Phys, 1978. Vol.
4. pp. 49-52.
Prmted m the U.S.A.
COMIBINED RADIOTHERAPY-CHEMOTHERAPY LUNG CARCINOMA G. GORDON STEEL, Ph.D., Department
RICHARD P. HILL, Ph.D. Research,
and MICHAEL J. PECKHAM, M.D.
Institute of Cancer Research Belmont, Surrey, England
Intramuscular implants of Lewis lung tumor have been treated with combinations of cyclophosphamide and local irradiation. The results were assessed in terms of long-term tumor control and early skin damage. The cyclophosphamide treatment reduced the radiation dose required for cure from about 4200 to 1200 rad, and it was best to give the two treatments within a day of one another. Radiation-induced skin damage was not increased by cyclophosphamide treatment, and these results therefore indicate that the combination has a useful therapeutic advantage. Radiotherapy, control.
modality therapy, Lewis lung carcinoma,
Skin damage, Local tumor
curves. The intricacy of this term lies in how one should define the “expected” result. If potentiation occurs equally in tumor and normal tissues, there will be no improvement in therapeutic index, but improvements could arise if the antitumor potentiation exceeds normal tissue potentiation. The present project was designed to examine the second of these mechanisms, although the results also demonstrate Spatial Cooperation of chemotherapy and radiotherapy. In discussion, two ancillary projects that were specifically designed to study Potentiation will be mentioned. The work described here was based upon the studies of Karrer, Humphreys and Goldin’ and Johnson3 which demonstrated that the Lewis lung tumor is useful in the study of combined modality treatment of a metastasising tumor.
There are various ways in which the combination of radiotherapy and chemotherapy can give better results than either treatment alone. These may be grouped into the following three categories: Spatial cooperation. When one agent fails to deal adequately with disease in some particular anatomical site, the addition of a second agent may lead to an improvement in patient survival. Perhaps the most important basis for combined modality therapy is to use radiotherapy for bulky local disease and chemotherapy for metastases outside the radiation fields. Independent toxicity. The addition of two agents that have largely non-overlapping toxicities may allow greater tumor cell k.ill even though the antitumor effects of the agents may not combine additively. If each agent can be given close to its maximum tolerated level for use alone, and provided the antitumor effects are not antagonistic, it should be possible for the combination to achieve greater tumor response within tolera‘ble toxicity than for either agent used alone. Potentiation. This tenm is difficult to define briefly, but is used here to describe the situation in which treatment with one agent increases the sensitivity of a tissue to a second agerrt. If this occurs, the effect of the combination will be greater than the result expected on the basis of the single-agent dose-response
The Lewis lung tumor has been maintained by subcutaneous and intramuscular passage in C57 Black mice of the Institute of Cancer Research colony for the past 6 years. In these mice it has been found to display only weak antigenicity.* For the experiments reported here, the tumor was chopped finely and reduced to a mush by forcing it through needles of decreasing diameter. The mush was washed and mixed with 10 vols of tissue culture medium, and then
Acknowledgements-We are grateful for the support and encouragement of Prof. L. F. Lamerton and the technical
of Kay Adams.
Radiation Oncology 0 Biology 0 Physics
0.02 ml was injected into the gastrocnemius muscles of the left hind legs of recipient mice. All tumors grew, and from such inocula they began to produce a noticeable swelling about 6 days later. Irradiation was performed using a 1500 Ci source of ‘?Zo y-rays. The source was in the form of a vertical cylinder, 15 cm long and 9.5 mm dia., which could be wound down a loading tube into the centre of a well-shielded room. Mice were anaesthetised with nembutal and secured face-downwards on Perspex platforms using pieces of sticking-plaster over each hind foot. The tumor in each hind leg was accurately located over a 1 cm circle that was inscribed at the same point on each platform. The platforms fitted together in vertical stacks of 5; interlocking legs gave a constant inter-platform distance of 2 cm and kept each stack accurately vertical. It was possible to irradiate two such stacks at a time. For irradiation the inscribed circles were located at 25 cm from the centre of the source. The mice were shielded behind 13.5 cm of lead, as shown in Fig. 1. The lead was accurately positioned so that one face was in line with the centre of the source. A smaller lead toeguard was provided to reduce damage to the foot, and scattered radiation was reduced by a wedge of lead on each platform which also served to keep the body of each mouse well out of the radiation field. Build-up of radiation dose was secured by a 3 mm Perspex filter. Body
1978, Vol. 4, No. I and No. 2
Table 1. Skin reaction
+ ++ +++
2.0 2.5 3.0
Only the highest score was recorded. The reactions became noticeable towards the end of the second week after irradiation, reached a peak between 20 and 25 days, and then gradually declined. In each experiment all the mice were scored on the
same day, at 2-3 day intervals, and the mean reaction score between days 14 and 31 was taken as the measure of early skin response. The time-course of tumor recurrences was such that most tumors recurred within 40 days. The proportion of cures was assessed at 60 days post treatment, as was also done by Karrer and Humphreys.4 Mice lost without tumor before this time were excluded from the experiment. RESULTS In experiment 1, mice were selected at 7 days after tumor implantation and graded doses of irradiation were given. Cyclophosphamide (300 mg/kg intraperi-
