Europ. J. Canter Vol. 9, pp. 853-857. Pergamon Press 1973. Printed in Great Britain
RBE Values for Regrowth of C3H Mouse Mammary Carcinomas after Single Doses of Cyclotron Neutrons or X-rays J. F. FOWLER,* JULIANA DENEKAMP* and S. B. FIELD t *Canter Research Campaign, Gray Laboratory, Mount Vernon Hospital, Northwood, Middlesex, and t Medical Research Cyclotron Unit, Hammersmith Hospital, London, W.12, England Abstract--RBE values were obtainedfrom the delay in regrowth of first-generation transplanted mammary carcinomas after irradiation with single doses of X-rays or neutrons. A decrease of R B E with increasing dose to a minimum value of 2.6+0.6 at about 600 rads of neutrons was found. The results agree with those obtained previously for delay in regrowth and alsofor local control of tumours in multi-fraction experiments using small dosesper fraction. The RBE valuesfor this carcinoma were not significantly greater than RBE' sfor skin reactions at low dose~perfraction, as was predictedfrom their shrinkage pattern after large doses of X-rays and their known reoxygertation kinetics.
INTRODUCTION
It was considered advisable to investigate further whether the therapeutic advantage was indeed so small as to bc difficultto demonstrate in these turnouts, using a different design of experiment. In the present experiments the effects of singlesmall doseswcrc thereforeinvestigated,for comparison with the multiple exposures of about the same size of dose per fraction (as in paper I). A wider range of doses can bc used in rcgrowth than in tumour control experiments and resultsarc obtained in a shorter time. A n increase of R B E with de~e~ing dose per fraction is expected because of the relatively smaller intraccllular repair capacity after neutron irradiation, than after X-irradiation [2, 3]. In addition, for partially hypoxic tissues,such as tumours, at higher dose levels an increase of R B E with itwreoJ'ingdose per fraction is expected as the response of the hypoxic component becomes predominant.
THE PRESENTexperiments were carried out to compare RBE values obtained from tumour regrowth with those from local control of turnouts at 150 days, previously reported for single and fractionated exposures of cyclotron neutrons or 250 kV X-rays (1, designated paper I). In those experiments, 5 fractions of X-rays or neutrons were given in 9 days and the RBE values were found to be similar both for turnout control and for the production of skin reactions. Each neutron dose was about 330 fads. This result suggested that neutrons offered no therapeutic advantage over X-rays when used in this fractionation schedule for the treatment of C a l l mouse mammary tumours. When 9 fractions were given in 18 days however, each neutron dose being about 180 fads, the RBE for tumour control was 1.2 + 0.2 times greater than for skin reactions. This fractionation schedule, employing smaller neutron doses per fraction, may therefore have some therapeutic advantage, although the advantage was not statistically significant.
MATERIAL AND M E T H O D S
First-generation transplants of spontaneous mammary turnouts in male and female C3H mice from our inbred colonies were used as described in detail in Paper I (1). Two sets of
Accepted 12 October 1973. 853
854
J . F. _Fowler, Juliana Denekamp and S. B. Field
experiments were done, the first using 98 C allH e l l mice (obtained from the M R C Radiobiology Unit at Harwell in 1964 and bred at the Royal Postgraduate Medical School, Hammersmith Hospital), the second using 160C3H-He mice (obtained from M R C Carshalton in 1970 and inbred at the Gray Laboratory). In the first set two transplants were used and in the second set of experiments mice were drawn from 16 transplants. The techniques used for local irradiation of the tumours were as described in Paper I; but for the second set of experiments a 240 kVcp Pantak X-ray machine was used with the same filtration (0.25 m m Cu + 1 m m A1) and H V T (1.3 ram), giving a dose rate of 230 rad/min instead of 180rad/min. The scattered X-ray dose at the centre of the mouse's body was measured as 22 rads per kilorad received by the tumour. During irradiation all mice were surrounded by oxygen warmed to 25 + I°C as in Paper I, so that the results could be compared with those of Hill et al. [14]. Three perpendicular diameters of each tumour were recorded 2-5 times per week until they reached 6-7 m m average diameter when they were irradiated, and thereafter until the mean diameter of a tumour reached 12-15 mm, when the mouse was killed for ethical reasons. The method of using delay in regrowth as an end-point was as described by Thomlinson [4] and Thomlinson and Craddock [5]. The growth rates before and after irradiation and the delay in regrowth caused by a given X-ray dose were similar in the two sub-strains of mice.
