(cu/p + d).
(Eq. 2)
Table 1. Effects of treatments on tumor growth A. Radiolabeled antibody Tumor bed effect No. animals
Injection Control 100 uCi P96.5 200 uCi QUO54
11 10 9
Log V/V, at nadir
Days to nadir
Days to v/v, = 1
Td at v/v, = 1
-.24 -.2
28 18
0 36 28
2.8 4.4 7.9
No. animals 11 I* 7+
* Two tumors did not regress to V, and one tumor had not reached 4V0 at the end of the study. + One tumor did not regress to V0 and one animal died after Day 2 1. B. External beam Tumor bed effect Dose per fraction
No. fractions
No. animals
Log V/V, at nadir
Days to nadir
Days to v/v, = 1
Td at v/v, = 1
0 1.5 2 2 2 3 3 3 4.5 10
10 6 8 10 4 6 8 3 1
6 6 6 5 4 4 6 5 4 6
-.12 -.08 -.21 -.36 -.31 -.48 -.54 -.72 -.35
24 21 21 27 21 21 33 21 18
0 32 29 43 59 38 68 100 61 29
3.5 12 13 18 18 17 15 18 22
* One tumor did not regress to Vo. + One tumor had not reached 4V0. * None of the tumors in group had reached 4V0.
No. animals 6 5* 5* 5 4 4 5+ _* 4 6
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0-l 0
20
40
Total Dose (Gy)
Fig. 3. Growth delay as a function of total dose for two 9oYlabeled monoclonal antibodies and for different fractionated external beam treatments. Each point represents the mean of the individual tumor growth delay for each treatment and the vertical lines indicate plus or minus the standard error.
The parameter CY/~determines how the response changes with changes in dose per fraction. The method of Gutenberger et al. (7) was used to estimate the value of a/& It is assumed that the tumor growth delay, TGD, can be related to TE by the linking function: TGD = bl *(TE)b2.
(Eq. 3)
All the individual regrowth delay data were used in a nonlinear regression program? to determine the values of a/ p, bl, and bl which gave the best fit to Equation 3. A best-fit value of 3.7 Gy was obtained for LY/@If the model accurately described the data, then in a plot of the individual TGDs versus D*(3.7 + d), the individual points should be clustered about the line generated with the bestfit values of bl and b2 in Equation 3. When this was done, it was clear that the response for the single-fraction treatment was aberrant, being much lower than expected. When the analysis was repeated with only the multi-fraction (three or more) data, an a/p of 2.3 Gy was obtained (95% confidence limits; 0.4-4.2 Gy). Figure 4 shows a plot of TGD for the individual tumors that received multiple fractions (pluses) versus D* (2.3 + d). The multifraction points (pluses) do lie about the line generated by the best-fit parameters, suggesting that the linear-quadratic model with an a/P of 2.3 Gy does provide a good description of the fractionation-dependence of the responses to multi-fraction treatments. The TGDs for tumors receiving only the single-fraction treatment are also shown (Xs) in Figure 4. The single-fraction TGDs are less than would be expected from Equation 1, even if the upper limit of 4.2 Gy for (u//3is used. For continuous irradiation, the term d in Equation 2, associated with sublethal damage, is replaced by a term depending on the dose rate and rate of repair. In Figure
+ SAS Institute, Cary, NC.
Volume 24, Number I. 1992
4, we have also plotted the TGDs observed with antibodies assuming that this term is zero. Although there was considerable variability in the TGDs observed with antibodies, most of the points lay below (and to the right) the bestfit line. The antibody points would be even further to the right if sublethal damage contributed to the response. Thus, for an CY/~of 2.3 Gy, the poor response with antibodies can not all be attributed to expected dose-rate effects in this system. However, if the data is replotted with an a//3 of 1.O Gy, the antibody growth delays overlap the external beam growth delays. Since this is within the confidence limits on a//3, we cannot exclude the possibility that the poor response with antibodies seen in Figure 3 is attributable to a very large dose-rate effect in this tumor system. Tumor bed e&c&. To determine whether differences in damage to tumor stroma (tumor bed effects) were contributing to the observed differences in regrowth delay, the growth rate of each tumor at the time of return to VU was determined. Figure 5A shows the results for the tumors treated with antibody and Figure 5B shows the results for the same two external beam treatments as shown in Figure 2. The average doubling time at V0 for each treatment is given in Table 1. Figure 6 shows these average doubling times versus dose. For the external beam treatments, there was a general trend towards longer doubling times for larger doses per fraction, with the single fraction treatment producing the longest doubling time. However, the relatively large errors precluded an estimation of o(/P for this effect. The volume doubling times for the antibody-treated tumors were less than that for any of the external beam treatments.
lco-
50 -
70
90
110
130
D’(2.3+d)
Fig. 4. Growth delay for each tumor versus total dose times 2.3 plus dose per fraction. The pluses indicate tumors receiving multi-fraction treatments. The line indicates the best fit of these points to equation 3 (a/@ = 2.3 Gy). Xs indicate tumors receiving single-fraction treatment. Growth delays for tumors treated with labeled antibody are plotted (squares for p96.5, circles for QCI) assuming a dose per fraction of 0.
Comparison of radiolabeled antibody and external beam therapies 0 J. A. WILLIAMSefa/.
A . . . 0.
?? *
.I.
I
A
&A
*
.
.
.
??
.* .
.
