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Cellular responses determining the effectiveness of fast neutrons relative to X-rays for effects on experimental tumours
Europ..7. CancerVol. 7, pp. 181-190. Pergamon Press 1971. Printed in Great Britain
Cellular Responses Determining the Effectiveness of Fast Neutrons Relative to X-Rays for Effects on Experimental Yumours G. W. BARENDSEN
Radiobiological Institute TWO, 151 Lange Kleiweg, R~isw~k Z.H., The Netherlands Relations between the relative biological effectiveness of fast neutrons and the dose or daily dosefractions are presented for cultured cells, tumours and normal tissues. These relations are dependent on a variety orfactors, namely the neutron energy spectrum, the shapes of dose-survival curves, repah" of sub-lethal damage, the presence of anoxic cells, reoxygenation during intervals betweenfractions and the dependence of radiosensitivity on the age of cells in the# cycle. The relative influence of thesefactors is discussed. Comparison of the RBE values of fast neutrons derivedfrom published data for experimental tumours and normal tissues indicates that overall gain factors, RBE
INTRODUCTION THE AIM of a radiotherapeutical treatment is to administer radiation according to a schedule which, by the proper choice of various parameters, namely the spatial distribution of the dose in the patient, the sizes of the dose fractions, the intervals between fractions and the total dose, will just be tolerated by normal tissues while causing maximum damage to the turnout. In comparison with orthovoltage Xrays, the introduction of high energy X-rays and fast electrons has allowed considerable improvement to be made in the dose distribution in the patient. With fast neutrons this distribution presumably cannot be improved further, and this radiation can only provide an advantage if the relative biological effectiveness (RBE) is larger for damage to the tumour than the RBE for damage to the dose-limiting normal tissues. Values of the RBE may depend on the parameters mentioned, i.e. the sizes of the
dose fractions, the intervals between fractions and the total dose, as well as on the type of tumour and normal tissue irradiated and consequently a large amount of experimental and clinical data is required before possible advantages of fast neutrons can be adequately evaluated. In this paper the influence of a number of factors on the RBE will be discussed. Results of radiobiological studies, obtained during the past decade, have shown that radiation-induced damage to normal tissues and tumours in animals is determined to a large extent by responses of those constituent cells which are capable of unlimited proliferation, i.e. the clonogenic cells. Furthermore, responses of individual cells after irradiation in vivo have been shown with a number of experimental systems to be similar to responses of the same type of cells grown and assayed in vitro, provided various environmental conditions, e.g. oxygen concentration, are made equal.
181
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G. W. Barendsen
Investigations of cells cultured in vitro have demonstrated that a variety of factors can influence their radiosensitivity with respect to the capacity for proliferation, e.g. radiation quality, the distribution of dose in time, the presence of oxygen and other sensitizing compounds, the presence of protecting compounds, the proliferative state of the cells and their age in the cell cycle [1]. These factors, which in experiments in vitro have been studied in well controlled conditions, may cause large variations in the responses to fractionated irradiation of tumours and of normal tissues in patients, where cellular conditions are in general unknown and cannot be easily determined or controlled. Experimental data obtained with respect to the radiosensitivity of cells in culture, in experimental tumours or in various normal tissues, have often been used to emphasize similarities between the characteristics of dose survival curves of different types of mammalian cells. It should not be forgotten however that differences by a factor of 2-3 in Do values, which range from 70 to 200 rads, and differences in extrapolation numbers by at least a factor of 10, have been observed. These differences in intrinsic radiosensitivity of various types of mammalian cells may be augmented by certain conditions in turnouts or tissues due to some of the dose modifying factors mentioned and this can result in very large differences in the responses of these multicellular systems to a fractionated treatment [i]. In addition to factors influencing the radiosensitivity of individual cells, the responses of tumours and normal tissues after irradiation or during a fractionated treatment depend on the proliferative activity and changes in physiological conditions of those cells that have retained the capacity for unlimited proliferation. Irradiated cells will generally experience a phase of mitotic delay, after which proliferation is resumed. The rate of proliferation of parenchymal cells and the rate of removal of dead cells in irradiated tumours and in normal tissues m a y depend on various factors, which have not yet been sufficiently investigated, e.g. tumour architecture and the vascular system. Increased as well as decreased rates of cell production in comparison with pre-irradiation values have been observed in irradiated tumours, depending on the dose administered and the time interval after irradiation [2-7]. Furthermore changes in oxygenation status of cells have been demonstrated in tumours containing a fraction of severely hypoxie cells at the start of a treatment [6, 8-10]. Finally
the volume response of a tumour to irradiations may depend on factors concerned with the architecture of the turnout, e.g. the amount and type of stroma, the blood vessels and the surrounding tissues. As a consequence of all these factors which can cause variations in responses of tumours as well as of normal tissues, it is not inconsistent with radiobiological data that tumours in patients show large differences in their clinical radiosensitivity to fractionated treatments with X-rays or ~,-rays. The main problem in experimental tumour radiobiology is to evaluate the relative importance of the various factors mentioned and to detect systematic differences between responses of various types of tumours and various types of normal tissues. Comparison of the effectiveness of fast neutrons relative to X-rays for inducing damage to different types of cells, tumours and normal tissues, can provide data for this evaluation, because some of these factors play a smaller part with neutron irradiations. Dose-effect relations for X-rays and fast neutrons differ in two main aspects, namely a reduced shoulder of the survival curve for fast neutrons as compared with X-rays, with a consequent diminished contribution of repair of sub-lethal damage in fractionated treatments, and a relatively low oxygen enhancement ratio of fast neutrons, which does not depend greatly on neutron energy in the range between 5 and 30 MeV [11]. As a result of these differences, the relative biological effectiveness (RBE)* of a given dose of fast neutrons for effects on turnouts and normal tissues depends on the dose, the fractionation schedule, the presence of anoxic cells and on changes in oxygenation status. Finally, differences between effects of fast neutrons and X-rays have been observed with respect to the dependence of radiosensitivity on cell age in the division cycle. The influence of this factor may also depend on the dose fractionation schedule, because the degree of induced synchrony depends on the dose, and the desynchronization before the next dose is given depends on the time interval. All these factors, which can cause variations of the RBE of fast neutrons in dependence on the dose and dose fractionation schedule, have been listed in Table 1A. In part B of this table, three other factors have been listed which are important with respect to the final result of a *The RBE of a dose of neutron radiation is defined as the ratio of a dose of X-rays and the dose of neutron radiation required for producing quantitatively equal effects.
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The Effectiveness of Fast Neutrons Relative to X-Rays Table 1. Factors determining responses of tumours and normal tissues to single andfractionated irradiations i
Factors
Characteristics
A. Factors which influence the RBE of fast neutrons 1.
Neutron energy spectrum
2. Size of dose or dose fraction
RBE increases with decreasing neutron energy [11] RBE increases with decreasing dose, dependent on shapes of survival curves of cells [1]
3. Intra-cellular repair of sub-lethal RBE increases with decreasing dose fraction, ff damage
intervals between fractions are long enough to allow repair [1, 12, 13]
4.
Hypoxia of proportion of cells
Because (OER)neut . . . . *~ (OER)x-r~ys the RBE of neutrons is larger for damage to hypoxic cells [1, 4-6, 18]
5.
