The response of mouse skin to irradiation with neutrons from the 62 MV cyclotron at Clatterbridge, U.K.

Radiotherapy Elsevier

and Oncology,

12 (1988) 153-166

153

RTO 00469

M.C. Joiner’ and S.B. Field2 ‘Gray Laboratory

qf the Cuttcrr Research Contpoigtt. Mom Cyclotrott Unit. Hatmterstttith (Received 9 November

Key words:

Neutron;

Vmott Hospital, Northwood, A4iddlcse.v HA6 ZRN, L1.K. ottd ‘MRC Hospitd, Duccttte Road, Londott WI2 OHS. U.K.

1987. revision received 29 January

RBE: Mouse skin; Linear-quadratic

1988, acccptcd I February

1988)

model

Summary

Acute skin reactions on mouse feet were used to measure the effect of 62 MeV -Be neutrons from the cyclotron at Clatterbridge, U.K. The results were compared with the response to 16 McV d-Be neutrons from the cyclotron at Hammersmith, 4 MeV d-Be neutrons from the van de Graaff accelerator at the Gray Laboratory, and 250 kVp X-rays. Up to 16 equal radiation fractions were given alone, or 16 fractions foliowcd by “top-up” doses of 4 MeV d-Be neutrons to study the effect of neutron doses < 1 Gy per fraction. For equivalent skin reacitons, 9-16% more dose (total neutron + gamma) was needed with p(62)-Be neutrons compared with d(l6)-Be neutrons. This did not vary significantly between 1 and 16 fractions. The top-up studies indicated that this figure might rise to approximately 1432% at very low doses of neutrons, the value depending on the method of analysis of the data. The data indicate that the “standard” clinical protocol of 1.47 Gy per fraction (N + y dose) in 12 fractions given at Hammersmith with d( 16)-Be neutrons would correspond to a dose of 1.64 Gy per fraction (N + y) at Clatterbridge using a similar regime of p(62)-Be neutrons. d(4)-Be neutrons were more effective than d(l6)-Be neutrons by a factor of 1.6 over the whole range of dose per fraction studied (0.05-14.5 Gy per fraction of d(4)-Be neutrons). Relative to X-rays, the RBE for p(62)-Be neutrons was 1.6 f 0.02 for a single X-ray dose of 30 Gy, rising to 2.9 f 0.04 for an X-ray dose per fraction of 4.6 Gy $ven 16 times. The full-course fractionation data and the top-up data together indicate an extrapolated hmiting RBE at vanishingly small doses per fraction of 4.2-4.8 depending on the method of analysis. The X-ray data were well-fitted by a linear-quadratic (LQ) model of dose-fractionation, with a/P = 8.6 f 1.5 Gy. The LQ model also provides a fairly good description of the neutron responses, cx/pbeing large ( > 24) reflecting predominantly linear underlying dose-responses for all the neutron beams. This in turn reflects the small variation observed in the relative effectiveness between the 3 neutron beams with changes in dose per fraction.

Address for correspotdet~ce: M.C. Joiner, Gray Laboratory Middlesex HA6 2RN, U.K.

0167-8140/88/$03.50

0 1988 Elsevier Science Publishers

of the Cancer Research Campaign,

B.V. (Biomedical Division)

Mount Vernon Hospital. Northwood.

154 Introduction In order to provide neutron radiotherapy with depth-doses comparable with modern photon therapy, it is necessary to use the highest practical neutron energies. Only by this approach will it become possible to study further the role of neutron therapy in cancer treatment. A high energy neutron facility in the U.K. has been provided by the installation of a cyclotron at Clatterbridge Hospital, Merseyside. The cyclotron was manufactured by AB Scanditronix, Sweden and produces 62 MeV protons with an extracted beam current 30 ,uA. A beryllium target assembly is mounted within an isocentric treatment head (Elven Precision Ltd., Crawley, U.K.). Neutron dose rates at the isocentre are 20.4 Gy - min- ‘. This paper describes preclinical studies of the radiobiological effectiveness of this new neutron beam, assessed by measuring acute skin reactions on the mouse foot. Comparison of the early reactions in mice, rats, pigs and humans have demonstrated previously that the neutron RBE for skin is independent of species [14,16,17] and RBE values for skin have been found to be useful in optimising doses for use in clinical trials [4,5]. For these reasons, skin has been used as a “biological dosimeter”. The study described here compared the effects on mouse skin of neutrons at Clatterbridge (p(62)-Be) with the earlier clinical facility at Hammersmith (d(lG)-Be). Also included in this inter-. comparison were 250 kVp X-rays, and d(4)-Be neutrons from the Gray Laboratory accelerator which were also used in additional top-up experiments to study low doses per fraction at each facility.