Control
100 P96 5
200 cm
C0ntVd
10XlSGy
3X4 5Gy
4
I
Fig. 5. Tumor volume doubling times at treatment time for control tumors and at the time regrowing tumors exceeded V,,. A) For the two antibody treatments. B) For the same two external beam treatments as in Figure 2. Each point represents an individual tumor.
DISCUSSION There have been conflicting reports on the relative efficacy of radiolabeled antibody and external beam radiotherapies. Buchsbaum et al. (3) have observed in a human colon carcinoma xenograft model that to produce the same delay in growth to two times the treatment volume about five times as much tumor dose is required from an ‘3’I-radiolabeled antibody as from single fraction external beam treatment. This is roughly comparable to what we have observed in the human glioma xenograft
1A
I * ‘0
c 0 I 0
I
I
20 Total Dose
I
40 (GyJ
Fig. 6. Average tumor doubling times at V, versus total tumor dose. Plus or minus the standard error is indicated by the vertical line whenever larger than the symbol.
115
model with “Y-radiolabeled antibody for regrowth to the treatment volume. In contrast, Wessels et al. (29) and Knox et al. ( 12) reported greater responses for radiolabeled antibody than for external beam treatments. Wessels et al. (29) observed in a renal cell carcinoma xenograft model delays in growth to two or four times the treatment volume following ‘3’I-radiolabeled antibody treatments which were greater than following single or four fraction external beam treatments. Knox et al. ( 12) reported that treatment of a mouse B-cell lymphoma with 13’-labeled anti-idiotype monoclonal antibody was more effective than multi-fraction external beam given in 10 fractions over 2 weeks. These conflicting conclusions emphasize that meaningful comparisons of these two forms of radiotherapy will require a fuller (quantitative) understanding of the properties of each tumor system. Among the factors that could contribute to differences in responses to these two therapies are hypoxia, fractionation or dose rate effects, tumor bed effects, and dose distribution effects. The relative importance of these factors will depend on what measure of tumor response is used. All studies of radiolabeled antibodies versus external beam to date have been based on the delayed growth of treated tumors. The current study and that of Buchsbaum et al. (3) and Wessels et al. (29) used growth delay to measure tumor response. Knox et al. ( 12) used a cumulative measurement of changes in tumor size that extended only up to 12 days (the last day of multi-fraction treatment) that would favor any treatment (such as the antibody treatment) which would deliver the majority of the dose in the first half of the period. Comparison of single-fraction external beam to radiolabeled antibodies may be confounded by the presence of hypoxic cells which would render the tumor less responsive to the single-fraction treatment; the impact of these initially hypoxic cells is reduced by reoxygenation during fractionated treatments (27). In our study, the poor response with the single fraction stood out from the multifraction responses (Fig. 4). With radiolabeled antibodies, hypoxia is less likely to be a factor than with single-fraction because the bulk of the dose is delivered over several days, comparable to the duration of the shorter multifraction treatments, allowing for reoxygenation, and because the oxygen enhancement ratio decreases as the dose per fraction decreases (20). Thus, the poor response observed by us and by Wessels et al. (29) for single fraction might be because of tumor hypoxia. The effectiveness of radiation in killing cells generally decreases as the radiation is split into smaller fractions or the dose rate is lowered. The ‘incomplete repair model’ relates the killing of target cells for multifraction highdose-rate or continuous low-dose-rate treatments to the linear and quadratic components of killing by single highdose-rate treatments, characterized by the parameters (Y and p, respectively (25). The size of the dose-rate effect and the decrease in killing with decreased dose per fraction of high-dose rate radiation are inversely related to (Y/Pof
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I. J. Radiation Oncology 0 Biology 0 Physics
the target cells (24). We observed an CY/@ of 2.3 Gy for multifraction external beam treatments. This a/p value is smaller than has been generally observed for the responses of experimental tumors (36). However, U251 cells, like other glioblastomas ( 17) exhibit a large shoulder on their in vitro high-dose-rate survival curve (1 S), and would thus be expected to have a small a//3. Based on the small CY/&much and possibly all of the differences between fractionated external beam and antibody in delaying tumor regrowth in this system could be caused by a large dose-rate effect on survival of the tumor cells. For cells with a very large value of cu/p, we would expect, on the basis of the incomplete repair model, the efficacy of RIT to approach that of multifraction treatment for doses per fraction 4 a/& After clearance of dead cells and regrowth of the tumor from surviving cells, the growth rate of the tumor will depend on whether there is any long-lasting damage to the tumor stroma (1). In Figure 6 it can be seen that neither of the antibody treatments produced as large a decrease in the growth rate of the regrowing tumors as any of the external beam treatments. Thus, like physical implants (10, 2 l), radiolabeled antibody therapy appears to spare the tumor bed (and probably other surrounding normal tissue) relative to external beam treatments, although it should be pointed out that a larger ratio of normal tissue to tumor was irradiated in our external beam treatments than would be likely in a clinical situation. Nevertheless, our results suggest that irradiation of surrounding normal tissue from antibody concentrated in the tumor is not likely to be a limitation in the use of radiolabeled antibody therapy in multicyclic treatments (19) or in the combination of radiolabeled antibodies with external beam irradiation. We cannot say at this point how much of this tumor bed sparing is due to a dose-rate effect in the target
Volume 24, Number I. 1992
cells and how much is due to dose heterogeneity. The tumor bed effect appears to be larger following the 200 uCi QCI054 treatment than following the 100 uCi P96.5 treatment although both produced similar tumor doses. This difference might reflect the difference in total injected dose or a difference in tumor dose distribution. Begg and Terry reported an a/p of 6.7 Gy for the tumor bed effect in a mouse adenocarcinoma (2). Although an a/P could not be calculated for the external beam treatments, there was a clear tendency for an increase in effect with larger dose per fraction. In contrast to the regrowth delay, the 10 Gy single fraction produced the largest tumor bed effect. This result further suggests that in this system the regrowth delay depends mainly on the survival of the tumor cells, some of which were hypoxic at the start of treatment, but that regrowth of the tumor beyond the treatment size depends on the response of the well-oxygenated tumor bed. The differences in tumor volume doubling times shown in Figure 6 may contribute to the differences in regrowth to the treatment volume shown in Figure 3. If larger-fold increases in volume are used as the endpoint to measure growth delay, the results will depend more on differences in tumor bed effects and less on differences in killing of tumor cells. The increased specificity of absorbed dose delivery by radiolabeled antibodies is counterbalanced by decreased effectiveness when compared on a per dose basis with external beam therapy. Because of the constraints associated with irradiating normal brain, the limits of treatment of human gliomas by external beam photon therapy alone have probably been reached. Concurrent or sequential radiolabeled antibody therapy with external beam radiotherapy may allow increased tumor absorbed dose with relative sparing of surrounding normal brain.
