interval between agents, was used asan example of a factor which may influence ... plot (envelope 1) as determined by the methods of Steel & Peckham (1979).
Br. J. Catncer (1980) 41, Suppl. IV, 294
TUMOUR SIZE: A FACTOR INFLUENCING THE ISOEFFECT ANALYSIS OF TUMOUR RESPONSE TO COMBINED MODALITIES D. W. SIEMANN Froan the Multimodality Research Section, University of Rochester Cancer Center, Box 704, 601 Elmwood Avenue, Rochester, New York 14642, U.S.A. Summary.- Isoeffect analysis of complete dose response curves can be used to study the interaction of agents in combined modality protocols. When such an analysis is applied to data from in vivo tumour model systems, the effects of the agents on factors such as tumour vasculature, growth or reoxygenation pattern also need to be considered. In this study the change in tumour size, which can occur during a long timeinterval between agents, was used as an example of a factor which may influence the position of data points on an isoeffect plot. Assays of in vivo tumour growth delay and in vitro clonogenic survival were performed to demonstrate that the radiation response curves of EMT-6/Ro tumours were size dependent. These curves were used to illustrate that data points obtained from a combined modality treatment may fall outside of the envelope of additivity of an isoeffect plot, as a direct consequence of tumour growth. This finding indicates that it may not be possible to interpret the results from isoeffect analyses of in vivo data on the basis of cellular interactions between agents, and suggests that instead isoeffect analyses be applied primarily to assess the overall response of the tumour system.
ONE objective of the use of radiotherapy in combination with chemotherapy is to attempt to achieve an improved therapeutic result through an enhancement of the tumour response. Of fundamental importance to assessing the effectiveness of such a combination is a means of analysing the nature of the interaction between the two agents. This problem has recently been extensively reviewed by Steel & Peckham (1979). These authors used the method of constructing isoeffect plots (isobolograms) from single agent response curves to define explicitly the concept of additivity. When performing experiments to carry out an isoeffect analysis, a range of doses of both agents must be given. Since tumours of different sizes will arise during a combined modality treatment involving a long time interval between agents, tumours which have grown during this time interval may be inherently more resistant to the second agent. Such sizedependent changes in sensitivity of in vivo tumour models have been reported
previously for both radiation and chemotherapeutic agents (Fu et al., 1976; Stanley et al., 1977; Stanley et al., 1978). The aim of the present study, therefore, was to examine the influence of changes in tumour size during the treatment interval on the isoeffect analysis of the interaction between agents. MATERIALS AND METHODS
Theoretical considerations.-Fig. 1 shows hypothetical tumour cell survival curves for agents A and X at a given tumour size and the resulting isoeffect plot at a cell survival level of 3 x 10-2 constructed using the methods described by Steel & Peckham (1979). If agents A and X are administered simultaneously (To) then data points lying inside the envelope of additivity (envelope 1) would be interpreted as indicating an additive cellular interaction between agents A and X. If the time interval between the two modalities is long, the range of doses of agent A (given at time zero) will result in tumours of varying sizes at the time of treatment with agent X. Thus, if the response to the second agent is size-dependent (i.e. a larger tumour
INFLUENCE OF TUMOUR SIZE ON ISOEFFECT ANALYSIS
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being more resistant) then the response to this agent at different tumour sizes must be considered for a clear understanding of the interaction between the agents. This is demonstrated by the dashed line in Fig. 1, representing a more resistant response to agent X and giving rise to the second envelope of additivity. Thus, data points resulting from an additive cellular interaction between agents A and X would apparently lie in the protective or sub-additive regions of the initial isoeffect plot (envelope 2 vs envelope 1) simply because of an inherent change in sensitivity due to a change in tumour size. This example indicates that tumour growth, during the time interval between agents, has made the interaction between the agents appear to be sub-additive or protective. Similarly, tumour shrinkage to a size that is more sensitive to the second modality, could lead to a third envelope of additivity, lying in the supra-additive region of the isoeffect plot. Changes in tumour size may, therefore, be an important factor making it difficult to interpret the nature of the interaction between agents in an isoeffect analysis. Because of these considerations, experiments were