Reoxygenation of hypoxic cells Decreases the effect of hypoxic cells [5, 6, 8-10] during intervals between fractions
6. Dependence of radiosensitivity on This dependence is smaller for fast neutrons as cell age
compared with X-rays [14]
B. Factors which do not influence the RBE of fast neutrons 1. Proliferation of cells in intervals Rate can increase or decrease during and after between dose fractions
fractionated treatment [2, 3, 7]
2. Non-lethal damage
Causes death in part of the progeny of surviving cells and slow rate of regrowth of tumours [15, 16]
3. Mitotic delay
Influence is presumably small in practical treatment schedules
radiotherapeutical treatment, but which do not cause further variations of the RBE of fast neutrons in dependence on the fractionation schedule employed. The first of these factors, proliferation of cells in intervals between dose fractions reduces the effect of a given treatment, i.e. a larger dose is required in order to kill all cells. The limited experimental data available indicate that changes in the rate of proliferation and of cell loss do not depend on the type of radiation employed if doses are compared which cause equal damage to the proliferative capacity of the clonogenic cells [1, 5]. Nonlethal damage has also been demonstrated to be caused to an equal degree by different radiations provided doses are compared which cause the same degree of cell reproductive death. Mitotic delay, as far as indicated by the limited data available, is caused with the same RBE as cell lethality. Thus if a treatment with fast neutrons and a treatment with X-rays produce equal effects with respect to cell killing, then cell proliferation, impaired cell production due to non-lethal damage or mitotic delay will not cause other differences between the effects of these treatments with respect e.g. to the probability of cure or the growth rate of recurrent tumours.
RBE VALUES OF FAST NEUTRONS FOR REPRODUCTIVE DEATH IN CULTURED CELLS With in vitro systems, RBE values can be determined for ceils in well controlled conditions, whereby variations due to, e.g. partial hypoxia or changes in age distributions of the cells can be eliminated. Survival curves for cultured asynchronous mammalian cells of human kidney origin (T-1 g cells) have been determined with fast neutrons of different energies by the clone technique [11]. From these curves RBE values relative to 250 kVp X-rays have been derived for various levels of the neutron dose. The results are presented in Fig. 1. These curves illustrate some general features of the dependence of the RBE on the neutron dose and energy. The logarithmic scales are used because some fundamental radiobiological considerations indicate that relatively simple shapes of the curves might be obtained [ 17]. The curves of Fig. 1 show that the largest RBE values are derived for the lowest neutron energy and conversely. The mean value of "10 M e V " assigned to the neutron spectrum obtained by bombarding Be with 20 MeV SHe-ions, is quite arbitrary because the spec-
184
G. W. Barendsen Neutron producHon
1. 15 MeV ~0.4 MeV deuterons on 3H)
10-
2. " 7 M e V " (16MeV deuterons on Be) 3. "10 MeV" (20 MeV 3He on Bq)
4
~
4. "~ M~v" (r~,s~onor 23%)
0
100
1000 dose or tast neutrons (fads)
Fig. 1. Relative biological effectiveness as a function of the dose of fast neutrons relative to 250 kVp X-rays. Data derivedfrom survival curvesfor cultured cells of human kidney origin [11]. Curve 1: 15 MeV neutrons produced by the 8H(d, n)4He reaction. Curve 2 : " 7 MeW' neutrons produced by the 9Be(d, n)X0Breaction. Curve 3:"10 MeV" neutrons produced by the 9Be(SHe, n)nC reaction. Curve 4: "1 Me V" neutrons produced by fission of ~.35U.
trum is complex and extends from very low values up to about 33 MeV. The mean value for the energy spectrum of neutrons produced by bombarding Be with 16 MeV deuterons, which extends from 0 to 18 MeV, is 7 MeV according to Bewley [18]. However, the fact that the RBE values of the "10 MeV" neutrons are larger than the values for "7 MeV" neutrons cannot be assigned a special significance with regard to the dependence of the RBE on neutron energy, because the contributions of the different parts of the energy spectrum cannot yet be adequately evaluated. In addition to the dependence on neutron energy, the RBE is shown to increase with decreasing level of damage, i.e. with decreasing size of the doses compared. This is due to the fact that survival curves obtained with fast neutrons generally exhibit a smaller shoulder width as compared with survival curves obtained with X-rays or ,/-rays [1]. In cases where fractionated irradiation is given with intervals which are sufficiently long to allow for complete repair of sub-lethal damage, the RBE value for a given dose in this Fig. 1 also applies to the dose per fraction. The problem of whether at very low doses or dose fractions a constant RBE value is approached has been discussed elsewhere [1, 17]. For the application of fast neutrons in radiotherapy, the region between 50rads and 250 rads of fast neutrons is presumably the most important, because very small dose fractions would require a very large number of fractions to attain the total dose necessary for cure, while
dose fractions in excess of 250 rads would be expected to be too large to be administered. In radiotherapy, doses of radiation are generally applied in fractionated treatments in a number of weeks. The intervals of one or a few days can be expected to be sufficient for repair of sub-lethal damage to be completed and the extent of this repair is related to the shape of the shoulder region of the survival curves [13]. Consequently the RBE of a given total dose of fast neutrons increases with increasing number of fractions, i.e. with decreasing dose per fraction and to a first approximation the RBE is determined by the size of the dose fraction employed. In addition to the smaller dependence of the effect of a given dose of neutrons, compared with X-rays, on dose fractionation, the oxygen enhancement ratio (OER).cut .... is generally smaller than (OER) x-rays by a factor 1.6-1.8 [18]. The maximum radiotherapeutic gain with respect to the "effective" dose to the tumour as compared to normal tissue, which can be obtained from this difference, would be attained for single acute doses, applied to tumours in which all cells were severely hypoxic, assuming that all ceils in normal tissues were well oxygenated. This hypothetical oxygen effect gain factor (OEG)m~x is equal to (OER)x-,~ys/(OER),e,t,o.s. In practice the gain factor will be smaller than (OEG)max because not all cells in tumours are severely hypoxic and because reoxygenation may occur during intervals between fractions, diminishing the influence of hypoxic cells.
T h e Effectiveness o f Fast Neutrons Relative to X - R a y s
10-
185
15 MeV neutrons (0.4 MeV deuterons on 3['0
a. b. c. d.
cultured cells normal tissues
- ~ - ~ " * . . . . . . _ . . _ ' ~ ' " - ~ . - ~
rhabdomyosarcorna
a
1
I0
c
osteosarcorna
.
.
.
.
.
.
160
......
~ b
1,Joo
dose per fraction of fast neutrons (rads)
Fig. 2. Relations between the relative biological effectiveness of 15 Me V neutrons and the neutron dose or daily dosefraction, relative to 250 k Vp X-rays. Curve"a" representsdata obtainedfrom survival curvesfor cultured cells of human kidney origin [11]. Curve "b" represents data for normal epithelial tissues [21]. Curve "c" represents data for a mouse osteosarcoma. From van Putten et al. [20]. Curve "d" represents data for a rat rhabdomyosarcoma [4, 5].
RBE VALUES MEASU~ErJ FOR RESPONSES OF EXPERIMENTAL T U M O U R S AND N O R M A L TISSUES Fast neutrons will only provide an advantage relative to X-rays or T-rays for the treatment of cancer if the RBE value for damage to the tumour is larger than the RBE for producing damage in those normal tissues which are dose-limiting for the treatment considered. For two types of experimental tumours, a rhabdomyosarcoma transplantable in WAG/ Rij rats and an osteosarcoma transplantable in (CBA/Rij × C57BL/Rij)F1 hybrid mice, effects of fast neutrons with an energy of about 1 4 - 1 5 M e V produced by the 8H(d, n)4He reaction, have been studied with a sufficiently wide range of doses and dose fractions, to allow estimates of the RBE as a function of dose or daily dose fractions [4, 5, 19, 20]. In Fig. 2 these data are represented by curves " d " and " c " respectively. Curve " a " of this figure represents the dependence of the RBE of these neutrons on the dose for damage to cultured cells of h u m a n kidney origin, i.e. curve " a " is identical to curve 1 of Fig. 1. Curve " b " represents the dependence of the RBE on the dose or daily dose fraction for damage to skin and intestinal epithelium, derived from experiments with rats and mice [1, 21, 22]. This curve " b " can only be considered as an approximation, because the data are limited and not accurate enough to evaluate whether small differences exist between RBE values for the various systems investigated. Moreover, most of the data on which curve " b " is based have been measured for doses in excess of 500 rads and for low doses,
curve " b " has been extrapolated parallel to curve " a " obtained from experiments on cell cultures. A discussion of the relations between the curves of Fig. 2 can be given by referring to the factors summarized in Table 1. In the region between 70 and 350 rads of 15 MeV neutrons, all curves of Fig. 2 are within experimental errors parallel and the RBE increases with decreasing dose or magnitude of the daily dose fractions. This variation can be ascribed to the differences between shapes of the survival curves for neutrons and X-rays (factor A2) and to differences in the influence of repair of sub-lethal damage (factor A3) in the 24-hr intervals employed. The fact that the curves in Fig. 2 are within experimental errors parallel in the region between 70 and 350 rads suggests that the influence of these factors on the RBE is approximately equal for the cultured cells, the epithelial cells of skin and intestinal tract and the cells of the two tumours investigated. It is important to note however that, for cells of the haemolymphopoietic system, X-ray survival curves generally exhibit a smaller shoulder as compared with epithelial cells. As a consequence the differences in the shapes of the survival curves for X-rays and fast neutrons are smaller and the RBE of fast neutrons for damage to these cells generally depends less on the dose or dose per fraction as compared with epithelial cells. Moreover, the values of the RBE of fast neutrons for damage to the haemolymphopoietic system are significantly lower than for epithelial cells [21, 23]. For instance, the RBE of 14 MeV
G. fir. Barendsen
186
"7 MeW'neutrons
10-
"(16 MeV deuterons on Be)