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IUi~SULT$ Figures 1 and 2 show the tumour growth curves plotted as mean diameters versus time after irradiation for each of the nineteen dose groups including unirradiated controls, for the Hammersmith Hospital and Mount Vernon Hospital experiments respectively. Representative standard errors of the mean diameter for a group of mice are shown as vertical bars. They were calculated for each measurement day but are not all shown in the figures. The numbers of tumour-bearing mice in each dose group are shown in brackets. Where a fraction less than unity appears (e.g. 9/10 for 634 rads of neutrons) the regrowth curve shown is for 9 of the I0 tumours irradiated, because one grew so rapidly that it was considered as significantly different from the remainder of the group and the mouse was excluded from the analysis. Values for RBE (i.e. ratio of doses to give the same effect), with approximate estimates of the
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errors, can be obtained by finding the time at which the mean diameter of the regrowing tumours reaches an arbitrary size [6, 7]. Two sizes were chosen for analysis in the present work: 7.8 m m (250 m m a) and 9.8 m m (500 mmS). Figure 3 shows a graph of the times at which the mean diameters reached 9.8 m m plotted against dose. The vertical error bars represent the intersections with the 9.8 m m abcissae of envelopes drawn through the ends of all the standard error bars on the regrowth curves. They are thus only approximately 70% confidence limits and are not necessarily symmetrical. The agreement between the first and second sets of experiments is good except for the 390 rad neutron point, so that the first set of experi-
R B E Valuesfor Regrowth o f C 3 H Mouse Mammary Carcinomas
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ments involving 98 mice would by itself have been inadequate. Curves have been drawn by eye between the data points. The deviations in Fig. 3 represented by the dotted lines suggest break-points or plateaux at doses consistent with 10% of hypoxic cells (see discussion), but because of the scatter of the data they cannot be considered significant. In Fig. 4 R_BE values derived from Fig. 3 (and from a similar plot where regrowth to 7.8 m m diameter was used) are plotted against neutron dose per fraction as proposed by Field [8]. The vertical lines show the range of RBE values obtained graphically, in the absence of a a rigorous statistical test for such data. Envelopes were drawn through the ends of the error bars in Fig. 3, and from these the range of (D REGROWTH 10 7.O~am (~ 9.8mm
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Fig. 4. R B E vs neutron dose. Circles show the present results. The error bars represent the whole range of spread of experimental values. In addition the following data from (1) Paper I are shown: • = 90% probability of tumour control at 150 days; • = 50% probability of contrvl (vertical line gives standard error); • = 10% control. Thick vertical columns show range of values obtainedfrom regrowth curves in Paper L The data from Paper I are shown for single neutron doses (on right) and for 9F]18d and 5F]9d (on leJ'~). Dotted line: RBE for skin reactions in several species [9].
855
doses to produce a given regrowth delay was expressed as a percentage of the mean dose. A "root mean square standard error" of the RBE value was obtained from the square root of the sum of the squares of these percentages for corresponding neutron and X-ray doses. This gives a "maximum likely range" of values of R.BE, and is similar to the range obtained by dividing the maximum X-ray dose by the minimum neutron dose and vice versa. In Fig. 4 the RBE results from Paper I are also plotted against the neutron dose per fraction used in those experiments. The RBE's from the 10, 50 and 90% probabilities of tumour control are shown; also the ranges of RBE obtained from regrowth of those tumours which recurred. The groups of RBE's for the two low doses shown are from the 9-fractionsin-18-days and 5-fractions-in-9-days schedules respectively. Good agreement is demonstrated between the RBE results for multiple small fractions reported in Paper I and for the single doses of similar size used here. For a normal-tissue comparison, RBE values for skin reactions in several species of mammal are shown dotted in Fig. 4 (from Fig. 2 in [9]). DISCUSSION
No discrepancies between the RBE's for tumour growth delay and local control have been found in our results using this mammary carcinoma either with recurring tumours in the previous experiments (Paper I) or with similar tumours given smaller single doses in the present work. Field et al. [6] found a similar close agreement of RBE's measured by regrowth or local control in the rat fibrosarcoma RIBs; Barendsen and Broerse [10, 11] however did not, in their rat rhabdomyosarcoma. The variance in results is probably somewhat greater in this first-generation transplant system than in serially transplanted tumours, especially for small single doses. It appears that if more than one transplant has to be used, then a large number as in the second set of experiments gives more reliable results than just two transplants, probably because of the difficulty of ensuring an even distribution into dose groups. A recent estimate of RBE obtained by comparing the rate of loss of 12sIUdR-labelled tumour cells in vivo has confirmed the present rather low values of RBE for neutron doses of 400 to 600 rads in these C3H mammary tumours (Begg and Fowler [12] using a method developed by Pittner et al., [13]). Hill et al. [14], using oestrogen-induced mammary carcinomas in castrated male C3H