Materials and methods Mice arId irradiation

A total of 1020 adult female albino mice (CFLP) aged 12-16 weeks were used in these experiments. Mice were irradiated unanaesthetised whilst breathing air at room temperature, in order to simulate the conditions of clinical radiotherapy. Dosegroups of 4 or 5 mice were given radiation to the

left hind foot and were allocated to one of 4 different radiation arms as shown in Table I. Immediately after the last irradiation, all mice were returned to a single centre (Hammersmith) for subsequent observation and scoring of the skin reactions. For irradiations with p(62)-Be and d(4)-Be neutrons and for X-rays, the total treatment was administered as a single radiation dose or as 2 fractions with a 6-day interval, 4 fractions with 2-day interfraction intervals, 8 daily fractions, or 16 fractions given as 2 fractions per day separated by 7 h. In addition, 30 groups of 4 mice were given 16 fractions as above, followed one day later by a top-up dose of d(4)-Be neutrons as described by Joiner [21]. For the d( 16)-Be neutron irradiations, the total treatment was administered as a single radiation dose or as 2 fractions with a 3-day interval, 4 daily fractions or 8 fractions given as 2 fractions per day separated by 7 h. In addition, 25 groups of 4 mice were given 8 fractions as above, followed one day later by a d(4)-Be neutron top-up dose. The range of radiation doses given in all the treatment schedules is summarised in Table I. During irradiation, the mice were carefully restrained in Perspex boxes (lead boxes for X-irradiation) with breathing holes. The left hind foot protruded and was gently held in position within the radiation field by three Perspex posts. Details of this system have been described by Douglas and Fowler [12] and Joiner et al. [23]. Neutron irradiations at Clatterbridge

p(62)-Be neutrons were produced by bombarding a beryllium target with 62 MeV protons from a cyclotron accelerator. Each mouse foot was irradiated at one of 5 positions along the edges of an open square field 16.5 x 16.5 cm. A mean dose rate of 0.6 Gy - min- ’ was achieved for a mean target current of 30 PA. Neutrograms of the radiation field demonstrated that the dose uniformity along each foot was approximately &4%. The variation in mean dose at the different positions was f 1.6%. However, for all irradiation schedules, mice were either moved through consecutive irradiation positions at each subsequent fraction (8 and 16 fraction

155 TABLE 1 Radiation

beams intercompared.

Radiation

source

--__-

62 MeV p-Be neutrons: Cyclotron

at U.K.

Clutterbridge,

16 MeV d-Be neutrons: Cyclotron at

U .K .

Hummersmith.

4 MeV d-Be neutrons: van de Graaff’ accelerator at Gray Laboratory,

Northwood.

U.K.

250 kVp X-rays: Hummersmith,

U.K.

No. of fractions

N + ;‘dosc per fraction (Gy)

d(4)-Bc neutron top-up dose (Gy)

Single dose

15.0 -30.0

None None None None None 2.3-14.5

2 4 8 16 16 Single dose 2

8.5 -18.0 4.5 -10.0 2.25- 5.5 1.2 - 3.0 0.1 - 1.2 12.9 -26.4 6.58-13.15

4

3.64- 7.12

8 8

1.98- 3.84 0.16- 0.94

Single dose

8.64-17.28

2 4 8 16 16

4.612.361.210.610.06-

Single dose 2 4 8 16 16

schedules) or moved to a different position halfway through irradiation (1, 2 and 4 fractions). This reduced the maximum to minimum variation in total fractionated dose for the mice in each dose group to < 0.3% for 5 mice per dose group (1,2,4, 8 and 16 fraction groups) or < 0.1% for 4 mice per dose group (groups receiving 16 fractions + subsequent top-up of d(4)-Be neutrons). All radiation dosimetry at Clatterbridge was carried out according to the European Protocol [3], and doses are quoted as the total dose (i.e. neutron plus gamma dose). The gamma contamination in these experiments was estimated to be 2 f 1% of the total N + y dose (S.W. Blake and D.E. Bonnett, pers. commun.). The high-energy neutron facility at Clatterbridge has been described in detail previously [2,27].

20.0 13.0 8.0 5.0 3.0 0.4

None

None None None 5.5-14.5

9.33 4.78 2.42 1.21 0.52

None None None None None 2.3-14.5

-38.0 -23.0 -14.0 - 8.75 - 5.0 - 2.5

None None None None None 2.3-14.5

Neutron iwadiarions at Hatmtermrirlt d( 16)-Be neutrons were produced by the reaction of 16 MeV deuterons with a beryllium target. The physical characteristics of this beam and its use as both a clinical radiotherapy machine and experimental facility have been described previously 141. The radiation field and position of the mouse feet were the same as those at Clatterbridge. Target to skin distance was 100 cm giving a mean dose rate of 0.45 Gy - min -’ for a target current of 60 /iA. The maximum to minimum variation in total fractionated dose for the mice in each dose group was estimated to be < 1% as a result of changing the position of the feet within the square field halfway through irradiation or at consecutive fractions. Doses are quoted (unless stated otherwise) as the total N + y dose, according to the European Protocol. The gamma contamination was estimated to

156 be 3.2% of the total N + y dose (C.J. Parnell, pers. commun.). Neutron irradiations at the Gray Laboratory

The irradiation system for mice feet at the Gray Laboratory has been described in detail by Joiner et al. [23,25]. d(4)-Be neutrons were produced by a van de Graaff accelerator from the reaction of 4 MeV deuterons with a thick beryllium target. Average dose rates of 0.6 Gy - min-’ were achieved, with a gamma contamination of 13.2% of the total N + y dose. The top-up doses are quoted as neutron dose alone, without the gamma component, which is consistent with our normal practice [ 11,231. X-irradiations

X-ray treatment was carried out using techniques described by Douglas and Fowler [ 121. 250 kVp X-rays were generated with a Marconi X-ray set and filtered with 0.25 mm Cu + 1 mm Al, giving an HVL of 1.3 mm Cu. Dose rate was 2.0 Gy min-’ for the 1,2,4,8 and 16 equal fraction treatments. It was reduced to 0.41 Gy - min-’ for the dose groups given 16 smaller dose fractions of 0.4-2.5 Gy per fraction prior to a d(+Be neutron dose. This allowed sufficient radiation exposure time to avoid significant dose-errors due to the time taken in opening the shutter.