**%, o. b. c. d.
.I
cultured ceils normal tissues RIB5 flbrosarcoma l),mphocytic leukaemia
10
100
1000
dose per fraction of fast neutrons (rads)
Fig. 3. Relations between the relative biological effectivenessof "7 MeV" neutrons and the neutron dose or daily dosefraction relative to 250 k Vp X-rays. Curve "a" representsdata obtainedfrom survival curvesfor cultured cells of humankidney origin [ 11]. Curve"b" representsdata obtainedfor reactionsof normalepithelial tissues of animals. From Field [23]. Curve "c" represents data for the RIB5 rat fibrvsarcoma. FromField [23]. Curve "d" pertains to a mouse lymphocytic leukaemia. From Berry et al. [28].
neutrons for lethality of haemopoietic stem cells in mice ranges between 0.9 and 1 "2 [21]. It can be concluded that, for cells in different types of tumours and for cells in different types of normal tissues, considerable differences can exist with respect to the width of the shoulder of survival curves. With respect to the influence of such differences on effects of dose fractionadon in radiotherapy, two possibilities can be envisaged. The first possibility is that the response of the dose-limiting tissue is characterized by a cell survival curve with a shoulder which has a greater width than the shoulder of the survival curve for the tumour cells. In such a case fractionation of a given total dose would have a greater sparing effect for the normal tissue as compared with the tumour. Consequently the use of a type of radiation which enhances this difference, would be indicated, i.e. X-rays or y-radiation should give optimal treatment results. A second possibility which can be envisaged is that the shoulder width of the survival curve of the tumour cells is larger than that of cells of the dose-limiting normal tissue. In this case, fractionation of a given total dose of X- or 7-rays in a large number of fractions would have a greater sparing effect on the tumour as compared with the dose-limiting normal tissue. A treatment with fractionated doses of X- or y-rays would then result in a relatively small effect on the tumour, i.e. the tumour would be judged to be radioresistant. In this case the use of fast neutrons, which would eliminate a large part of the shoulder of the survival curve
of tumour cells, might provide an advantage as compared with X- or 7-rays, although the shoulder of the survival curve for the cells in normal tissues would also be eliminated. At present methods are not yet available however to distinguish in clinical practice those tumours for which survival curves of the cells are characterized by large shoulders. Curve " d " of Fig. 2 shows at doses in excess of 350 rads a distinct increase of the RBE with the dose. Experiments described in detail elsewhere have shown that this rhabdomyosarcoma contains a proportion of about 15% of severely hypoxic cells [4]. Furthermore (OER) x-r~y~/(OER) neu,o~, was measured at approximately 1.7 [4]. At 800 rads of 15 MeV neutrons, the ratio of the RBE for damage to the rhabdomyosarcoma relative to the RBE for damage to normal epithelium is approximately 1.9. This indicates that for large doses or daily dose fractions the total gain factor can almost completely be explained by factor A4 of Table 1. With fractionated doses of 300 rads of X-rays it has been demonstrated that reoxygenation, mentioned as factor A5 of Table 1, occurs during intervals of 24 hr and this presumably accounts for the fact that the influence of the presence of a proportion of anoxic cells on the RBE of 15 MeV neutrons is diminished for fracfionated treatments [5]. It can be derived from Fig. 2 that with decreasing dose per fraction the overall gain factor decreases from 1.9 at 800 rads to 1-3 at 400 rads and approximately 1.2 at 100 rads of 15 MeV neutrons. Curve c of Fig. 3 for damage to the reproduc-
The Effectiveness of Fast Neutrons Relative to X-Rays five capacity of cells in a mouse osteosarcoma has a shape which at neutron doses smaller than 400 rads is not significantly different from curve " d " and the increase in RBE with decreasing dose or daily dose fraction can be ascribed to the factors A2 and A3 mentioned in Table 1. For this osteosarcoma it has been demonstrated that reoxygenation of severely hypoxic cells occurs relatively slowly [19]. Consequently it might have been expected that for this tumour the RBE for fractionated treatments with daily doses of 100 fads or more would be considerably larger than for normal tissues. As discussed in detail elsewhere, this is not observed, because the oxygen-effect gain factor OEG=(OER) x-,~,8/(OER) n,u,ron8 is equal to only approximately 1.2 [20]. This low value can also explain why the RBE does not increase at doses in excess of 400 rads. With respect to the influence of the dependence of radiosensitivity on cell age, i.e. factor A6 in Table 1, on the RBE of fast neutrons, no quantitative experimental data have been published for tumours. For cultured cells irradiated with "fission spectrum" neutrons, the variation of the response throughout the cycle was less than for X-rays [14]. As a consequence the RBE showed variations by a factor of about 1.3 in dependence of cell age [14]. It is important to note that this is the maximum variation between well synchronized sub-populations of Chinese-hamster cells where differences in sensitivity, as a function of cell age, are larger than in many other cell types. Moreover with "fission spectrum" neutrons this difference in RBE is likely to be larger than for higher energy neutrons. In tumours treated with relatively small daily dose fractions, the degree of synchronization will presumably be too small to cause significant changes in the RBE of 15 MeV neutrons for different fractionation regimes. A special case in which such changes might occur is provided by low-dose rate treatments as applied in interstitial and intracavitary treatments of cancer. Data obtained with the rat rhabdomyosarcoma, irradiated at a dose rate of about 100 rads/hr of y-rays and a dose rate of 25 rads/ hr of 15 MeV neutrons, indicate that for treatments in excess of 12 hr large cells accumulate, probably in late S or in G,. The RBE of 15 MeV neutrons for the induction of cell reproductive death in this tumour by lowdose rate irradiation was calculated at about 4. This value is larger than those obtained for fractionated treatments, but definite proof that this is due to induced synchrony is not yet obtained [24].