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J.; Ruehl, W.; Finston, R.; Day-Lolline, P.; Goris, M. L. Determinants of the antitumor effect of radiolabeled monoclonal antibodies. Cancer Research 50:4935-4940;1990. Langmuir, V. K.; Sutherland, R. M. Radiobiology of radioimmunotherapy; current status. Antibody Immunoconj. Radiopharm. 1:195-211;1988. Lee, Y.; Bullard, D. E.; Zalutsky, M. R.; Coleman, R. E.; Wikstrand, C. J.; Friedman, H. S.; Colapinto, E. V.; Bigner, D. D. Therapeutic efficacy of antiglioma mesenchymal extracellular matrix 13 I -1odinsradiolabeled murine antibody in a human glioma xenograft model. Cancer Res. 48:559566;1988. Lee, Y.; Bullard, D. E.; Humphrey, P. A.; Colapinto, E. V.; Friedman, H. S.; Zalutsky, M. R.; Coleman, R. E.; Bigner, D. D. Treatment of intracranial human glioma xenografts with 13 1-I-labeled anti-tenascin monoclonal antibody 8 lC6. Cancer Res. 48:2904-2910;1988. Leichner, P. K.; Yang, N. C.; Frenkel, T. L.; Loudenslager, D. M.; Hawkins, W. G.; Klein, J. L.; Order, S. E. Dosimetry and treatment planning for 90-Y labeled antiferritin in hepatoma. Int. J. Radiat. Oncol. Biol. Phys. 14:1033-1042;1988. Malaise, E. P.; Fertile, B.; Deschavanne, P. J.; Chavaudra, N.; Brock, W. A. Initial slope of radiation survival curves is characteristic of the origin of primary and established cultures of human tumor cells and fibroblasts. Radiation Res. 111:319-333;1987. Marin, L. A.; Smith, C. E.; Langston, M. Y.; Quashie, D. V.; Dillehay, L. E. Response of glioblastoma cell lines to low-dose-rate radiation. Int. J. Radiat. Oncol. Biol. Phys. 21:397-402;1991. Order, S. E.; Stillwagon, G. B.; Klein, J. L.; Leichner, P. K.; Siegelman, S. S.; Fishman, E. K.; Ettinger, D. S.; Haulk, T.; Kopher, K.; Finney, K.; Surdyke, M.; Self, S.; Leibel, S. Iodine- 13 1 antiferritin, a new treatment modality in hepatoma: a Radiation Therapy Oncology Group study. J. Clin. Oncol. 3:1573-1582;1985. Palcic, B.; Skarsgard, L. D.; Reduced oxygen enhancement ratio at low doses of ionizing radiation. Radiat. Res. 100: 328-339; 1984. Ruifrok, A. C. C.; Levendag, P. C.; Lakeman, R. F.; Deurloo, I. K.; Visser, A. G. Dose-effect relation of interstitial lowdose-rate radiation (Ir19’) in an animal tumor model. Int. J. Radiat. Oncol. Biol. Phys. 18:31-36;1990. Salazar, 0. M., Rubin, P., Fieldstein, M. L.; Pizzutiello, R. High dose radiation therapy in the treatment of malignant gliomas: final report. Int. J. Radiat. Oncol. Biol. Phys. 5: 1733-1748;1979. Steel, G. G. Species-dependent growth patterns for mammalian neoplasms. Cell Tissue Kinet. 13:45 l-453; 1980. Steel, G. G.; Down, J. D.; Peacock, J. H.; Stephens, T. C. Dose-rate effects and the repair of radiation damage. Radiother. Oncol. 5:32 l-33 1; 1986.