daily score” was calculated for a constant period covering the time of appearance to disappearance of the reactions for the lower doses, by summing the reactions for each day and dividing by the number of days. A mean reaction and “standard error” were then derived for each dose group of 4 mice. Although this procedure does not have strict statistical validity, since the scoring scale is non-parametric, it is used to enable smooth mono$zmc dose-response curves to be constructed, from which isoeffect doses can be derived for the different treatment schedules. Standard parametric statistical tests can be applied subsequently to these isoeffective dose values. The period from 10 to 32 days after a single d(4)-Be neutron dose was chosen arbitrarily as a reference for analysis. In some of the schedules, notably dose groups receiving a d(4)-Be neutron topup dose, the reaction appeared a few days later. The time of assessment was adjusted for these dose groups to the time course for reaction after single d(4)-Be neutron doses, by matching the time course curves for the appearance of the reactions and using the corresponding 23-day period for averaging [9].

Results Full course j?actionation

Skin reaction scoring system

The method for scoring the skin reactions on the foot and the numerical scale used here are described by Douglas and Fowler [12] and based on the method developed originally by Fowler et al. [19]. The use of this system to study RBE relationships has been documented extensively (e.g. [13,25,26]). The advantages and problems associated with the interpretation of measurements made using this arbitrary ordinal scoring scale have also been discussed [I I]. Briefly, the skin reactions were scored 3 times a week between 7 and 40 days after the commencement of radiation treatment. The time course for development of damage was found to be “smooth”, and similar for X-rays and the different neutron arms, as shown previously [lo], making comparisons possible. For each mouse, an “average

Figure 1 shows dose-response curves for skin reactions obtained on mouse feet after irradiation with the d(l6)-Be neutron beam (Fig. la) and the p(62)-Be neutron beam (Fig. lc). For both neutron beams, consistent data were obtained showing a monotonically increasing skin reaction against total dose, given as 1, 2, 4, 8 or 16 equal fractions. For equal effectiveness, the doses with p(62)-Be neutrons were about lo-20% greater than those with the d(l6)-Be beam. This is rather less than we had predicted from the change in RBE for skin and gut as a function of neutron energy [ 171 and from a comparison of clinical data between Hammersmith [4] and the Fermi Laboratory [6]. As a result, several of the higher dose-groups of mice irradiated at Clatterbridge were slightly overdosed (Fig. lc).

3 d(16)-

Be

a

2 ! ls ._ t; 0 L

0 0

I 10

, 20

I 30

I 40

501

0

5

10

15

20

25

d

0 0

10

20

30

40 (N+y

50

0

) total

20

40

60

80

dose(Gy1

Fig. 1. Average skin reaction pMed against total dose for the three different neutron beams, and X-rays. divided into I. 2.4. 8 or 16 equal dose fractions. About 12% more dose is needed as p(62)-Be neutrons compared with d( 16)-Be neutrons for equal reactions. There is only a slight increase in totali dose with increasing fractionation for the three neutron beams, in contrast to X-rays which give a large increase in total dose with increasing fractionation, and the least effect per gray.

Figure 1 also shows that a small, but significant, dose-sparing was present for both neutron beams, when single radiation doses were subdivided into equal dose fractions. For the d(l6)-Be beam, this was apparent up to 8 fractions. Unfortunately, 16 fractions were not given due to a limitation on the beam time available, but previous skin data summarised by Joiner et al. [25] suggest that there would be a further increase in total dose of only about 2-3%, i.e. about 0.5 Gy, if 8 fractions were further subdivided into 16. The data from the p(62)-Be beam (Fig. lc) demonstrate a bigger increase in total dose when a single treatment is divided into 2 and 4 fractions, but there was no indication of a further increase beyond 4 fractions. The response of skin to d(4)-Be neutrons is shown in Fig. lb. Although again there is a slight

dose-sparing with increasing fractionation, this is considerably less than with the other two neutron beams and the doses needed to produce a given reaction are about 75% of those needed with p(62)Be or d(l6)-Be neutrons (Figs. la,c). Figure Id summarises the X-ray results, which confirm the well-known large increase in total dose with increasing fractionation for photons. This difference in capacity for recovery between doses is the principal reason for the increasing RBE with decreasing dose per fraction [16]. Top-up studies The data from the top-up studies are presented graphically in Fig. 2, with skin reaction plotted against the final top-up dose of d(4)-Be neutrons

158

3

0.94

(N+7 Idose Per fraction d(16)-Be t 0fractions

0.31

- N dose per frahon d(4)- Be Iii fractions

0.15

_I_

0.45 0.35 0.25 0.15 0.05 OGy

d (4)-Be neutrons alone ‘b

(N -k-7 Idose per fraction p(62)-

3

12

1.0 0.75 0.5 0.3 0.1 OGy f

Be 16fractions

per

- fraction X-ray dose

2

-2.5

1.5

1 0.4

I

I I I 12 14

OGY

- 16 fractions

2/ .dY 1‘

alone d

C 0

0

”

2

”

4

”

6

’

”

8

n

’

I

I

n

n

’

0 16 10 12 14 Final d (41- Be neutron top-up

L 2

I 4

I

I 6

I

I 8

I

I 10

I

I 16

dose (Gy)