performed to examine the influence of tumour size variations in the combined modality treatment of an animal tumour model. Tumour system.-EMT-6/Ro tumours, a subline derived from the original tumour characterized by Rockwell et al. (1972) were transplanted into the left calf of 8-14 week old BALB/cKa mice (Bio Breeding Labs, Ottawa, Canada) by injecting 2 x 105 cells intramuscularly. Irradiation and drug treatments.-The irradiation was given locally to unanaesthetized mice using a Cs137 source operating at a dose rate of 4-34 Gy/min, as previously described (Siemann et al., 1977). For anoxic irradiation the mice were either killed 10 min before irradiation or anaesthetized with sodium pentobarbitol (007 mg/g) and the blood supply to their tumour bearing legs occluded prior to and during irradiation. Both experimental conditions resulted in the same level of clonogenic tumour cell survival. Adriamycin was dissolved for 15 min in physiological saline and given as a single intraperitoneal injection as previously described (Siemann & Sutherland, 1980). Cetl survival assay.-To determine the
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number of clonogenic cells per EMT-6/Ro tumour, the animal was killed immediately after irradiation and a suspension of single cells was prepared using a combined mechanical and enzymatic procedure. Briefly, the enzymatic procedure involved separating the tumour cells in a collagenase, pronase, DNAase "cocktail" (Brown et al., 1979; Koch, private communication) for 30 min. Known numbers of cells then were plated and after 12 days colonies of over 50 cells were counted. Tumour growth delay assay.-After treatment tumour size was determined 3 times a week by passing the tumour-bearing leg through a series of increasing diameter holes in a plastic rod. This measurement was converted to a tumour weight using a calibration curve (Siemann & Sutherland, 1979). Growth delay was determined as the mean difference in time for the tumours of the control and treated mice to grow to 0-8 g. Similar results were obtained when median values were used instead of means.
longer time interval between agents led to a worse overall response but also implied that a sub-additive or inhibitive interaction between adriamycin and radiation had occurred. However, since the tumours had grown during the 120 h time interval, it was conceivable that the position of these combination data points could have resulted from size dependent changes in sensitivity to the second modality as discussed above. Therefore, the effect on the radiation response of an increase in tumour size from 0-2 g to 04 g (i.e. the size increase observed over 120 h after the lowest dose of adriamycin) was determined using both an in vitro clonogenic cell survival assay and an in vivo tumour growth delay assay. Single dose tumour cell survival curves for EMT-6/Ro tumours irradiated at various tumour sizes under aerobic or anoxic
RESULTS I
The effects of combinations of single and multiple doses of adriamycin and radiation on tumour response were studied (Siemann & Sutherland, 1979; Siemann & Sutherland, in preparation). Two sequential combinations of single doses of adriamycin and radiation were compared: (1) adriamycin followed 2 h later by radiation and (2) adriamycin followed 120 h later by radiation. The starting tumour size in these experiments was 0-2 g and complete tumour response curves for each agent were determined at this size. From these single agent response curves the additivity envelope (envelope 1; Fig. 2) for an isoeffect of 5-5 days of tumour growth delay was constructed. The combination data obtained for adriamycin and radiation separated by 2 h (A) were then plotted on this isoeffect plot. For this time interval all experimental data points were within the additivity envelope (1). However, when the adriamycin and radiation treatments were separated by 120 h all the data points ( A) were outside envelope 1 in the sub-additive region. These results indicated that the
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FIG. 4.-Tumour growth delay, following single doses of radiation for EMT-6/Ro tumours irradiated under aerobic or anoxic conditions. Each data point shown represents the mean difference in time for the tumours of 5-10 control and treated mice to grow to 0-8 g. Dashed lines indicate the possible region of the break in the tumour growth delay curve. an increase in tumour weight by a factor of 2 (from 0-2-0-4 g) can significantly alter the response of tumours irradiated under aerobic conditions even at very low doses. When additivity envelope 2 was calculated using the radiation dose response curve of 0-4 g tumours (the largest tumours following the lowest dose of adriamycin) all the combination data points (A) were within this enveope (Fig. 2). Ideally, additivity envelopes should be constructed at each tumour size. However, this example clearly indicates that the greatest deviation from the original additivity envelope will occur at the lowest dose of the first agent (which allows the maximum growth). The present findings imply that the interaction between adriamycin and radiation is, after all, additive for both the short and long time interval between agents when changes in tumour size (due to tumour growth) are accounted for.