toneally) was given on day 18, as was done by Johnson.3 The tumor control and skin reaction data are shown in Fig. 2. The TCDso (radiation dose for 50% tumor control) was roughly 4200 rad and there was a
Tumor over locating circle
Fig. 1. Diagram of irradiation set-up. This is a plan view showing the main lead shields (15 cm tall) behind which was a stack of 5 Perspex plates, one of which is shown, to each of which was strapped one mouse. The body guards were 1.5 cm tall, located on each plate by grooves. Dose-rate measurements were made using a calibrated Baldwin-Farmer dosimeter. The dose-rate at the tumor was 300-350 rad per min (decreasing during the course of the experiments). The dose to the mouse abdomen was less than 1.5% of the tumor dose. The early skin reaction over the exposed limbs was evaluated using an arbitrary scoring system, similar to that described by Field.’ This is outlined in Table 1.
Radlatlon dose, rod
Fig. 2. Proportion of tumors controlled and mean skin reaction in an experiment in which irradiation was performed at day 7 postimplantation and cyclophosphamide (300mg/kg) was given at day 18.
considerable dose-sparing effect of cyclophosphamide administration: approximately 1300 rad. In contrast, there was no apparent influence of cyclophosphamide on the radiation-induced skin damage; it would be possible to put a smooth curve through the skin reaction data as a function of radiation dose, irrespective of cyclophosphamide dosage, and at the doses that were given with and without cyclophosphamide the skin reactions were almost identical. In a series of subsequent experiments the timing of the administration of radiation and cyclophosphamide was varied. The cyclophosphamide dose was kept at 300 mg/kg, and in all cases the first treatment was at 7 days postimplantation. When radiation and cyclophosphamide were given on the same day, the drug was given l-2 hr after the y-rays. Figure 3 shows the results of these experiments. A line has been drawn to indicate the approximate locus of the 50% tumor control probability.
G. G. STEEL et al.
administration was far from optimum. Results were always worse when, for any given radiation dose, the cyclophosphamide administration was delayed; when irradiation was delayed the effect was less serious. In order to check the normal tissue reactions associated with short intervals between radiation and cyclophosphamide administration, the final experiment consisted of six groups of non-tumorbearing mice given 3, 4 or 5 krads of y-rays, with cyclophosphamide given 1 day before or one day afterwards. The results are shown in Fig. 4. The groups given drug before radiation always had the least skin reaction, but these results are probably again consistent with little effect of cyclophosphamide on radiation-induced skin damage.
Fig. 4. Skin reaction data for groups of mice given cyclophosphamide (3OOmg/kg) 1 day before or 1 day after irradiation.lJ,CY 1daybeforeR;A,CY 1dayafterR;O,noCY.
Fig. 3. Proportion of tumors controlled for various radiation doses and intervals between radiotherapy and chemotherapy (300 mg/kg cyclophosphamide). Each symbol represents the response of 10 tumor bearing mice, 7 days postimplantation at the time of the first treatment; the black segment indicates the proportion of tumors controlled at 60 days. It is clear that the timing of administration of cyclophosphamide had an important influence on the radiocurability of these tumors and that best results were obtained when the treatments were given close together. The one experiment in which radiation doses of 15OOrad were employed showed a smooth trend in favour of radiation being delivered after the cyclophosphamide. Th’e subsequent experiment with 5 groups centered on -2 days and 6 groups on 1000 rad showed no significant time-variation and implied a TCDK, of about 1400 ra.d. We conclude that the initial choice of an 11 day gap between radiation and drug
DISCUSSION Lewis lung tumors that are allowed to grow for 7 days after intramuscular implantation constitute a therapeutic problem that is readily solved by combined modality treatment. Although radiation alone is capable of sterilising the primary implantation site, many tumors have metastasised by this time (as shown by Hill and Stanley’) and chemotherapy is necessary to achieve a good level of tumor control. Cyclophosphamide is a very effective agent against Lewis lung tumors, but is nevertheless incapable of curing many palpable hind leg tumors in a single dose. The combination of a tolerated dose of cyclophosphamide with a modest dose of radiation to the primary site has enabled 100% tumor control to be achieved. This is therefore an example of what in the Introduction was called Spatial Cooperation of therapeutic modalities. It is of considerable therapeutic importance to know whether chemotherapy modifies the normal tissue damage produced by irradiation. We have so far only examined skin reaction, although ex-
Radiation Oncology 0 Biology
periments on lung damage are in progress. The present work has shown that whether cyclophosphamide was given 1 day before, I day after, or 11 days after irradiation, there was no significant exacerbation of skin damage. To this extent, these agents therefore also provide an example of what was called Independent Toxicity. When the chemotherapy was delayed after irradiation, a progressively higher radiation dose was required for 50% tumor control. The slope of the full line on the right hand side of Fig. 3 is approximately 190 rad/day. The work of Shipley, Stanley, Courtenay and Field6 has shown that the D,, of hypoxic cells in the Lewis lung tumor is 310rad. The requirement of 190 more rad per day in the interval between irradiation and chemotherapy is equivalent to a cell population doubling time of
= 1.1 days.