Fig. 2. Skin reaction against the final top-up dose of d(4)-Be neutrons (neutron dose alone), given after 8 or 16 equal very small dose fractions of all the radiations studied. The data points on each curve represent the same very small dose per fraction as shown, but given graded single top-up doses of d(4)-Be neutrons to produce a full dose-response curve. The leftwards displacement of these curves thus reflects the increasing underlying effect with increasing small dose per fraction.

given at the Gray Laboratory. These top-up doses are quoted as neutron dose alone, without the gamma component. Each separate dose-effect curve shows the response to 16 fractions of a specific small dose of p(62)-Be neutrons (panel c), X-rays (panel d), or d(4)-Be neutrons (panel b), or 8 fractions of a specific dose of d( 16)-Be neutrons (panel a). All these schedules were followed by graded d(4)-Be neutron top-up doses in order to elicit the full range of skin reactions. The leftwards shift (to lower top-up doses) of each dose-effect curve away from the reference curve for a single dose of d(4)Be neutrons (i.e. no priming treatment), represents the underlying effect of the 16 or 8 priming fractions [21]. This leftwards shift is termed the equivalent remembered dose (ERD) and is measured in units of grays of d(4)-Be neutron dose alone, since this is ho-w the top-up doses are expressed. As the prim-

ing dose per fraction is increased the ERD increases. For the neutron dose fractions (panels a,b,c) the ERD is approximately linear with the dose per fraction displayed at the top of each dose-effect curve. This reflects the essentially linear underlying doseresponse relationship for neutrons, with a consequent lack of dose-sparing with increasing fractionation for doses less than about 1 Gy per fraction. The relationship between ERD and the (N + y) dose per fraction of each neutron beam, or dose per fraction of X-rays, is given in Fig. 3. Each data point shows the mean and standard error of the ERD measured over the complete dose-effect curve, for each dose per fraction. The data are actually shown as the ERD perfraction [23] since this allows a direct comparison of the results from the d(16)Be beam (Fig. 2a) where only 8 fractions were given in the top-up studies, with all other beams where

159 0.5

d(l6)-Be

r

.01.8r

T 16F + Top-up

Y

16F

6 5 . 1.2

0.5 (N+r)

1.0 1.5 dose per fraction

2.0

2.5

(Gy)

Fig. 3. Equivalent Remembered Dose (ERD) per fraction, derived from Fig. 2 (see text) as a function of (N + 7) dose per fraction of each neutron beam, or dose per fraction of X-rays. ERD is measured in units of grays of d(4)-Be neutrons alone. This plot may be used directly to obtain RBE values (see text). Data points are mean f S.E.M.

16 priming fractions were given. ERD per fraction is clearly linear with dose per fraction for the 3 neutron beams at doses below 1.2 Gy per fraction and straight lines were fitted to the data by weighted least-squares linear regression, forcing the lines of best-fit through the origin. The curved line through the X-ray data was fitted using the linear-quadratic (LQ) model (see Discussion) and demonstrates that a continued increase in X-ray total dose would be expected with decreasing dose per fraction (increasing number of fractions) below 2.5 Gy. Comparison of dosesfor the p(62)-Be and d( 16) -Be neutron beams

The principal aim of this study was to compare the responses to neutron irradiation at Clatterbridge (p(62)-Be) and Hammersmith (d( 16)-Be). This was required in order to relate the extensive clinical experience gained previously at Hammersmith [4] to dose regimes for the higher neutron energy at the new clinical facility in Clatterbridge. Figure 4 shows the ratio of (N + y) doses at these centres (p(62)-Be/d( 16)-Be) needed to give the same skin reactions, as a function of the d( 16)-Be neutron dose per fraction. We have termed this ratio the

!? 1.0 0.1 x

.2 d(l6)-Be

4

.6

1

8F

2

4F

4

N+? dose per fraction

6

2F

,o

1F

20

30

(Gy)

Fig. 4. Ratio of N + 7 dose needed for equal effects using p(62)-Be and d(lh)-Be neutrons. Data points are mean f S.E.M. Horizontal bars show the rurlge of doses per fraction within each fractionation schedule, contributing lo the dose ratio measurements. Although 16 fraction-irradiations were not done with d( 16)-Be neutrons at Hammersmith, a 16fraction dose ratio (0) was estimated using the p(62)-Be dara and assuming no further dose increment between 8 and 16 fractions for the d( I6)-Be beam. The solid line is fitted by eye to the data, assuming an NDR of 1.28 below I Gy per fraction. The two aberrant data points at the lowest doses could indicate higher NDRs as shown by the dotted line (see text).

neutron dose ratio (NDR). The data points from 1 to 20 Gy per fraction are the mean value f standard error of the N R, derived from Fig. 1 by comparing the doses for each beam needed to give equal skin reactions for the same schedule of fractions over the whole range of effect covered by the dose-effect curves. The horizontal bars on each point show the range of dose per fraction that this range of effect represents. This simple approach of presenting the dose ratio as a single mean value for each fractionation schedule is justified here because these data demonstrate no significant change in NDR with dose per fraction between 1 and 20 Gy. The results in Fig. 4 below 1 Gy per fraction were derived from the top-up data summarised in Fig. 3. Equivalent ERD values imply equivalence of the pretreatments [21,23] and so the NDR was derived initially by comparing each d(l6)-Be neutron data point with the best-fit line through the p(62)-Be neutron data points. This procedure gives an estimate of a dose ratio at a given dose per fraction of