187
Finally it can be concluded from the RBE values derived for these experimental systems that for treatments with daily dose fractions of between 70 and 200 rads of 15 MeV neutrons, the overall gain factor does not exceed a value of 1 "2. This might however still provide a significant advantage in clinical applications if the dose distributions in the patient obtained with 14 MeV neutrons are not considerably worse than those obtained with high energy X-rays. With respect to the factors listed in Table 1B, it can be noted that for the rat rhabdomyosarcoma proliferation of cells in intervals between daily dose fractions of 50, 70 and 100 rads of 15 MeV neutrons and of 200 and 300 rads of X-rays applied during a three-week treatment, plays an important part in determining the responses of these tumours [5]. After a single dose of 2000 fads of X-rays, Hermens has demonstrated that an increased rate of production of cells can occur due to a shortening of the cell cycle [2]. However, for daily doses of 15 MeV neutrons and 300 kV X-rays respectively, which cause equal effects with respect to cell lethality, the influence of repopulation due to proliferation of cells was not significantly different, i.e. factor B1 of Table 1 does not cause changes in the RBE which are independent of the RBE for cell lethality [5]. Furthermore, after treatments which reduce the fraction of clonogenic cells by at least a factor 104, it has been shown for this experimental tumour that the rate of growth, after the time interval required to regrow to its pre-irradiation volume, is slower than for unirradiated tumours of the same volume. This can be ascribed to the non-lethal damage mentioned as factor B2 in Table 1 [1, 3-5, 25]. This slower growth of recurrent tumours is observed both after treatments with X-rays and 15 MeV neutrons, but it does not significantly influence the RBE [5]. Investigations of non-lethal damage detected as "small colony formation" for cells cultured in vitro, have also shown that the RBE for this type of damage is not significantly different from the RBE for cell lethality, but only depends on the level of damage induced [1, 15, 16]. With respect to mitotic delay, measurements for cultured mammalian cells treated with a-particles have indicated that the RBE for this effect is similar to the RBE for reproductive death. Consequently with respect to this factor, fast neutrons would not be expected to provide an advantage or disadvantage over Xor v-rays. Moreover, the influence of partial synchrony due to mitotic delay is presumably
188
G. W. Barendsen
small for dose fractions of between 200 and 400 rads of X-rays or equivalent doses of neutrons applied with 24-hr intervals [26]. A discussion of the influence of various factors, as given for results of experiments with 15 MeV neutrons, can also be presented for data obtained with “7 MeV” neutrons from the cyclotron at Hammersmith. In Fig. 3, curves “c” and “d” represent RBE values published for a rat fibrosarcoma (RIBS) and a mouse lymphocytic leukaemia [23, 27, 281. Curve “b” of Fig. 3 represents a relation of the RBE as a function of the dose or daily dose fraction for normal epithelium of skin and intestine in mice and rats. This curve represents only an average and no distinction has been made between data for skin and intestinal epithelium [29]. Curve “a” of Fig. 3, derived from data for cultured cells of human kidney origin, is included for comparison. Curve “bl’ of Fig. 3 for normal epithelial tissues is within experimental errors parallel to curve “a” for cultured cells, but the values of curve “b” are approximately a factor of l-2 larger than corresponding values of curve “a”. Both curves show a distinct increase of the RBE with decreasing dose or daily dose fraction which can be ascribed to factors A2 and A3 of Table 1. Values of the RBE of “7 MeV” neutrons for damage to normal tissues are for equal doses larger than RBE values of 15 MeV in agreement with the general neutrons, characteristic listed as factor Al of Table 1. Curve ‘cc” of Fig. 3 represents RBE values for growth delay of a solid fibrosarcoma (RIB’), known to contain a proportion of severely hypoxic cells. The shape of this curve is similar to the curve “c” of Fig. 2 for the rat rhabdomyosarcoma, i.e. at large doses in excess of 700 rads the RBE increases due to the influence of hypoxia, factor A4 of Table 1, while, at small influences daily dose fractions, reoxygenation the RBE significantly (factor A5 of Table 1). Unfortunately no data are available yet for doses lower than 200 rads. Comparison of curve “c” and “b” of Fig. 3 indicates that at
200 rads of “7 MeV” neutrons an overall gain factor of l-3 is obtained while at 500 rads this factor is about l-2. These values are smaller than the oxygen-enhancement gain factor of about 1.7, measured for this neutron beam with various systems [ 181. Curve “d” of Fig. 3 represents RBE values for reproductive death of cells of a lymphocytic leukaemia growing in mice in conditions in which all cells are severely hypoxic [28]. The RBE values are a factor of about 1-7-l *9 larger than corresponding values of curve “b”. This overall gain factor is almost entirely due to hypoxia of the cells, factor A4 of Table 1, because if these cells are oxygenated, the RBE values are decreased to values very close to curve “b” [28]. Th e increase in RBE with decreasing dose for these severely hypoxic cells indicates that the survival curve for X-rays is not an exponential but differs in shape from the neutron survival curve (factor A2 of Table 1).
CONCLUDING REMARKS From the RBE values of fast neutrons derived from published data for tumours and normal tissues as a function of the dose and dose fractionation it can be concluded that overall gain factors (RBE~tumour)/RBE~normsl tiaaue)) of between l-1 and l-3 might be obtained for fast neutrons relative to X-rays. The magnitude of this factor depends on a variety of parameters and also on the types of tumours and the types of tissues which are dose-limiting. Even if clinical applications of fast neutrons would eventually provide only a relatively small advantage over X-rays or y-rays for only a limited group of tumours in man, the insights gained from the comparison of effects induced by the different types of radiations with respect to the influence of repair of sub-lethal damage and of anoxic cells relative to other factors e.g. repopulation in intervals between fractions might provide a basis for the design of optimal treatment schedules for fast neutrons as well as for X-rays and y-rays.
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G. W. BARENDSEN, Responses of cultured cells, tumours and normal tissues to
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of different linear energy transfer. In Current Topics in Radiation (Edited by M. EBERT and A. HOWARD), Vol. IV, p. 332. North-
Holland, Amsterdam, ( 1968). A. F. HERMENSand G. W. BARENDSEN, Changes of cell proliferation characteristics in a rat rhabdomyosarcoma before and after X-irradiation. Europ. J. Cancer 5, 173
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(1969). R. J. BERRY, “Small clones” in irradiated tumour cells in vivo. Brit. J. Radiol. 40, 285 (1967).
The Effectiveness of Fast Neutrons Relative to X-Rays 4.
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19. 20. 21. 22. 23. 24.
25. 26.