856
J. F. Fowler, Juliana Denekamp and S. B. Field
mice of a different substrain and a modified form o f t u m o u r regrowth analysis, found RBE's of 2.6 and 2.3 for neutron doses of 1600 and 560 rads respectively, rising again to 2.8 at 300 rads (RBE's corrected from electron to X-ray doses by the factor 0.85). These values are in good agreement with the present results, but they did not investigate small single doses in detail. A minimum value of RBE in Fig. 4 occurs between 500 and 700 rads of neutrons. This indicates the dose at which the response of the hypoxic cells in the tumours begins to be significant as the dose is increased; it is consistent with a proportion of 5-10% of hypoxic cells present before irradiation, assuming a conventional two-component cell survival hypothesis with Do = 130 rads, Dq = 400 rads and O E R = 3 for the X-ray response. A similar estimate of about 10% comes from separate experiments on local control of tumours for 150 days: the difference in doses required to control 50% of tumours irradiated breathing warmed oxygen (as here) or clamped-off to make them anoxic being about 800 rads. Both estimates depend to some extent on the values assumed for the parameters, but for the same assumptions regrowth and control give consistent results. Figure 5 shows the same results as in Fig. 4 plotted against the reciprocal of neutron dose. This was suggested by Alper [15], who showed that the slope ADq of the steepest part of such curves, to the right of the trough in Fig. 5, is an indication of the minimum size of the shoulder of the survival curve for those cells RBE
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Fig. 5. R B E vs reciprocal of neutron dose. Circles show present results, as in Fig. 4. Squares and triangles show tumour control results and vertical columns the regrowth results from Paper I, as in Fig. 4. Dotted line : R B E for skin reactions, as in Fig. 4.
when irradiated with X-rays. However, the analysis is only valid for RBE values derived from the exponential region of survival curves, i.e. for doses of neutrons greater than about 300 fads. Also an error is introduced by the presence
of hypoxic cells; in the present case this is apparent for doses greater than about 500 rads. Thus ADq can only be derived for neutron doses between about 300 and 500 rads and will therefore be very imprecise. If doses outside these limits are used, this will tend to cause the estimate of A a q tO be less than the true value. No useful value of ADq is obtainable. It can be seen in Figs. 4 and 5 that the present single-dose RBE's are slightly greater than those for the five fractions in 9 days (Paper I) by about 10%. Although this difference is not significant it might represent a small amount of hypoxia affecting the single-dose experiments, whilst sufficient reoxygenation occurs during the 5-fraction-in-9-day schedules to give a reduction in tumour RBE. With nine fractions in 18 days the multi-fraction RBE was equal to or 10% higher than that for a single dose, implying inadequate reoxygenation with the smaller X-ray dose-fractions used. Inadequate reoxygenation was also found for two and five daily fractions in the rat fibrosarcoma RIB s by Field et al. [7]. The lack of therapeutic gain with neutrons (the tumour RBE's being similar to those for skin) for small doses per fraction [15a] is, in our opinion, correlated with the histology of the tumours and their immediate shrinkage after single doses of X-rays. Experimental carcinomas in general shrink rapidly after a substantial single dose of X-rays [17] and these tumours are no exception. This behaviour may correlate with a better blood supply than in sarcomas [16]. If shrinkage occurs sufficiently early during the course of the fractionated treatment, more reoxygenation is likely to occur [18]. In other histological types of experimental tumour, however, neutrons have been found to be advantageous even at fairly small doses per fraction, giving higher RBE's for delay in tumour regrowth than for injury to skin. Examples are the rat fibrosarcoma RIBs (7) and another sarcoma (type F) in CBA mice which has 50% of its cells hypoxic and shrinks less readily after irradiation than the C3H mouse mammary carcinomas [20]. Acknowledgements--We have pleasure in thanking Miss Lily Huschtscha, Miss Anthea Page, Mr. P. W. Sheldon, Miss Susan Harris and Mr. K. Butler for their skilled assistance; Mr. D. D. Vonberg and the cyclotron staff for providing the neutrons; Dr. E. W. Emery and staff of the Medical Physics Department in the Royal Postgraduate Medical School, Hammersmith Hospital for facilities for the first set of Xirradiations; Mr. F. S. Stewart and Dr. B. D. Michael for the second; and the M.R.C. and C.R.C. for support.