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25. Thames, H. D. An “incomplete-repair” model for survival after fractionated and continuous irradiations. Int. J. Radiat. Biol. 47:319-339;1985. 26. Thames, H. D.; Hendry, J. H. Fractionation in radiotherapy. London, UK: Taylor & Francis; 1987. 27. van Putten, L. M.; KaIlman, R. F. Oxygenation status of a transplantable tumor during fractionated radiotherapy. JNCI 40:441-451;1968. 28. Walker, M. D.; Alexander, E.; Hunt, W. E.; MacCarty, S.; Mahaley, M.; Mealy, J.; Norell, H. A.; Owens, G.; Ransohoff, J.; Wilson, C. B.; Gehan, E. A.; Strike, T. A. Evaluation of BCNU and/or radiotherapy in the treatment of anaplastic gliomas. J. Neurosurg. 49:333-343;1978. 29. Wessels, B. W.; Vessella, R. L.; Palme, D. F. II, Berkopec, J. M.; Smith, G. Y.; Bradley, E. W. Radiobiological comparison of external beam irradiation and radioimmunotherapy in renal cell carcinoma xenografts. Int. J. Radiat. Oncol. Biol. Phys. 17:1257-1263;1989. 30. Williams, J. A.; Klein, J. L.; Wharam, M. D.; We&s, B. W.; Wanek, P. M.; Order, S. E. Radioimmunotherapy of human glioma xenografts in viva. Conference on Radioimmunodetection and Radioimmunotherapy of Cancer. Princeton, NJ, September 8-10, 1988. 31. Williams, J. A.; Klein, J. L.; Wharam, M. D.; Wessels, B. W.; Order, S. E. Targeting of human glioma xenografts in vivo utilizing radiolabeled antibodies (Abstr.). Int. J. Radiat. Oncol. Biol. Phys. 1S(Supp1. 1):2 15;1988. 32. Williams, J. A.; Klein, J. L.; Wharam, M. D.; Wessels, B. W.; Order, S. E. Quantitative intercomparison ofYttrium90 radiolabeled monoclonal antibodies targeting human glioma in vivo (Abstr.). J. Neurosurg. 70:3 12A; 1989. 33. Williams, J. A.; Edwards, J. A.; Klein, J. L.; Dillehay, L. E.; Wharam, M. D.; We&s, B. W.; Order, S. E. Targeting and therapy of human glioma xenografts in vivo utilizing radiolabeled antibodies. (Abstr.) Int. J. Radiat. Oncol. Biol. Phys. 17(Suppl. 1): 119; 1989. 34. Williams, J. A.; Wessels, B. W.; Wharam, M. D.; Order, S. E.; Wanek, P. M.; Poggenburg, K. A.; Klein, J. L. Targeting of human glioma xenografts in vivo utilizing radiolabeled antibodies. Int. J. Radiat. Oncol. Biol. Phys. 18: 1367-1375;1990. 35. Williams, J. A.; Edwards, J. A.; Wessels, B. W.; Dillehay, L. E.; Wanek, P. M.; Poggenburg, K. A.; Wharam, M. D.; Order, S. E.; Klein, J. L. Targeting and therapy of human glioma xenografts in vivo utilizing radiolabeled antibodies. Int. J. Radiat. Oncol. Biol. Phys. 19:633-642;1990. 36. Williams, M. V.; Denekamp, J.; Fowler, J. F. A review of (Y/Pvalues for experimental tumors: implications for clinical studies of altered fractionation. Int. J. Radiat. Biol. Oncol. Phys. 11:87-96;1985.
Ini J Rudiarwn Oncology BkJ Phw Printed in the U.S.A. All nghts reserved. .
0 Biology Original Contribution QUANTITATIVE COMPARISON OF RADIOLABELED ANTIBODY THERAPY AND EXTERNAL BEAM RADIOTHERAPY IN THE TREATMENT OF HUMAN GLIOMA XENOGRAFTS . JEFFERY A. WILLIAMS, Division
of Radiation
M.D.,
Oncology,
JAMES A. EDWARDS, B.S. AND LARRY E. DILLEHAY,
The Johns Hopkins
Oncology
Center,
600 N. Wolfe St., Baltimore,
PH.D.
MD 2 1205
Using “Yttrium radiolabeled antibodies, radioimmunotherapy was compared to fractionated external beam radiotherapy in the treatment of human glioma xenografts. Antibody treatments required administration of an approximately threefold greater total dose compared to external beam treatments to achieve the same tumor regrowth delay. Following multi-fraction external beam radiation treatments, tumor regrowth delay demonstrated a large fractionation effect (a/p = 2.3 Gy, 95% confidence limits 0.4-4.2 Gy), suggesting that much of the ineffectiveness of the antibody treatments could be caused by a large dose-rate effect in this system. Despite the large fractionation effect, the regrowth delay was small for a large single-fraction external beam irradiation, possibly because of tumor hypoxia. When compared to external beam radiation, radiolabeled antibody treatments resulted in a comparatively diminished tumor bed effect, suggesting radioimmunotherapy spares normal tissue surrounding the tumor. Radioimmunotherapy,
Glioma, Fractionated external beam, Xenograft, 9oY, Tumor bed effect.