160

d( 16)-Be neutrons. Error estimates are standard errors obtained by combining the variances contributed by the d(l6)-Be data points and the leastsquares regression analysis of the p(62)-Be data, using Fieller’s theorem. The results suggest that the NDR increases below 1 Gy per fraction. However, these NDR values have considerable uncertainties and in fact Fig. 3 suggests that the two highest values of NDR in Fig. 4 at the lowest doses may be due to aberrant ERD values for d(l6)-Be neutrons which are higher than the linear fit to all the d(l6)-Be ERD data, rather than to any change in effectiveness with dose for p(62)-Be neutrons where the lowest dose data points lie exactly on their best straight line. Figure 3 shows that it is most likely that the ERD is linear with dose for the 3 neutron beams, thus implying a constant NDR below 1 Gy per fraction as given by the ratio of the slopes of the lines. This reasoning leads to a fixedNDR for d(l6)-Be and p(62)-Be neutrons of 1.28 f 0.05 (S.E.M.) below 1 Gy per fraction as shown by the solid line in Fig. 4. Intercomparison of all 3 neutron beams

The effectiveness of all 3 neutron beams is compared in Fig. 5, by plotting the RBE* relative to 250 kVp X-rays, as a function of X-ray dose per fraction. In this case to calculate RBE, the dose for each neutron beam has been expressed as neutron dose alone, excluding the additional gamma component, since this approach has been argued to be the least error-prone method for determining the biological effectiveness as a function of neutron energy [15]. Figure 5 demonstrates the increasing biological effectiveness with lower neutron energy. These data also indicate an essentially constant ratio of neutron dose for isoeffect of about 1.6 between the d( 16)-Be and the d(4)-Be beams over the complete range of dose per fraction studied.

I A

1’

“11111 .6 .6

.4

1

X-ray

2

4

6

dose per Fraction

810

I 2

4

(Gy)

Fig. 5. RBE three neutron and 3 as the the same skin

as a function of X-ray dose per fraction for the beams. These data were calculated from Figs. I ratio of X-ray dose to neutron dose u/one to give reaction (see text). Data points are mean f S.E.M. ); d(lWBe neutrons (A); p(62)-Be neutrons fitted directly to these data by non-linear least squares regression (see Appendix).

Discussion

The results of these experiments and particularly the comparison between p(62)-Be and d(l6)-Be neutrons summarised in Fig. 4, enable estimates to be made of the p(62)-Be doses at Clatterbridge which would be equivalent, in terms of skin tolerance, to the doses given previously in the clinical protocols at Hammersmith using d(l6)-Be neutrons. The most-used clinical treatment at Hammersmith was 16.45 Gy total neutron dose (European Dosimetry Protocol) given in 12 equal fractions over 26 days [1,4], i.e. 17.6 Gy total N + y dose, if m 7% is taken as a representative figure for the proportion of the total N + y dose given as photon contamination at depth in the body at Hammersmith. Thus the equivalent treatment at Clatterbridge, using p(62)-Be neutrons, would be 17.6 multiplied by the NDR derived from Fig. 4 at the dose per fraction of 1.47 Gy (17.6/12), i.e. 17.6 x 1.12 = 19.7 Gy total N + y dose. Comparison with other tissues

*RBE (Relative Biological Effectiveness) = Dose of X-rays to give the same effect. Dose of neutrons

Other biological systems that have been irradiated at Clatterbridge with p(62)-Be neutrons include

TABLE 11 The ratio of neutron present.

+ gamma dose (p(62)-Bc/d( 16)-Bc) for isoeffect (the NDR) for various tissues. All references arc

System

Dose range of observations

Author

NDR range

Bewley et al.

I.2

1.8 -9

Hornsey et ai.

1.2 -1.13

Mouse kidney

0.19-9.6

Joiner

I.J?-1.35

Mouse skin

1.3 -23.6 0.16- 0.94

Present data Present data

1.09-1.16 1.14-1.32

unpubli&&at

(GY) Cells in vitro Intestinal

crypts (mouse)

4-8

mouse small intestine (S. Hornsey, pers. commun.), mouse kidney (Joiner, in preparation) and V79 cells in vitro (D. Bewley pers. commun.). NDR values for these systems are summarised in Table II. For cells in vitro, an NDR of 1.2 was found for isosurvival, at d( 16)-Be neutron doses in the range 4 to 8 Gy. This compares with about 1.14 for similar doses per fraction in skin (Fig. 4). In the gut, an NDR of 1.13-1.2 was found for single doses, 2 and 4 fractions of radiation, covering the d( 16)-Be neutron dose per fraction range of 9 to 1.8 Gy. In skin, we found the mean NDR to be 1.13 over the same dose range. Thus the early-reacting tissues studied so far (skin and gut) and cells in vitro indicate a dose-increase for p(62)-Be neutrons of 9-20% compared with d(l6)-Be neutrons over a wide dose range (Table II). For late effects in the mouse kidney, we have also compared directly the effects of the p(62)-Be and d( 16)-Be neutron beams (Joiner, in preparation). For equal effectiveness as measured by loss of renal clearance, reduction in haematocrit and increase in urine output, an almost-constant NDR for p(62)Be/d( 16)-Be neutrons of 1.35-l .42 was found in the d(l6)-Be neutron dose per fraction range 0.2 to 9.6 Gy. For the “standard” clinical protocol using the d(l6)-Be beam at Hammersmith (1.47 Gy per fraction) the NDR would be 1.40. These data suggest that late renal injury might be spared by moving from the low to high neutron energy or alternative-

ly, that kidney should not be included in a low energy neutron field. Fitting the LQ model to the data