G. W. BARENDSEN and J. j . BROERSE, Experimental radiotherapy of a rat rhabdomyosarcoma with 15 MeV neutrons and 300 kV X-rays. I. Effects of single exposures. Europ..77. Cancer 5, 373 (1969). G. W. BARENDSEN and J. J. BROERSE, Experimental radiotherapy of a rat rhabdomyosarcoma with 15 MeV neutrons and 300 kV X-rays. II. Effects of fracfionated treatments, applied five times a week for several weeks. Europ. 07. Cancer 6, 89 (1970). R . H . THOMLINSON,Changes of oxygenation in tumours in relation to irradiation. In Frontiers of Radiation Therapy and Oncology (Edited by J. M. VAETH), Vol. 3, p. 109. Karger, Basel (1968). E. FRINDEL, F. VASSORT and M. TUmANA, Effects of irradiation on the cell cycle of an experimental ascites tumour of the mouse. Int. J. Radiat. Biol. 17, 329 (1970). L . M . VAN PUTWENand R. F. KALLMAN,Oxygenation status of a transplantable tumor during a fractionated radiation therapy. 07. nat. Cancer Inst. 409 441 (1968). R . F . KALLMAN, Repopulation and reoxygenation as factors contributing to the effectiveness of fractionated radiotherapy. In Frontiers of Radiation Therapy and Oncology (Edited byJ. M. VAETH), VO1. 3, p. 96. Karger, Basel (1968). A.E. HowEs, An estimation of changes in the proportions and absolute numbers of hypoxic cells after irradiation of transplanted C3H mouse mammary tumours. Brit. 07. Radiol. 42, 441 (1969). J.J. BROERSE,G. W. BARENDSENand G. R. VAN KERSEN, Survival of cultured human cells after irradiation with fast neutrons of different energies in hypoxic and oxygenated conditions. Int. 07. Radiat. Biol. 13, 559 (1967). M . M . ELKIND, Cellular aspects of tumor therapy. Radiology 74, 529 (1960). G. W. BARmNDSEN, Possibilities for the application of fast neutrons in radiotherapy: recovery and oxygen enhancement of radiation induced damage in relation to linear energy transfer. Europ. 07. Cancer 2, 333 (1966). W . K . SINCLAIR,Radiation survival in synchronous and asynchronous Chinese hamster cells in vitro. In Biophysical Aspects of Radiation Quality, 2nd Panel Report, p. 39. IAEA, Vienna (1968). A. WESTRA and G. W. BARENDSEN, Proliferation chracteristics of cultured mammalian cells after irradiation with sparsely and densely ionizing radiations. Int. 07. Radiat. Biol. 11, 477 (1966). A . H . W . NIAS, Clone size analysis: a parameter in the study of cell population kinetics. Cell Tissue Ifinet. 1, 153 (1968). H . H . RossI, The effects of small doses of ionizing radiation. Phys. in Med. Biol. 15, 255 (1970). D . K . BEWLEY,Fast neutron beams for therapy. In Current Topics in Radiation Research (Edited by M. EBERT and A. HOWARD), Vol. VI, p. 249. NorthHolland, Amsterdam (1970). L . M . VAN PUTTEN, Tumour reoxygenation during fractionated radiotherapy: studies with a transplantable mouse osteosarcoma. Europ. 07. Cancer 4, 173 (1968). L. M. VAN PUTTEN, P. LELIEVELDand J. J. BROERSE, Response of a poorly reoxygenating mouse osteosarcoma to X-rays and fast neutrons. Europ. 07. Cancer, this issue. J . J . BROERSE, G. W. BARENDSEN, G. FRERIKSand L. M. VAN PUTTEN, RBE values of 15 MeV neutrons for effects on normal tissues. Europ. 07. Cancer 7, 171 (1971). H . R . WITHERS,The response of stem cells of intestinal mucosa to irradiation with 14 MeV neutrons. Brit. 07. Radiol. 43, 796 (1970). S.B. FIELD,The relative biological effectiveness of fast neutrons for mammalian tissues. Radiology 93, 915 (1969). H. B. KAL, Impairment of cell reproductive integrity in an experimental rhabdomyosarcoma by fast neutron and gamma-irradiation at low dose rates. Proc. IVth Int. Congress of Radiation Research, Evian (1970). In press. W. K. SINCLAIR, X-ray-induced heritable damage (small colony formation) in cultured mammalian cells. Radiat. Res. 21, 584 (1964). J. M. YOUNO and J. F. FOWLER, The effect of X-ray induced synchrony on two-dose cell survival experiments. Cell Tissue Kinet. 2, 95 (1969).
189
190
G. IV. Barendsen 27.
28.
29.
S. B. FIELD, T. JONES and R. H. THOMLINSON,The relative effects of fast neutrons and X-rays on tumour and normal tissue in the rat. II. Fractlonatlon: recovery and reoxygenation. Brit. 07. Radiol. 41~ 597 (1968). R . J . BERRY, D. K. BEWLEY and C. J. PARNELL,Reproductive capacity of mammalian tumour ceils irradiated in vivo with cyclotron-produced fast neutrons. Brit. 07. Radiol. 38, 613 (1965). S . B . FIELD and S. HORNSEY, R.BE values for cyclotron neutrons for effects on normal tissues and tumours as a function of dose and dose fractionation. Europ.
y. Cancer7, 161 (1971).
Cellular Responses Determining the Effectiveness of Fast Neutrons Relative to X-Rays for Effects on Experimental Yumours G. W. BARENDSEN
Radiobiological Institute TWO, 151 Lange Kleiweg, R~isw~k Z.H., The Netherlands Relations between the relative biological effectiveness of fast neutrons and the dose or daily dosefractions are presented for cultured cells, tumours and normal tissues. These relations are dependent on a variety orfactors, namely the neutron energy spectrum, the shapes of dose-survival curves, repah" of sub-lethal damage, the presence of anoxic cells, reoxygenation during intervals betweenfractions and the dependence of radiosensitivity on the age of cells in the# cycle. The relative influence of thesefactors is discussed. Comparison of the RBE values of fast neutrons derivedfrom published data for experimental tumours and normal tissues indicates that overall gain factors, RBE
INTRODUCTION THE AIM of a radiotherapeutical treatment is to administer radiation according to a schedule which, by the proper choice of various parameters, namely the spatial distribution of the dose in the patient, the sizes of the dose fractions, the intervals between fractions and the total dose, will just be tolerated by normal tissues while causing maximum damage to the turnout. In comparison with orthovoltage Xrays, the introduction of high energy X-rays and fast electrons has allowed considerable improvement to be made in the dose distribution in the patient. With fast neutrons this distribution presumably cannot be improved further, and this radiation can only provide an advantage if the relative biological effectiveness (RBE) is larger for damage to the tumour than the RBE for damage to the dose-limiting normal tissues. Values of the RBE may depend on the parameters mentioned, i.e. the sizes of the
dose fractions, the intervals between fractions and the total dose, as well as on the type of tumour and normal tissue irradiated and consequently a large amount of experimental and clinical data is required before possible advantages of fast neutrons can be adequately evaluated. In this paper the influence of a number of factors on the RBE will be discussed. Results of radiobiological studies, obtained during the past decade, have shown that radiation-induced damage to normal tissues and tumours in animals is determined to a large extent by responses of those constituent cells which are capable of unlimited proliferation, i.e. the clonogenic cells. Furthermore, responses of individual cells after irradiation in vivo have been shown with a number of experimental systems to be similar to responses of the same type of cells grown and assayed in vitro, provided various environmental conditions, e.g. oxygen concentration, are made equal.
181
182
G. W. Barendsen
Investigations of cells cultured in vitro have demonstrated that a variety of factors can influence their radiosensitivity with respect to the capacity for proliferation, e.g. radiation quality, the distribution of dose in time, the presence of oxygen and other sensitizing compounds, the presence of protecting compounds, the proliferative state of the cells and their age in the cell cycle [1]. These factors, which in experiments in vitro have been studied in well controlled conditions, may cause large variations in the responses to fractionated irradiation of tumours and of normal tissues in patients, where cellular conditions are in general unknown and cannot be easily determined or controlled. Experimental data obtained with respect to the radiosensitivity of cells in culture, in experimental tumours or in various normal tissues, have often been used to emphasize similarities between the characteristics of dose survival curves of different types of mammalian cells. It should not be forgotten however that differences by a factor of 2-3 in Do values, which range from 70 to 200 rads, and differences in extrapolation numbers by at least a factor of 10, have been observed. These differences in intrinsic radiosensitivity of various types of mammalian cells may be augmented by certain conditions in turnouts or tissues due to some of the dose modifying factors mentioned and this can result in very large differences in the responses of these multicellular systems to a fractionated treatment [i]. In addition to factors influencing the radiosensitivity of individual cells, the responses of tumours and normal tissues after irradiation or during a fractionated treatment depend on the proliferative activity and changes in physiological conditions of those cells that have retained the capacity for unlimited proliferation. Irradiated cells will generally experience a phase of mitotic delay, after which proliferation is resumed. The rate of proliferation of parenchymal cells and the rate of removal of dead cells in irradiated tumours and in normal tissues m a y depend on various factors, which have not yet been sufficiently investigated, e.g. tumour architecture and the vascular system. Increased as well as decreased rates of cell production in comparison with pre-irradiation values have been observed in irradiated tumours, depending on the dose administered and the time interval after irradiation [2-7]. Furthermore changes in oxygenation status of cells have been demonstrated in tumours containing a fraction of severely hypoxie cells at the start of a treatment [6, 8-10]. Finally
the volume response of a tumour to irradiations may depend on factors concerned with the architecture of the turnout, e.g. the amount and type of stroma, the blood vessels and the surrounding tissues. As a consequence of all these factors which can cause variations in responses of tumours as well as of normal tissues, it is not inconsistent with radiobiological data that tumours in patients show large differences in their clinical radiosensitivity to fractionated treatments with X-rays or ~,-rays. The main problem in experimental tumour radiobiology is to evaluate the relative importance of the various factors mentioned and to detect systematic differences between responses of various types of tumours and various types of normal tissues. Comparison of the effectiveness of fast neutrons relative to X-rays for inducing damage to different types of cells, tumours and normal tissues, can provide data for this evaluation, because some of these factors play a smaller part with neutron irradiations. Dose-effect relations for X-rays and fast neutrons differ in two main aspects, namely a reduced shoulder of the survival curve for fast neutrons as compared with X-rays, with a consequent diminished contribution of repair of sub-lethal damage in fractionated treatments, and a relatively low oxygen enhancement ratio of fast neutrons, which does not depend greatly on neutron energy in the range between 5 and 30 MeV [11]. As a result of these differences, the relative biological effectiveness (RBE)* of a given dose of fast neutrons for effects on turnouts and normal tissues depends on the dose, the fractionation schedule, the presence of anoxic cells and on changes in oxygenation status. Finally, differences between effects of fast neutrons and X-rays have been observed with respect to the dependence of radiosensitivity on cell age in the division cycle. The influence of this factor may also depend on the dose fractionation schedule, because the degree of induced synchrony depends on the dose, and the desynchronization before the next dose is given depends on the time interval. All these factors, which can cause variations of the RBE of fast neutrons in dependence on the dose and dose fractionation schedule, have been listed in Table 1A. In part B of this table, three other factors have been listed which are important with respect to the final result of a *The RBE of a dose of neutron radiation is defined as the ratio of a dose of X-rays and the dose of neutron radiation required for producing quantitatively equal effects.