INTRODUCTION Human malignant gliomas are rarely cured. Conventional postoperative external beam radiotherapy, however, extends median survival (28). When radiotherapy is conventionally fractionated, higher total doses yield further improved median, but not overall, survival (22). Hyperfractionated external beam radiotherapy improves neither median nor overall survival (5). More recently, radiolabeled antibodies have been used in the experimental treatment of human gliomas (4, 14, 15,30-35). P96.5, a murine IgG2a monoclonal antibody, specifies a neuroectodermally-derived cell surface antigen of molecular weight 97,000 (8) and targets experimental glioma in vivo (30-35). In nude mice bearing human glioma xenografts, intravenous administration of “Y-radiolabeled P96.5 causes striking accumulation of injected dose in tumor with resultant regression and growth delay (30-35). Such radiolabeled antibody therapy differs, however, from external beam radiotherapy in several important respects: 1. The dose rate to the tumor is both low and continuously decreasing after the initial tumor uptake ( 13, 16).
Reprint
requests
to: Larry E. Dillehay, Ph.D. work was supported by The Preuss Foundation for Brain Tumor Research, National Institute of Health grant PO1 CA4379 1, and Hybritech.
Acknowledgements-This
2. The total dose to the tumor is delivered over a comparatively short time (days rather than weeks) (19). 3. Both geometric and biological factors may render absorbed dose (Gy) non-uniform within the tumor. Absorbed radiation dose at the periphery of spherical tumors exhibiting uniform uptake of radiolabeled antibody may be less than the dose rate at the center (11). Differences in regional blood flow, antigen density or availability, or antibody specificity may cause nonuniform distribution of antibody and resultant inhomogeneity of tumor absorbed dose (6,9, 13). Despite these important differences, the comparative efficacies of external beam and radiolabeled antibody therapy have not been measured in the treatment of human gliomas. We therefore compared growth delay following varying regimens of 9oY radiolabeled antibody therapy or external beam radiotherapy in a human glioma xenograft-nude mouse model. METHODS
AND MATERIALS
Growth of human U-251 glioma xenografts in nude mice, labeling and biodistribution of ’’‘In and 9oY radiolabeled antibodies, dosimetry, and measurement of xe-
Accepted
for publication
3 February
1992.
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nograft growth delay following 9oY radiolabeled antibody treatments were performed as previously described (34, 35). Treatments were performed when tumors grew to 0.3-0.4 grams. External beam phantom dosimetry Tumor-bearing nude mice phantoms were constructed of polystyrene and mounted a fixed distance from the collimated 13’Cssource of a laboratory irradiator.* Using surface-mounted thermoluminescent dosimeters, the tumor phantom absorbed dose (Gy) per unit time of irradiation was measured. Human glioma xenograji therapy ‘oY-radiolabeled antibody therapy. Groups of at least nine tumor-bearing animals received intravenous injections of either 2% bovine serum albumin in phosphatebuffered saline (PBS), unlabeled P96.5, 100 uCi ‘oY-radiolabeled P96.5, or 200 uCi “Y-radiolabeled QC1054 (a monoclonal IgG anti-ferritin) as previously described (34, 35). External beam radiotherapy. Pairs of tumor-bearing nude mice were restrained in ventilated 50 ml conical centrifuge tubes and mounted in the 13’Csirradiator with tumor-bearing flanks facing the collimated source in a geometry identical to that used with the phantoms. The collimator shielded the animals anteriorly and posteriorly with 0.5 cm margins from the edges of the tumors. No collimation was used dorsally or ventrally, allowing irradiation of the inferior abdomen, rectum, and flank muscle and skin. Groups of at least four tumor-bearing animals received either no irradiation (control) or daily fractionated external beam irradiations. Tumor volumetrics Measurements of tumor length (L), width (W), and height (H) were made every 3 days and the product L X W X H, proportionate to tumor volume, was calculated. The logarithm of the ratio of tumor volume (V) to the initial tumor volume at the time of treatment (VCJ was plotted versus time for each animal as was the average of Log(V/V,,) for each treatment group. Tumor regression was defined as any decrease in an individual tumor volume below its initial value (V/V, < 1). The nadir in tumor volume for each treatment was defined as the lowest value of the average Log(V/VO). The delay in regrowth to the original volume (V/V, = 1) for each treatment was estimated from the plot of average Log(V/VO) versus time. The tumor bed effect (1) was quantitated by estimating the volume doubling time for the control tumors at the time of treatment and for the treated tumors at the time they regrew to V = VO.The three parameters of the Gompertz equation (23)
* Mark I, Model 68, J. L. Shepherd and Associates, San Fernando, CA.