The response of mouse skin to different radiations has been shown previously to be well-described by the LQ modei [25]. This model also applies to a wide range of other normal tissues [ 181. If E represents an arbitrary numerical constant representing the underlying effect responsible for damage, then the LQ model can be summarised by the equation E = n(ccd + @P) = &I + JdD

(1)

where d is the dose per fraction, and 11and D are the number of fractions and total dose needed to achieve the effect E. A common use of the LQ model is to relate the dose per fraction to the total dose (or number of fractions) needed to achieve isoeffective tissue damage, when a radiation treatment is split into multiple equal dose fractions. This was also the purpose of the NSD and CRE concepts, but the LQ model is now considered to be more appropriate [18]. The formula for predicting dose changes can be derived simply from Eqn. (1): D2 -Dl

=

4

+

CdP)

d2

+

&‘P)

(2) *

162 From this, it can be seen that the important parameter in the LQ model is the ol/j3ratio. For photons, a//I is generally 6-12 Gy for early-responding tissues like skin and l-3 Gy for late-responding tissues [18]. For neutron irradiation, these values are much higher and expected to be in the region of 40-100 Gy for skin [25]. Dale [7,8] has discussed the LQ model in some detail and has described, with clinical examples, how the cr/fl ratio may be used to determine any equivalent irradiation schedule involving both fractionated and protracted (low dose rate) radiotherapy. The LQ model is also useful in comparing differeni radiations as in the present studies, since the RBE or NDR for two radiations may be predicted at any dose per fraction, using a simple equation containing a/P ratios for both radiations and an

additional LQ parameter, a&tz. This is the ratio of the 01value in Eqn. (1) for the two radiations (1 and 2) and is actually the limiting value of RBE or NDR at vanishingly low doses per fraction [24]. We initially determined a//3 values for the 4 radiations compared in this study, as well as the ratio of cx values, using a weighted non-linear least squares regression of total dose against number of fractions (see Appendix) for an isoeffect equal to a skin reaction of 1.5. The parameters obtained for the different radiations are shown in Table III (top), and have been calculated for doses expressed both as neutron + gamma dose and as neutron dose alone. The CIvalues for the 4 radiations decrease progressively with increasing neutron energy, i.e. with decreasing LET. The ratio of a between p(62)-Be

TABLE III Parameters in the LQ model for the four radiations studied. Neutrons

250 kVp X-rays

d(4)-Be Neutron dose alone Isoeffect doses at SR = 1.5

62 f 19 8.96 +

0.79

+ y dose

Isoeffect doses at SR = 1.5

p(62)-Be

Infinite @lP a/ax

Neutrm

d( 16)-Be

a@

72 f 22

a/u,

7.78 f

0.69

a/B

28.8 f

9.6

U/G

8.46 f

0.55

(indeterminate errors) 6.41 f 0.65 Infinite (indeterminate errors) 6.21 f 0.62

90 f 57 4.81 f

0.46

92 f 59

9.7 f 1.9 1 9.7 f 1.9

4.71 *

0.45

1

27.7 f

6.8

8.6 f 1.5

4.16 f

0.23

1

28.3 f

6.9

8.6 f 1.5

4.07 f

0.22

1

Neutron dose alone

Direct fitting of RBE versus dose

24.7 f 5.4 4.79 f 0.33

Neutron + y dose

Direct fitting of RBE versus dose

alp ula,

33.2 f 11.0 7.34 f

0.48

25.5 f 5.6 4.64 f 0.32

Values in the top half of the table were derived from the data expressed either as neutron dose alone or as the neutron + gamma dose, by weighted non-linear least squares regression of isoeffect doses at a skin reaction level of 1.5 using Eqn. (Al) [28]. Values in the bottom half of the table were derived by fitting RBE versus dose data directly using Eqn. (A2) [24]. a/a, shows the value of a normalised to that for X-rays and is thus the limiting RBE for each neutron beam at vanishingly small doses per fraction. The limiting dose ratio for any2 neutron beams at vanishingly small doses is then the ratio of their a/a, values. Errors are standard errors of the mean.

(N+y)

dose

perfraction

(Gy)

Fig. 6. Underlying dose-response curves for neutrons and X-rays. These were constructed by measuring isoeffect total doses for the different fraction numbers, at a skin reaction of 1.5. Single doses are plotted at a Proportion of Full Effect = 1.0. the 2-fraction dose per fraction at a value of 0.5, 4-fraction dose per fraction at 0.25, etc., according to Douglas and Fowler [12]. The top-up data were included by calculating an equivalent fraction number for each small dose per fraction (see Appendix). The left hand panels show the full curves and the right hand panels show the low-dose regions enlarged.