183
The Effectiveness of Fast Neutrons Relative to X-Rays Table 1. Factors determining responses of tumours and normal tissues to single andfractionated irradiations i
Factors
Characteristics
A. Factors which influence the RBE of fast neutrons 1.
Neutron energy spectrum
2. Size of dose or dose fraction
RBE increases with decreasing neutron energy [11] RBE increases with decreasing dose, dependent on shapes of survival curves of cells [1]
3. Intra-cellular repair of sub-lethal RBE increases with decreasing dose fraction, ff damage
intervals between fractions are long enough to allow repair [1, 12, 13]
4.
Hypoxia of proportion of cells
Because (OER)neut . . . . *~ (OER)x-r~ys the RBE of neutrons is larger for damage to hypoxic cells [1, 4-6, 18]
5.
Reoxygenation of hypoxic cells Decreases the effect of hypoxic cells [5, 6, 8-10] during intervals between fractions
6. Dependence of radiosensitivity on This dependence is smaller for fast neutrons as cell age
compared with X-rays [14]
B. Factors which do not influence the RBE of fast neutrons 1. Proliferation of cells in intervals Rate can increase or decrease during and after between dose fractions
fractionated treatment [2, 3, 7]
2. Non-lethal damage
Causes death in part of the progeny of surviving cells and slow rate of regrowth of tumours [15, 16]
3. Mitotic delay
Influence is presumably small in practical treatment schedules
radiotherapeutical treatment, but which do not cause further variations of the RBE of fast neutrons in dependence on the fractionation schedule employed. The first of these factors, proliferation of cells in intervals between dose fractions reduces the effect of a given treatment, i.e. a larger dose is required in order to kill all cells. The limited experimental data available indicate that changes in the rate of proliferation and of cell loss do not depend on the type of radiation employed if doses are compared which cause equal damage to the proliferative capacity of the clonogenic cells [1, 5]. Nonlethal damage has also been demonstrated to be caused to an equal degree by different radiations provided doses are compared which cause the same degree of cell reproductive death. Mitotic delay, as far as indicated by the limited data available, is caused with the same RBE as cell lethality. Thus if a treatment with fast neutrons and a treatment with X-rays produce equal effects with respect to cell killing, then cell proliferation, impaired cell production due to non-lethal damage or mitotic delay will not cause other differences between the effects of these treatments with respect e.g. to the probability of cure or the growth rate of recurrent tumours.
RBE VALUES OF FAST NEUTRONS FOR REPRODUCTIVE DEATH IN CULTURED CELLS With in vitro systems, RBE values can be determined for ceils in well controlled conditions, whereby variations due to, e.g. partial hypoxia or changes in age distributions of the cells can be eliminated. Survival curves for cultured asynchronous mammalian cells of human kidney origin (T-1 g cells) have been determined with fast neutrons of different energies by the clone technique [11]. From these curves RBE values relative to 250 kVp X-rays have been derived for various levels of the neutron dose. The results are presented in Fig. 1. These curves illustrate some general features of the dependence of the RBE on the neutron dose and energy. The logarithmic scales are used because some fundamental radiobiological considerations indicate that relatively simple shapes of the curves might be obtained [ 17]. The curves of Fig. 1 show that the largest RBE values are derived for the lowest neutron energy and conversely. The mean value of "10 M e V " assigned to the neutron spectrum obtained by bombarding Be with 20 MeV SHe-ions, is quite arbitrary because the spec-
184
G. W. Barendsen Neutron producHon
1. 15 MeV ~0.4 MeV deuterons on 3H)
10-
2. " 7 M e V " (16MeV deuterons on Be) 3. "10 MeV" (20 MeV 3He on Bq)
4
~
4. "~ M~v" (r~,s~onor 23%)
0
100
1000 dose or tast neutrons (fads)
Fig. 1. Relative biological effectiveness as a function of the dose of fast neutrons relative to 250 kVp X-rays. Data derivedfrom survival curvesfor cultured cells of human kidney origin [11]. Curve 1: 15 MeV neutrons produced by the 8H(d, n)4He reaction. Curve 2 : " 7 MeW' neutrons produced by the 9Be(d, n)X0Breaction. Curve 3:"10 MeV" neutrons produced by the 9Be(SHe, n)nC reaction. Curve 4: "1 Me V" neutrons produced by fission of ~.35U.
trum is complex and extends from very low values up to about 33 MeV. The mean value for the energy spectrum of neutrons produced by bombarding Be with 16 MeV deuterons, which extends from 0 to 18 MeV, is 7 MeV according to Bewley [18]. However, the fact that the RBE values of the "10 MeV" neutrons are larger than the values for "7 MeV" neutrons cannot be assigned a special significance with regard to the dependence of the RBE on neutron energy, because the contributions of the different parts of the energy spectrum cannot yet be adequately evaluated. In addition to the dependence on neutron energy, the RBE is shown to increase with decreasing level of damage, i.e. with decreasing size of the doses compared. This is due to the fact that survival curves obtained with fast neutrons generally exhibit a smaller shoulder width as compared with survival curves obtained with X-rays or ,/-rays [1]. In cases where fractionated irradiation is given with intervals which are sufficiently long to allow for complete repair of sub-lethal damage, the RBE value for a given dose in this Fig. 1 also applies to the dose per fraction. The problem of whether at very low doses or dose fractions a constant RBE value is approached has been discussed elsewhere [1, 17]. For the application of fast neutrons in radiotherapy, the region between 50rads and 250 rads of fast neutrons is presumably the most important, because very small dose fractions would require a very large number of fractions to attain the total dose necessary for cure, while
dose fractions in excess of 250 rads would be expected to be too large to be administered. In radiotherapy, doses of radiation are generally applied in fractionated treatments in a number of weeks. The intervals of one or a few days can be expected to be sufficient for repair of sub-lethal damage to be completed and the extent of this repair is related to the shape of the shoulder region of the survival curves [13]. Consequently the RBE of a given total dose of fast neutrons increases with increasing number of fractions, i.e. with decreasing dose per fraction and to a first approximation the RBE is determined by the size of the dose fraction employed. In addition to the smaller dependence of the effect of a given dose of neutrons, compared with X-rays, on dose fractionation, the oxygen enhancement ratio (OER).cut .... is generally smaller than (OER) x-rays by a factor 1.6-1.8 [18]. The maximum radiotherapeutic gain with respect to the "effective" dose to the tumour as compared to normal tissue, which can be obtained from this difference, would be attained for single acute doses, applied to tumours in which all cells were severely hypoxic, assuming that all ceils in normal tissues were well oxygenated. This hypothetical oxygen effect gain factor (OEG)m~x is equal to (OER)x-,~ys/(OER),e,t,o.s. In practice the gain factor will be smaller than (OEG)max because not all cells in tumours are severely hypoxic and because reoxygenation may occur during intervals between fractions, diminishing the influence of hypoxic cells.