Volume 24. Number I. 1992
Log V/V, = P,( 1 - eP(P2(t-P3))),
(Eq. 1)
were found which gave the best least-squares fit to the data for each tumor of Log(V/V,) versus time (t) for growth between V/V, = 1 and V/V, = 4. The tumor volume doubling time at V/V, = 1 was then calculated to be 0.3/(P1 *Pz). Some tumors could not be analyzed for the reasons indicated in Table I. RESULTS
In vivo thermoluminescent dosimetry Direct measurement of absorbed radiation dose in tumor by microthermoluminescent dosimetry revealed average absorbed doses (Gy) of 37.7 + 4.5 (s.e.m.) and 20.4 + 1.3 following administration of 100 uCi of T-radiolabeled P96.5 and QCIO54, respectively. These values were corroborated by the MIRD formalism of absorbed dose calculation (35). Human glioma xenograft therapy Radiolabeled antibody. Following intracardiac administration of buffer or unlabeled P96.5 (Controls) no tumors regressed, and the average time to grow to four times the initial volume was 6.1 -t 0.9 days. Following administration of 100 uCi “Y-radiolabeled P96.5, 9 of 10 tumors regressed, the average of Log V/V, reached a minimum at 57% of the initial treatment volume and subsequently reached the initial treatment volume 36 days after injection (Fig. 1 and Table 1). Following administration of 200 uCi “Y-radiolabeled QCI054, 7 of 9 tumors regressed, the average of Log V/V0 reached a minimum at 63% of the initial treatment volume and subsequently reached the initial treatment volume 28 days after injection (Fig. I and Table 1). Fractionated external beam radiotherapy. Figure 2 illustrates the growth of tumors following two of the external beam treatments. The treatment with a larger dose per fraction, three 4.5 Gy fractions, produced a larger regrowth delay than that with ten 1.5 Gy fractions, even though the total dose was smaller. The regrowth delay (time for the average of Log V/V, to return to 0) following each treatment is listed in Table 1. The time for regrowth to V/V, = 1 was also estimated for each tumor and the mean and standard error for each treatment (including antibody treatments) plotted versus the total dose in Figure 3. For treatments with the same dose per fraction, the regrowth delay increased with total dose. It can also be seen in Figure 3 that regrowth delays increased with increasing dose per fraction, except for the treatment with a single 10 Gy fraction. Comparison of radiolabeled antibody to external beam. In Figure 3 it can be seen that much larger doses were required to produce the same regrowth delay with anti-
113
Comparison of radiolabeled antibody and external beam therapies 0 J. A. WILLIAMS et a/.
Fig. 1. Effect of two radiolabeled antibody treatments on tumor growth. Each symbol is the average of Log V/V, for the tumors
Fig. 2. Effect of two fractionated external beam treatments on tumor growth. Each symbol is the average of Log V/V0 for the
in the group. Plus or minus the standard error is indicated by the vertical line whenever larger than the symbol.
tumors in the group. Plus or minus the standard error is indicated by the vertical line whenever larger than the symbol.
bodies as with the external beam treatments. We wanted to determine whether this ineffectiveness could be caused by a large fractionation/dose-rate effect, that is, that the antibodies effectively deliver irradiation in a large number of small fractions, which might be ineffective in this tumor. We first determined whether the response of the tumors to external beam could be described by the FDF formu-
lation of the linear-quadratic tumor-cell-kill model (26) of independently-acting fractions (complete repair of sublethal damage between fractions). In this model, for treatments that deliver a total dose D in fractions of size d, the response depends on the total effect, TE, defined by: TE = D*(cu/p + d).
(Eq. 2)
Table 1. Effects of treatments on tumor growth A. Radiolabeled antibody Tumor bed effect No. animals
Injection Control 100 uCi P96.5 200 uCi QUO54
11 10 9
Log V/V, at nadir
Days to nadir
Days to v/v, = 1
Td at v/v, = 1
-.24 -.2
28 18
0 36 28
2.8 4.4 7.9
No. animals 11 I* 7+
* Two tumors did not regress to V, and one tumor had not reached 4V0 at the end of the study. + One tumor did not regress to V0 and one animal died after Day 2 1. B. External beam Tumor bed effect Dose per fraction
No. fractions
No. animals
Log V/V, at nadir
Days to nadir
Days to v/v, = 1
Td at v/v, = 1
0 1.5 2 2 2 3 3 3 4.5 10
10 6 8 10 4 6 8 3 1
6 6 6 5 4 4 6 5 4 6
-.12 -.08 -.21 -.36 -.31 -.48 -.54 -.72 -.35
24 21 21 27 21 21 33 21 18
0 32 29 43 59 38 68 100 61 29
3.5 12 13 18 18 17 15 18 22
* One tumor did not regress to Vo. + One tumor had not reached 4V0. * None of the tumors in group had reached 4V0.
No. animals 6 5* 5* 5 4 4 5+ _* 4 6
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0-l 0
20
40
Total Dose (Gy)
Fig. 3. Growth delay as a function of total dose for two 9oYlabeled monoclonal antibodies and for different fractionated external beam treatments. Each point represents the mean of the individual tumor growth delay for each treatment and the vertical lines indicate plus or minus the standard error.
The parameter CY/~determines how the response changes with changes in dose per fraction. The method of Gutenberger et al. (7) was used to estimate the value of a/& It is assumed that the tumor growth delay, TGD, can be related to TE by the linking function: TGD = bl *(TE)b2.
(Eq. 3)
All the individual regrowth delay data were used in a nonlinear regression program? to determine the values of a/ p, bl, and bl which gave the best fit to Equation 3. A best-fit value of 3.7 Gy was obtained for LY/@If the model accurately described the data, then in a plot of the individual TGDs versus D*(3.7 + d), the individual points should be clustered about the line generated with the bestfit values of bl and b2 in Equation 3. When this was done, it was clear that the response for the single-fraction treatment was aberrant, being much lower than expected. When the analysis was repeated with only the multi-fraction (three or more) data, an a/p of 2.3 Gy was obtained (95% confidence limits; 0.4-4.2 Gy). Figure 4 shows a plot of TGD for the individual tumors that received multiple fractions (pluses) versus D* (2.3 + d). The multifraction points (pluses) do lie about the line generated by the best-fit parameters, suggesting that the linear-quadratic model with an a/P of 2.3 Gy does provide a good description of the fractionation-dependence of the responses to multi-fraction treatments. The TGDs for tumors receiving only the single-fraction treatment are also shown (Xs) in Figure 4. The single-fraction TGDs are less than would be expected from Equation 1, even if the upper limit of 4.2 Gy for (u//3is used. For continuous irradiation, the term d in Equation 2, associated with sublethal damage, is replaced by a term depending on the dose rate and rate of repair. In Figure
+ SAS Institute, Cary, NC.