conditions are shown in Fig. 3. A straight line has been fitted by linear regression to each set of data for doses > 12.5 Gy and the slopes were not found to be significantly different (P < 0.05). The slope of the anoxic data gives a Do= 5-67 + 0-58 Gy ( ± s.e.), with no differences in cell survival for tumours ranging from 0 1-1'0 g. However, under aerobic conditions the survival ratio at 20 Gy indicates an estimated hypoxic fraction of 6% for 0 1-02 g tumours and 20% for 0 3-0-5 g tumours This hypoxic fraction increases to 30-40% when tumours are 0 8-1 0 g (data not shown). Fig. 4 shows growth delay as a function of radiation dose for 0-2 g (V) and 0-4 g (0) tumours irradiated under aerobic conditions in unanaesthetized mice, and DISCUSSION 0-2-0-4 g (0) tumours irradiated under clamped (anoxic) conditions in anaesthetiThe hypothetical example described zed animals. As in the cell survival study, above shows that an increase in tumour
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size during the treatment interval to a state inherently more resistant to the second agent could result in combination data points lying in the sub-additive regions of an isoeffect plot calculated at the initial tumour size (Fig. 1). Such a result was observed in experiments assessing the interaction between adriamycin and radiation in the EMT-6/Ro tumour model (Fig. 2; Siemann & Sutherland, in preparation). Clearly in theory and in practice the longer time interval between agents leads to a "worse" (sub-additive or inhibitive) overall response. Yet, the combination data points lying to the right of the initial additivity envelope do not necessarily signify inhibition or antagonism between the agents. Instead, this may arise strictly as a consequence of tumour growth during the treatment interval. In Fig. 2 the position of the combination data is probably due to an increase in the fraction of hypoxic tumour cells (Fig. 3). Clearly tumour size variation is only one of many important variables which may change in vivo during a combined modality treatment protocol. Other factors may include changes in tumour vasculature, reoxygenation etc. Although it may at times be possible to eliminate some of these factors, they make it extremely difficult to use in vivo analyses to assess or interpret the nature of the interaction between agents at a cellular or molecular level. In general, it appears that
isoeffect analyses of in vivo data must be carefully considered and probably only interpreted as assessing the overall effect or total response of a tumour system. This work was supported by NIH grant CA20329. The author thanks Mr D. Kirkpatrick for his technical assistance and Drs R. P. Hill, E. M. Lord, D. B. Plewes, R. M. Sutherland and K. T. Wheeler for their discussions and constructive criticisms of the manuscript.
REFERENCES BROWN, J. M., YU, N. Y. & WORKMAN, P. (1979) Pharmacokinetic considerations in testing hypoxic cell radiosensitizers in mouse tumours. Br. J. Cancer, 39, 310. FU, K. K., PHILLIPS, T. L. & WHARAM, M. D. (1976) Radiation response of artificial pulmonary metastases of the EMT6 tumor. Int. J. Radiat. Oncol. Biol. Phy8., 1, 257. ROCKWELL, S. C., KALLMAN, R. F. & FAJARDO, L. F. (1972) Characteristics of a serially transplanted mouse mammary tumor and its tissueculture-adapted derivative. J. Natl Cancer In8t., 49, 735. SIEMANN, D. W., HILL, R. P. & BIUSH, R. S. (1977) The importance of the pre-irradiation breathing times of oxygen and carbogen (5% CO2 95% 02) on the in vivo response of a murine sarcoma. Int. J. Radiat. Oncol. Biol. Phy8., 2, 903. SIEMANN, D. W. & SUTHERLAND, R. M. (1980) In vivo tumor response to single and multiple exposures of adriamycin. Eur. J. Cancer (submitted). STANLEY, J. A., PECKHAM, M. J. & STEEL, G. G. (1978) Influence of tumour size on radiosensitization by misonidazole. Br .J. Cancer, 37, 220. STANLEY, J. A., SHIPLEY, W. U. & STEEL, G. G. (1977) Influence of tumour size on hvpoxic fraction and therapeutic sensitivity of Lewis Lung tumour. Br. J. Cancer, 36, 105. STEEL, G. G. & PECKHAM, M. J. (1979) Exploitable mechanisms in combined radiotherapy-chemotherapy: the concept of additivity. Int. J. Radiat. Oncol. Biol. Phy8., 5, 85.