and d( 16)-Be neutrons suggests a limiting dose ratio (i.e. NDR at very low doses per fraction) for isoeffect of 1.33 f 0.10 for dose expressed as neutrons alone, or 1.32 f 0.10 for N + y dose. These values, derived from all the data, are in close agreement with the value of 1.28 f 0.05 obtained earlier from the top-up data alone (Fig. 3). It is always difficult to obtain statistically reliable estimates of the dose-squared fi term for neutron responses, since the shape of these responses tends to be dominated by the “single-hit” linear component 01.This is reflected in the large uncertainty in the a/j? values for the neutron beams, in contrast to X-rays. The generally large values of or//3seen, however, do reflect the much smaller changes in total dose with fractionation seen with all the neutron

beams compared with photons. The cl//? ratio for d(l6)-Be neutrons was particularly large in these experiments. However, Fig. 6 shows that while this represents the best value for all the data (full course fractions and top-up data) the full course fractionation data and top-up data do not fit together particularly well for d(l6)-Be neutrons. The full course fractionation data alone, expressed in terms of N + y dose, give C& = 4.92 f 0.44 and a//? = 47.2 f 8.4 Gy in the dose per fraction range 2.9 to 17.8 Gy. The other 3 sets of data plotted in Fig. 6 for p(62)-Be neutrons, d(4)-Be neutrons and 250 kVp X-rays, are all fitted reasonably well by a single LQ equation over the complete dose range. Joiner and Johns [24] have shown that RBE versus dose relationships (e.g. Fig. 5) may be used di-

164 rectly to obtain estimates of the a//_?ratios for the two radiations involved in such comparisons, and also the third parameter, al/ctz, which represents the limiting (constant) RBE or NDR at vanishingly small doses per fraction (or alternatively the ratio of the initial slopes to the “survival curves”). This approach, described in the Appendix, usually gives smaller confidence limits on the estimates of these LQ parameters than the analysis of isoeffect doses described above. This is because all the original measurements (Figs. 1 and 2) are effectively contributing in the RBE values (Fig. 5) rather than just the subset of the measurements in the middle of the range of response which determine isoeffect doses. The results of this analysis are shown in Table III (bottom). As expected, the parameter values are not significantly different from those obtained from the analysis of isoeffective doses (Table III, top), but the errors are reduced with a particularly noticeable improvement in the resolution of the cl//? values. Clearly, a linear dose component of underlying effect dominates the response to all these neutron beams and so their relative effectiveness with respect to each other is determined largely by the ratio of a values in the LQ model. The Mvalues determined by fitting the RBE data directly would indicate a limiting p(62)-Be/d(l6)-Be N + y dose ratio of 1.14 f 0.1 from this method. This lower value, although not significantly different from either the value obtained directly from the top-up data alone (1.28 f 0.05) or from LQ analysis of isoeffect doses (1.32 f O.l), reflects the much larger errors on the RBE estimates from the top-up experiments since in the direct fitting of RBE the data points were weighted by the inverse of their errors (see Appendix).

Conclusions

(1) For equal effects in mouse skin in the dose range 1.5 to 19 Gy (16 fractions to single doses) 9-16% more dose is needed with the p(62)-Be Clatterbridge beam compared with d( 16)-Be neutrons at Ham-

mersmith. There is little further sparing of dose with fractionation beyond 4 fractions for either of these neutron beams so that the dose ratio is fairly uniform over this dose range. This result is also reflected in the high al/p ratios for p(62)-Be neutrons and for the other two neutron beams studied. (2) At lower doses per fraction (< 1 Gy), the data from the top-up studies indicate that the ratio of p(62)-Be to d(l6)-Be N + y dose may increase. However, it is most likely that the dose ratio for these two neutron beams is constant below 1 Gy per fraction at 1.28 f 0.05 (S.E.M.). Two other different analyses of the full course fractionation data and top-up data together, using the LQ model, give limiting dose ratio estimates of 1.32 f 0.10 and 1.14 f 0.10 (S.E.M.). These two values are not significantly different. (3) These mouse skin data indicate that the “standard” clinical treatment of 1.47 Gy per fraction (N + y dose) in 12 fractions given previously with d(l6)-Be neutrons at Hammersmith would correspond to a dose of 1.65 Gy per fraction (N + y dose) for the p(62)-Be neutron beam at Clatterbridge. This value applies to the surface only. The predicted dose would need to be corrected further to take account of any change in beam hardness with increasing depth of treatment. Acknowledgements

This study was supported jointly by the Medical Research Council and the Cancer Research Campaign. A substantial number of people helped with the experiments, which were performed on four machines in three institutes. We are deeply indebted to all of them. We should like to thank particularly Mr. T. Saxton, Dr. D. Bonnett, Mr. S. Blake, Mr. W. Ranken, Ms. J. Finch and Mr. A. Taunton at Clatterbridge; Dr D. Bewley, Dr. J. Parnell, Dr. M. Law, Mr. B. Page, Mr. K. Butler, Mr. R. Ahier and Miss C. Morris at Hammersmith; Mr. B. Hall, Dr. B. Michael, Dr. M. Folkard, Dr. B. Vojnovic, Miss F. Adam and Miss H. Johns at the Gray Laboratory.