Volume 24, Number I. 1992
4, we have also plotted the TGDs observed with antibodies assuming that this term is zero. Although there was considerable variability in the TGDs observed with antibodies, most of the points lay below (and to the right) the bestfit line. The antibody points would be even further to the right if sublethal damage contributed to the response. Thus, for an CY/~of 2.3 Gy, the poor response with antibodies can not all be attributed to expected dose-rate effects in this system. However, if the data is replotted with an a//3 of 1.O Gy, the antibody growth delays overlap the external beam growth delays. Since this is within the confidence limits on a//3, we cannot exclude the possibility that the poor response with antibodies seen in Figure 3 is attributable to a very large dose-rate effect in this tumor system. Tumor bed e&c&. To determine whether differences in damage to tumor stroma (tumor bed effects) were contributing to the observed differences in regrowth delay, the growth rate of each tumor at the time of return to VU was determined. Figure 5A shows the results for the tumors treated with antibody and Figure 5B shows the results for the same two external beam treatments as shown in Figure 2. The average doubling time at V0 for each treatment is given in Table 1. Figure 6 shows these average doubling times versus dose. For the external beam treatments, there was a general trend towards longer doubling times for larger doses per fraction, with the single fraction treatment producing the longest doubling time. However, the relatively large errors precluded an estimation of o(/P for this effect. The volume doubling times for the antibody-treated tumors were less than that for any of the external beam treatments.
lco-
50 -
70
90
110
130
D’(2.3+d)
Fig. 4. Growth delay for each tumor versus total dose times 2.3 plus dose per fraction. The pluses indicate tumors receiving multi-fraction treatments. The line indicates the best fit of these points to equation 3 (a/@ = 2.3 Gy). Xs indicate tumors receiving single-fraction treatment. Growth delays for tumors treated with labeled antibody are plotted (squares for p96.5, circles for QCI) assuming a dose per fraction of 0.
Comparison of radiolabeled antibody and external beam therapies 0 J. A. WILLIAMSefa/.
A . . . 0.
?? *
.I.
I
A
&A
*
.
.
.
??
.* .
.
Control
100 P96 5
200 cm
C0ntVd
10XlSGy
3X4 5Gy
4
I
Fig. 5. Tumor volume doubling times at treatment time for control tumors and at the time regrowing tumors exceeded V,,. A) For the two antibody treatments. B) For the same two external beam treatments as in Figure 2. Each point represents an individual tumor.
DISCUSSION There have been conflicting reports on the relative efficacy of radiolabeled antibody and external beam radiotherapies. Buchsbaum et al. (3) have observed in a human colon carcinoma xenograft model that to produce the same delay in growth to two times the treatment volume about five times as much tumor dose is required from an ‘3’I-radiolabeled antibody as from single fraction external beam treatment. This is roughly comparable to what we have observed in the human glioma xenograft
1A
I * ‘0
c 0 I 0
I
I
20 Total Dose
I
40 (GyJ
Fig. 6. Average tumor doubling times at V, versus total tumor dose. Plus or minus the standard error is indicated by the vertical line whenever larger than the symbol.
115
model with “Y-radiolabeled antibody for regrowth to the treatment volume. In contrast, Wessels et al. (29) and Knox et al. ( 12) reported greater responses for radiolabeled antibody than for external beam treatments. Wessels et al. (29) observed in a renal cell carcinoma xenograft model delays in growth to two or four times the treatment volume following ‘3’I-radiolabeled antibody treatments which were greater than following single or four fraction external beam treatments. Knox et al. ( 12) reported that treatment of a mouse B-cell lymphoma with 13’-labeled anti-idiotype monoclonal antibody was more effective than multi-fraction external beam given in 10 fractions over 2 weeks. These conflicting conclusions emphasize that meaningful comparisons of these two forms of radiotherapy will require a fuller (quantitative) understanding of the properties of each tumor system. Among the factors that could contribute to differences in responses to these two therapies are hypoxia, fractionation or dose rate effects, tumor bed effects, and dose distribution effects. The relative importance of these factors will depend on what measure of tumor response is used. All studies of radiolabeled antibodies versus external beam to date have been based on the delayed growth of treated tumors. The current study and that of Buchsbaum et al. (3) and Wessels et al. (29) used growth delay to measure tumor response. Knox et al. ( 12) used a cumulative measurement of changes in tumor size that extended only up to 12 days (the last day of multi-fraction treatment) that would favor any treatment (such as the antibody treatment) which would deliver the majority of the dose in the first half of the period. Comparison of single-fraction external beam to radiolabeled antibodies may be confounded by the presence of hypoxic cells which would render the tumor less responsive to the single-fraction treatment; the impact of these initially hypoxic cells is reduced by reoxygenation during fractionated treatments (27). In our study, the poor response with the single fraction stood out from the multifraction responses (Fig. 4). With radiolabeled antibodies, hypoxia is less likely to be a factor than with single-fraction because the bulk of the dose is delivered over several days, comparable to the duration of the shorter multifraction treatments, allowing for reoxygenation, and because the oxygen enhancement ratio decreases as the dose per fraction decreases (20). Thus, the poor response observed by us and by Wessels et al. (29) for single fraction might be because of tumor hypoxia. The effectiveness of radiation in killing cells generally decreases as the radiation is split into smaller fractions or the dose rate is lowered. The ‘incomplete repair model’ relates the killing of target cells for multifraction highdose-rate or continuous low-dose-rate treatments to the linear and quadratic components of killing by single highdose-rate treatments, characterized by the parameters (Y and p, respectively (25). The size of the dose-rate effect and the decrease in killing with decreased dose per fraction of high-dose rate radiation are inversely related to (Y/Pof