T h e Effectiveness o f Fast Neutrons Relative to X - R a y s
10-
185
15 MeV neutrons (0.4 MeV deuterons on 3['0
a. b. c. d.
cultured cells normal tissues
- ~ - ~ " * . . . . . . _ . . _ ' ~ ' " - ~ . - ~
rhabdomyosarcorna
a
1
I0
c
osteosarcorna
.
.
.
.
.
.
160
......
~ b
1,Joo
dose per fraction of fast neutrons (rads)
Fig. 2. Relations between the relative biological effectiveness of 15 Me V neutrons and the neutron dose or daily dosefraction, relative to 250 k Vp X-rays. Curve"a" representsdata obtainedfrom survival curvesfor cultured cells of human kidney origin [11]. Curve "b" represents data for normal epithelial tissues [21]. Curve "c" represents data for a mouse osteosarcoma. From van Putten et al. [20]. Curve "d" represents data for a rat rhabdomyosarcoma [4, 5].
RBE VALUES MEASU~ErJ FOR RESPONSES OF EXPERIMENTAL T U M O U R S AND N O R M A L TISSUES Fast neutrons will only provide an advantage relative to X-rays or T-rays for the treatment of cancer if the RBE value for damage to the tumour is larger than the RBE for producing damage in those normal tissues which are dose-limiting for the treatment considered. For two types of experimental tumours, a rhabdomyosarcoma transplantable in WAG/ Rij rats and an osteosarcoma transplantable in (CBA/Rij × C57BL/Rij)F1 hybrid mice, effects of fast neutrons with an energy of about 1 4 - 1 5 M e V produced by the 8H(d, n)4He reaction, have been studied with a sufficiently wide range of doses and dose fractions, to allow estimates of the RBE as a function of dose or daily dose fractions [4, 5, 19, 20]. In Fig. 2 these data are represented by curves " d " and " c " respectively. Curve " a " of this figure represents the dependence of the RBE of these neutrons on the dose for damage to cultured cells of h u m a n kidney origin, i.e. curve " a " is identical to curve 1 of Fig. 1. Curve " b " represents the dependence of the RBE on the dose or daily dose fraction for damage to skin and intestinal epithelium, derived from experiments with rats and mice [1, 21, 22]. This curve " b " can only be considered as an approximation, because the data are limited and not accurate enough to evaluate whether small differences exist between RBE values for the various systems investigated. Moreover, most of the data on which curve " b " is based have been measured for doses in excess of 500 rads and for low doses,
curve " b " has been extrapolated parallel to curve " a " obtained from experiments on cell cultures. A discussion of the relations between the curves of Fig. 2 can be given by referring to the factors summarized in Table 1. In the region between 70 and 350 rads of 15 MeV neutrons, all curves of Fig. 2 are within experimental errors parallel and the RBE increases with decreasing dose or magnitude of the daily dose fractions. This variation can be ascribed to the differences between shapes of the survival curves for neutrons and X-rays (factor A2) and to differences in the influence of repair of sub-lethal damage (factor A3) in the 24-hr intervals employed. The fact that the curves in Fig. 2 are within experimental errors parallel in the region between 70 and 350 rads suggests that the influence of these factors on the RBE is approximately equal for the cultured cells, the epithelial cells of skin and intestinal tract and the cells of the two tumours investigated. It is important to note however that, for cells of the haemolymphopoietic system, X-ray survival curves generally exhibit a smaller shoulder as compared with epithelial cells. As a consequence the differences in the shapes of the survival curves for X-rays and fast neutrons are smaller and the RBE of fast neutrons for damage to these cells generally depends less on the dose or dose per fraction as compared with epithelial cells. Moreover, the values of the RBE of fast neutrons for damage to the haemolymphopoietic system are significantly lower than for epithelial cells [21, 23]. For instance, the RBE of 14 MeV
G. fir. Barendsen
186
"7 MeW'neutrons
10-
"(16 MeV deuterons on Be)
**%, o. b. c. d.
.I
cultured ceils normal tissues RIB5 flbrosarcoma l),mphocytic leukaemia
10
100
1000
dose per fraction of fast neutrons (rads)
Fig. 3. Relations between the relative biological effectivenessof "7 MeV" neutrons and the neutron dose or daily dosefraction relative to 250 k Vp X-rays. Curve "a" representsdata obtainedfrom survival curvesfor cultured cells of humankidney origin [ 11]. Curve"b" representsdata obtainedfor reactionsof normalepithelial tissues of animals. From Field [23]. Curve "c" represents data for the RIB5 rat fibrvsarcoma. FromField [23]. Curve "d" pertains to a mouse lymphocytic leukaemia. From Berry et al. [28].
neutrons for lethality of haemopoietic stem cells in mice ranges between 0.9 and 1 "2 [21]. It can be concluded that, for cells in different types of tumours and for cells in different types of normal tissues, considerable differences can exist with respect to the width of the shoulder of survival curves. With respect to the influence of such differences on effects of dose fractionadon in radiotherapy, two possibilities can be envisaged. The first possibility is that the response of the dose-limiting tissue is characterized by a cell survival curve with a shoulder which has a greater width than the shoulder of the survival curve for the tumour cells. In such a case fractionation of a given total dose would have a greater sparing effect for the normal tissue as compared with the tumour. Consequently the use of a type of radiation which enhances this difference, would be indicated, i.e. X-rays or y-radiation should give optimal treatment results. A second possibility which can be envisaged is that the shoulder width of the survival curve of the tumour cells is larger than that of cells of the dose-limiting normal tissue. In this case, fractionation of a given total dose of X- or 7-rays in a large number of fractions would have a greater sparing effect on the tumour as compared with the dose-limiting normal tissue. A treatment with fractionated doses of X- or y-rays would then result in a relatively small effect on the tumour, i.e. the tumour would be judged to be radioresistant. In this case the use of fast neutrons, which would eliminate a large part of the shoulder of the survival curve
of tumour cells, might provide an advantage as compared with X- or 7-rays, although the shoulder of the survival curve for the cells in normal tissues would also be eliminated. At present methods are not yet available however to distinguish in clinical practice those tumours for which survival curves of the cells are characterized by large shoulders. Curve " d " of Fig. 2 shows at doses in excess of 350 rads a distinct increase of the RBE with the dose. Experiments described in detail elsewhere have shown that this rhabdomyosarcoma contains a proportion of about 15% of severely hypoxic cells [4]. Furthermore (OER) x-r~y~/(OER) neu,o~, was measured at approximately 1.7 [4]. At 800 rads of 15 MeV neutrons, the ratio of the RBE for damage to the rhabdomyosarcoma relative to the RBE for damage to normal epithelium is approximately 1.9. This indicates that for large doses or daily dose fractions the total gain factor can almost completely be explained by factor A4 of Table 1. With fractionated doses of 300 rads of X-rays it has been demonstrated that reoxygenation, mentioned as factor A5 of Table 1, occurs during intervals of 24 hr and this presumably accounts for the fact that the influence of the presence of a proportion of anoxic cells on the RBE of 15 MeV neutrons is diminished for fracfionated treatments [5]. It can be derived from Fig. 2 that with decreasing dose per fraction the overall gain factor decreases from 1.9 at 800 rads to 1-3 at 400 rads and approximately 1.2 at 100 rads of 15 MeV neutrons. Curve c of Fig. 3 for damage to the reproduc-
The Effectiveness of Fast Neutrons Relative to X-Rays five capacity of cells in a mouse osteosarcoma has a shape which at neutron doses smaller than 400 rads is not significantly different from curve " d " and the increase in RBE with decreasing dose or daily dose fraction can be ascribed to the factors A2 and A3 mentioned in Table 1. For this osteosarcoma it has been demonstrated that reoxygenation of severely hypoxic cells occurs relatively slowly [19]. Consequently it might have been expected that for this tumour the RBE for fractionated treatments with daily doses of 100 fads or more would be considerably larger than for normal tissues. As discussed in detail elsewhere, this is not observed, because the oxygen-effect gain factor OEG=(OER) x-,~,8/(OER) n,u,ron8 is equal to only approximately 1.2 [20]. This low value can also explain why the RBE does not increase at doses in excess of 400 rads. With respect to the influence of the dependence of radiosensitivity on cell age, i.e. factor A6 in Table 1, on the RBE of fast neutrons, no quantitative experimental data have been published for tumours. For cultured cells irradiated with "fission spectrum" neutrons, the variation of the response throughout the cycle was less than for X-rays [14]. As a consequence the RBE showed variations by a factor of about 1.3 in dependence of cell age [14]. It is important to note that this is the maximum variation between well synchronized sub-populations of Chinese-hamster cells where differences in sensitivity, as a function of cell age, are larger than in many other cell types. Moreover with "fission spectrum" neutrons this difference in RBE is likely to be larger than for higher energy neutrons. In tumours treated with relatively small daily dose fractions, the degree of synchronization will presumably be too small to cause significant changes in the RBE of 15 MeV neutrons for different fractionation regimes. A special case in which such changes might occur is provided by low-dose rate treatments as applied in interstitial and intracavitary treatments of cancer. Data obtained with the rat rhabdomyosarcoma, irradiated at a dose rate of about 100 rads/hr of y-rays and a dose rate of 25 rads/ hr of 15 MeV neutrons, indicate that for treatments in excess of 12 hr large cells accumulate, probably in late S or in G,. The RBE of 15 MeV neutrons for the induction of cell reproductive death in this tumour by lowdose rate irradiation was calculated at about 4. This value is larger than those obtained for fractionated treatments, but definite proof that this is due to induced synchrony is not yet obtained [24].