This is consistent with the known growth rate of small implants of this tumor.’ It implies that the dependence of tumor control on the time interval between irradiation and chemotherapy is explicable simply in terms of tumor regrowth in the interval. The doses of agent required for tumor control are also consistent with available data on the sensitivity of Lewis lung tumor cells to these agents. For combined treatment on the same day, we required 300 mg/kg cyclophosphamide and approximately
4. No. I and No. 2
1200rad of -y-irradiation for 50% tumor control. Cell survival studies6 have shown that this chemotherapy gives a surviving fraction of about 10m6 and in airbreathing mice 1200 rad gives a surviving fraction of 8 x lo-‘. If approximately one cell implant must be left behind in order to observe 50% tumor control, this implies that the combination would be able to deal with about 10’ cells. The tumors at 7 days postimplantation are just palpable and we estimate that they weigh approximately 0.1 g and contain about 6 X 10’ cells,’ is reasonable agreement with the implications of the cell survival data. There is nothing in the present data that would lead us to postulate potentiation or antagonism between these two agents in the order and with the timing that we have used. However, Stanley, Shipley and Steel’ have shown that when lung metastases of Lewis lung tumor are treated first with cyclophosphamide and then with radiation at the nadir of tumor significant reoxygenation occurred. This volume, was therefore an approach to combination treatment that in very small tumors gave useful therapeutic advantages. Our experiments have not seriously examined whether at shorter intervals than one day it is possible to detect therapeutic synergism, but parallel work in this laboratory by Stephens, Peacock and Steel’has failed to demonstrate short-term potentiation in the response of the B16 mouse melanoma to combinations of cyclophosphamide and vincristine or radiation and 5fluorouracil.
REFERENCES 1. Field, S.B.: Early and late reactions in skin of rats following irradiation with X-rays or fast neutrons. Radiology 92: 381-384, 1%9. 2. Hill, R.P., Stanley, J.A.: Pulmonary metastases of the Lewis lung tumor, their cell kinetics and response to cyclophosphamide at different sizes. Cancer Treatment Rept. 61: 29-63, 1977. 3. Johnson, R.E.: Combined chemotherapy and irradiation in Ewings’ sarcoma. Front. Radiat. Ther. Oncol. 4: 195202, 1969. 4. Karrer, K., Humphreys,
S.R.: Continuous and limited courses of cyclophosphamide in mice with pulmonary metastases after surgery. Cancer Chemother. Reps 51: 439-449, 1%7. 5. Karrer, K., Humphreys, S.R., Goldin, A.: An experimental model for studying factors which influence metastasis of malignant tumors. Int. J. Cancer 2: 213223, 1967.
6. Shipley, W.U., Stanley, J.A., Courtenay, V.D., Field, S.B.: Repair of radiation damage in Lewis lung carcinema cells following in situ treatment with fast neutrons and X-rays. Cancer Res. 35: 932-938, 1975. 7. Stanley, J.A., Shipley, W.U., Steel, G.G.: Influence of tumour size on hypoxic fraction and therapeutic sensitivity of Lewis lung tumour. &it. J. Cancer 36: 105-l 13, 1977. 8. Steel, G.G., Adams, K.: Stem-cell survival and tumor control in the Lewis lung carcinoma. Cancer Res. 35: 1530-1535, 1975. 9. Stephens, T.C., Peacock, J.H., Steel, G.G.: Cell survival in B16 melanoma after treatment with combinations of cytotoxic agents: lack of potentiation. Brit. J. Cancer 36: 84-93, 1977.