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I. J. Radiation Oncology 0 Biology 0 Physics
the target cells (24). We observed an CY/@ of 2.3 Gy for multifraction external beam treatments. This a/p value is smaller than has been generally observed for the responses of experimental tumors (36). However, U251 cells, like other glioblastomas ( 17) exhibit a large shoulder on their in vitro high-dose-rate survival curve (1 S), and would thus be expected to have a small a//3. Based on the small CY/&much and possibly all of the differences between fractionated external beam and antibody in delaying tumor regrowth in this system could be caused by a large dose-rate effect on survival of the tumor cells. For cells with a very large value of cu/p, we would expect, on the basis of the incomplete repair model, the efficacy of RIT to approach that of multifraction treatment for doses per fraction 4 a/& After clearance of dead cells and regrowth of the tumor from surviving cells, the growth rate of the tumor will depend on whether there is any long-lasting damage to the tumor stroma (1). In Figure 6 it can be seen that neither of the antibody treatments produced as large a decrease in the growth rate of the regrowing tumors as any of the external beam treatments. Thus, like physical implants (10, 2 l), radiolabeled antibody therapy appears to spare the tumor bed (and probably other surrounding normal tissue) relative to external beam treatments, although it should be pointed out that a larger ratio of normal tissue to tumor was irradiated in our external beam treatments than would be likely in a clinical situation. Nevertheless, our results suggest that irradiation of surrounding normal tissue from antibody concentrated in the tumor is not likely to be a limitation in the use of radiolabeled antibody therapy in multicyclic treatments (19) or in the combination of radiolabeled antibodies with external beam irradiation. We cannot say at this point how much of this tumor bed sparing is due to a dose-rate effect in the target
Volume 24, Number I. 1992
cells and how much is due to dose heterogeneity. The tumor bed effect appears to be larger following the 200 uCi QCI054 treatment than following the 100 uCi P96.5 treatment although both produced similar tumor doses. This difference might reflect the difference in total injected dose or a difference in tumor dose distribution. Begg and Terry reported an a/p of 6.7 Gy for the tumor bed effect in a mouse adenocarcinoma (2). Although an a/P could not be calculated for the external beam treatments, there was a clear tendency for an increase in effect with larger dose per fraction. In contrast to the regrowth delay, the 10 Gy single fraction produced the largest tumor bed effect. This result further suggests that in this system the regrowth delay depends mainly on the survival of the tumor cells, some of which were hypoxic at the start of treatment, but that regrowth of the tumor beyond the treatment size depends on the response of the well-oxygenated tumor bed. The differences in tumor volume doubling times shown in Figure 6 may contribute to the differences in regrowth to the treatment volume shown in Figure 3. If larger-fold increases in volume are used as the endpoint to measure growth delay, the results will depend more on differences in tumor bed effects and less on differences in killing of tumor cells. The increased specificity of absorbed dose delivery by radiolabeled antibodies is counterbalanced by decreased effectiveness when compared on a per dose basis with external beam therapy. Because of the constraints associated with irradiating normal brain, the limits of treatment of human gliomas by external beam photon therapy alone have probably been reached. Concurrent or sequential radiolabeled antibody therapy with external beam radiotherapy may allow increased tumor absorbed dose with relative sparing of surrounding normal brain.
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Br. J. Cancer 4l(Supl. IV):93-97;1980. 2. Begg,A. C.; Terry, N. H. A. The sensitivity of normal stroma to fractionated radiotherapy measured by a tumor growth rate assay. Radiother. Oncol. 2:333-341;1984. 3. Buchsbaum, D. J.; Ten Haken, R. K.; Heidorn, D. B.; Lawrence, T. S.; Glatfelter, A. A.; Terry, V. H.; Guibault, D. M.; Steplewski, Z.; Lichter, A. S. A comparison of 13’1labeled monoclonal antibody 17-IA treatment to external beam irradiation on the growth of LS 174T human colon carcinoma xenografts. Int. J. Radiat. Oncol. Biol. Phys. 18: 1033-1041;1990. 4. Bullard, D. E.; Gillespie, G. Y.; Mahaley, M. S.; Bigner, D. D. Immunobiology of human gliomas. Sem. Oncol. 13: 96-109;1986. 5. Deutsch, M.; Green, S. B.; Strike, T. A.; Burger, P. C.; Robertson, J. T.; Selker, R. G.; Shapiro, W. R.; Mealy, J.; Ranshoff, J.; Paolette, P.; Smith, K. R.; Odom, G. L.; Hunt, W. E.; Young, B.; Alexander, E.; Walker, M. D.; Pistenma, D. A. Results of a randomized trial comparing BCNU plus radiotherapy, streptozotocin plus radiotherapy, BCNU plus hyperfractionated radiotherapy, and BCNU following misonidazole plus radiotherapy in the post-operative treatment
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