187
Finally it can be concluded from the RBE values derived for these experimental systems that for treatments with daily dose fractions of between 70 and 200 rads of 15 MeV neutrons, the overall gain factor does not exceed a value of 1 "2. This might however still provide a significant advantage in clinical applications if the dose distributions in the patient obtained with 14 MeV neutrons are not considerably worse than those obtained with high energy X-rays. With respect to the factors listed in Table 1B, it can be noted that for the rat rhabdomyosarcoma proliferation of cells in intervals between daily dose fractions of 50, 70 and 100 rads of 15 MeV neutrons and of 200 and 300 rads of X-rays applied during a three-week treatment, plays an important part in determining the responses of these tumours [5]. After a single dose of 2000 fads of X-rays, Hermens has demonstrated that an increased rate of production of cells can occur due to a shortening of the cell cycle [2]. However, for daily doses of 15 MeV neutrons and 300 kV X-rays respectively, which cause equal effects with respect to cell lethality, the influence of repopulation due to proliferation of cells was not significantly different, i.e. factor B1 of Table 1 does not cause changes in the RBE which are independent of the RBE for cell lethality [5]. Furthermore, after treatments which reduce the fraction of clonogenic cells by at least a factor 104, it has been shown for this experimental tumour that the rate of growth, after the time interval required to regrow to its pre-irradiation volume, is slower than for unirradiated tumours of the same volume. This can be ascribed to the non-lethal damage mentioned as factor B2 in Table 1 [1, 3-5, 25]. This slower growth of recurrent tumours is observed both after treatments with X-rays and 15 MeV neutrons, but it does not significantly influence the RBE [5]. Investigations of non-lethal damage detected as "small colony formation" for cells cultured in vitro, have also shown that the RBE for this type of damage is not significantly different from the RBE for cell lethality, but only depends on the level of damage induced [1, 15, 16]. With respect to mitotic delay, measurements for cultured mammalian cells treated with a-particles have indicated that the RBE for this effect is similar to the RBE for reproductive death. Consequently with respect to this factor, fast neutrons would not be expected to provide an advantage or disadvantage over Xor v-rays. Moreover, the influence of partial synchrony due to mitotic delay is presumably
188
G. W. Barendsen
small for dose fractions of between 200 and 400 rads of X-rays or equivalent doses of neutrons applied with 24-hr intervals [26]. A discussion of the influence of various factors, as given for results of experiments with 15 MeV neutrons, can also be presented for data obtained with “7 MeV” neutrons from the cyclotron at Hammersmith. In Fig. 3, curves “c” and “d” represent RBE values published for a rat fibrosarcoma (RIBS) and a mouse lymphocytic leukaemia [23, 27, 281. Curve “b” of Fig. 3 represents a relation of the RBE as a function of the dose or daily dose fraction for normal epithelium of skin and intestine in mice and rats. This curve represents only an average and no distinction has been made between data for skin and intestinal epithelium [29]. Curve “a” of Fig. 3, derived from data for cultured cells of human kidney origin, is included for comparison. Curve “bl’ of Fig. 3 for normal epithelial tissues is within experimental errors parallel to curve “a” for cultured cells, but the values of curve “b” are approximately a factor of l-2 larger than corresponding values of curve “a”. Both curves show a distinct increase of the RBE with decreasing dose or daily dose fraction which can be ascribed to factors A2 and A3 of Table 1. Values of the RBE of “7 MeV” neutrons for damage to normal tissues are for equal doses larger than RBE values of 15 MeV in agreement with the general neutrons, characteristic listed as factor Al of Table 1. Curve ‘cc” of Fig. 3 represents RBE values for growth delay of a solid fibrosarcoma (RIB’), known to contain a proportion of severely hypoxic cells. The shape of this curve is similar to the curve “c” of Fig. 2 for the rat rhabdomyosarcoma, i.e. at large doses in excess of 700 rads the RBE increases due to the influence of hypoxia, factor A4 of Table 1, while, at small influences daily dose fractions, reoxygenation the RBE significantly (factor A5 of Table 1). Unfortunately no data are available yet for doses lower than 200 rads. Comparison of curve “c” and “b” of Fig. 3 indicates that at
200 rads of “7 MeV” neutrons an overall gain factor of l-3 is obtained while at 500 rads this factor is about l-2. These values are smaller than the oxygen-enhancement gain factor of about 1.7, measured for this neutron beam with various systems [ 181. Curve “d” of Fig. 3 represents RBE values for reproductive death of cells of a lymphocytic leukaemia growing in mice in conditions in which all cells are severely hypoxic [28]. The RBE values are a factor of about 1-7-l *9 larger than corresponding values of curve “b”. This overall gain factor is almost entirely due to hypoxia of the cells, factor A4 of Table 1, because if these cells are oxygenated, the RBE values are decreased to values very close to curve “b” [28]. Th e increase in RBE with decreasing dose for these severely hypoxic cells indicates that the survival curve for X-rays is not an exponential but differs in shape from the neutron survival curve (factor A2 of Table 1).
CONCLUDING REMARKS From the RBE values of fast neutrons derived from published data for tumours and normal tissues as a function of the dose and dose fractionation it can be concluded that overall gain factors (RBE~tumour)/RBE~normsl tiaaue)) of between l-1 and l-3 might be obtained for fast neutrons relative to X-rays. The magnitude of this factor depends on a variety of parameters and also on the types of tumours and the types of tissues which are dose-limiting. Even if clinical applications of fast neutrons would eventually provide only a relatively small advantage over X-rays or y-rays for only a limited group of tumours in man, the insights gained from the comparison of effects induced by the different types of radiations with respect to the influence of repair of sub-lethal damage and of anoxic cells relative to other factors e.g. repopulation in intervals between fractions might provide a basis for the design of optimal treatment schedules for fast neutrons as well as for X-rays and y-rays.
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