R B E Values for Regrowth of C3H Mouse Mammary Carcinomas REFERENCES 1. J. F. FOWLER,J. DENEKAMP,A. L. PAGE, A. C. BEGO, S. B. FIELD and K. BUTLER,Fractionation with X-rays and neutrons in mice: response of skin and C3H mammary tumours. Brit. d. Radiol. 45, 237. 2. G. W. BARENDSEN,H. M. D. WALTER,J. F. FOWLER,and D. K. BEWLEY, Effects of different ionizing radiations on human cells in tissue culture. Rad. Res. 18, 106 (1963). 3. J. F. FOWLERand R. L. MORGAN, Pre-therapeutic experiments with the fast neutron beam from the Medical Research Council cyclotron. VIII: general review. Brit. J. Radiol. 36, 115 (1963). 4. R. H. THOMLINSON, An experimental method for comparing treatments of intact malignant tumours in animals and its application to the use of oxygen in radiotherapy. Brit. J. Cancer 14, 555 (1960). 5. R . H . THOMLXNSONand E. A. CRADDOCK,The gross response of an experimental tumour to single doses of X-rays. Brit. J. Cancer 21, 108 (1967). 6. S. B. FIELD, T. JONES and R. H. THOMLINSON, The relative effects of fast neutrons and X-rays on turnout and normal tissue in the rat. I--Single doses. Brit. J. Radiol. 40, 834 (1967). 7. S. B. FIELD, T. JONES and R. H. THOMLINSON, The relative effects of fast neutrons and X-rays on turnout and normal tissue in the rat. II---Fractionation: recovery and reoxygenation. Brit. J. Radiol. 41, 597 (1968). 8. S.B. FIELD, The RBE offast neutrons for mammalian tissues. Radiology 93, 915 (1969). 9. S. B. FIELD and S. HORNSEY,RBE values for cyclotron neutrons for effects on normal tissues and tumours as a function of dose and dose fractionation. Europ. J. Cancer 7, 161 (1971). 10. G. W. BAmZI~rOSENand J. J. BROE~E, Experimental radiotherapy of a rat rhabdomyosarcoma with 15 MeV neutrons and 300 kV X-rays. I--Effect of single doses. Brit. J. Cancer 5, 373 (1969). 11. G. W. BAmZ~OSENand J. J. BROE~E, Experimental radiotherapy of a rat rhabdomyosarcoma with 15 MeV neutrons and 300 kV X-rays. II--Effects of fractionated treatments, applied five times a week for several weeks. Europ. J. Cancer 6, 89 (1970). 12. A.C. B E ~ and J. F. FOWLER,A rapid method for the determination of tumour RBE. Brit. J. Radiol. In press. 13. W. PITTNER,W. PORSCHENand L. E. FEINENDEOEN,In vivo-Messung unterschiedlicher Strahlenempfindlichkeit yon Tumorzellen w~ihrend des Zellzyklnszehmarlderung mit x25i_Desoxyuridin" Strahlentherapie 145, 161 (1973). 14. R. P. HILL, P. J. CHESHIX~,P.J. LINDOPand S. B. FIELD,A comparison of the response of tumour and normal tissue in the mouse exposed to single doses of fast neutrons or electrons. Brit. J. Radiol. 43, 894 (1970). 15. ALPER, TIKVAH, Aspects of neutron therapy based on an analysis of relationships between RBE and dose. Brit. J. Radiol. 45, 39 (1972). 15a.J.F. FOWLER, Fast neutron therapy--physlcal and biological considerations. In Modern Trends in Radiotherapy, Vol. I, (Edited by T. J. DEELEYand C. A. P. WooD) pp. 145-170. Butterworths, London. 16. G. D. ZANELLXand J. F. FOWLER, 1973. Uptake of SeRb and 125IHSA in experimental tumours (submitted to CancerRes.). 17. J. DENEKAMP,The relationship between "Cell Loss Factor" and the immediate response to radiation in animal tumours. Europ. J. Cancer 8, 335 (1972). 18. J. DENEKAMP,Cell proliferation in rodent tumours. Ph.D. Thesis, University of London, p. 181 (1968). 19. A. E. HowEs, An estimation of changes in the proportions and absolute numbers of hypoxic cells after irradiation of transplanted C sH mouse mammary tumours. Brit. J. Radiol. 42, 441 (1969). 20. J. DEN~KAMP,The response of a mouse sarcoma to single and divided doses of X-rays and fast neutrons (submitted to Brit. J. Cancer).
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