165 References Bewley. D.K., Parnell, C.J. and Williams, J.R. Dosimetry of the neutron beams at Hammersmith and Edinburgh. Br. J. Radiol. 59: 529-530, 1986. Bonnett, D.E., Blake, SW., Shaw, J. and Bewley, D.K. The Clatterbridge high-energy neutron facility: specification and performance. Br. J. Radiol. 61: 3846. 1988. Broerse, J.J., Mijnheer. B.J. and Williams, J.R. European protocol for neutron dosimetry for external beam therapy. Br. J. Radiol. 54: 882-898, 1981. Catterall, M. and Bewley. D.K. Fast Neutrons in the Treatment of Cancer. Academic Press. London; Grunt & Stratton, New York, 1979. Catterall, M., Sutherland, 1. and Bewley, D.K. First results of a randomised clinical trial of fast neutrons compared with X or gamma rays in the treatment of advanced tumours of the head and neck. Br. Med. J. 2: 653-656, 1975. Cohen, L., Hendrickson, F.R.. Kurup. P.D.. Mansell. J.A.. Awschalom, M., Rosenberg, I. and Haken, R.K.T. Clinical evaluation of neutron beam therapy. Cancer 55: IO-I 7, 1985. 7Dale. R.G. The application of the linear quadratic dose effect equation to fractionated and protracted radiotherapy. Br. J. Radiol. 58: 515-528, 1985. 8 Dale, R.G. The application of the linear quadratic model to fractionated radiotherapy where there is incomplete normal tissue recovery between fractions, and possible implications for treatments involving multiple fractions per day. Br. J. Radiol. 59: 919-927, 1986. 9 Dcnekamp. J. Changes in the rate of repopulation during multifraction irradiation of mouse skin. Br. J. Radiol. 46: 38 I-387. 1973. IO Denekamp. J., Fowler. J.F.. Kragt, K.. Parnell. C.J. and Field S.B. Recovery and repopulation in mouse skin after irradiation with cyclotron neutrons as compared with 250 kV X-rays or I5 MeV electrons. Radiat. Res. 29: 71-84. 1966. II Denekamp. J., Joiner. M.C. and Maughan. R.L. Neutron RBE’s for mouse skin at low doses per fraction. Radiat. Res. 98: 317-331. 1984. I2 Douglas, B.G. and Fowler. J.F. The effect of multiple small doses of X-rays on skin reactions in the mouse foot and a basic interpretation. Radiat. Res. 66: 401-426. 1976. 13 Field, S.B. Early and late reactions in skin of rats following irradiation with X-rays or fast neutrons. Radiology 92: 381-384, 1969. I4 Field, S.B. An historical survey of radiobiology and radiotherapy with fast neutrons. Curr. Top. Radiat. Res. I I: l86, 1976. I5 Field, S.B. The expression of neutron dose. Br. J. Radiol. 60: 315, 1987. I6 Field, S.B. and Hornsey. S. RBE values for cyclotron neutrons for effects on normal tissues and tumours as a function of dose and dose fractionation. Eur. J. Cancer 7: 161-169, 1971.

I7 Field. S.B. and Hornscy. S. Neutron RBE for normal tissues. In: High LET Radiations in Clinical Radiotherapy. pp. 181-186. Edits: G.W. Barcnsdcn. J.J. Brocrsc and K. Pergamon Press. Oxford, 1979. 18 Fowlcr, J.F. What next in fractionated radiotherapy? Br. J. Cancer, 49: 285-300, 1984. 19 Fowler. J.F.. Kragt, K.. Ellis, RE., Lindop. P.J. and Berry. R.J. The effect of divided doses of I5 MeV electrons on the skin response of mice. Int. J. Radiat. Biol. 9: 241-252. 1965. 20 Joiner. M.C. Extension of the reciprocal dose analysis to the case of multiple fractions plus top-up. Radiother. Oncol. 7: 349-351, 1986. 21 Joiner. M.C. The design and interpretation of top-up cxperiments to investigate the effects of low radiation doses. Int. J. Radiat. Biol. 51: 115-130, 1987. 22 Joiner, M.C. and Denekamp. J. Evidence for a constant repair capacity over 20 fractions of X-rays. Int. J. Radiat. Biol. 49: !43-150. 1986. 23 Joiner. M.C.. Denckamp, J. and Maughan, R.L. The use of top-up experiments to investigate the effect of very small doses per fraction in mouse skin. lnt. J. Radial. Biol. 49: 565-580, 1986. 24 Joiner, MC. and Johns, H. Renal damage in the mouse: the effect of d(4)-Be neutrons. Radiat. Res. 109: 456-468, 1987. 25 .toincr. M.C., Maughan. R.L.. Fowler. J.F. and Denckamp. J. The RBE for mouse skin irradiated with 3 Mev neutrons: single arid fractionated doses. Radiat. Res. 95: 130-141. 1983. 26 Leith, J.T.. Schilling. W.A.. Lyman. J.T. and Howard, J. Comparison of skin responses of mice after sing12 or fractionatcd exposure to cyclotron-accelerated helium ions and 230 kV X-irradiation. Radiat. Res. 62: 195-215. lY75. 27 Shaw, J.E.. Bonnett, D.E. and Blake. S.W. The Clatterbridge high:energy neutron therapy facility: measurements of beam parameters for clinical use. Br. J. Radiol. 61: 47-53. 1988. 28 Tucker. S.L. Tests for the lit of the linear quadratic model to radiation isoefect data. Int. J. Radiat. Oncol. Biol. Phys. IO: 1933-1939, 1984. 29 Van Putten. W.L.J. Comment on a proposed test of the fit of the linear quadratic model to radiation isoeffect data. bit. J. Radiat. Oncol. Biol. Phys. 12: 290-291. 1986.

Appendix Fitting the LQ model to the data (A) Analysis of isoqffective doses_for each radiation For a chosen level of observed effect, the LQ model can also be expressed by the equation:

166 malised to or/E for X-rays, which was 0.00844 f 0.00072 (S.E.M.).

where D is the total dose given as n equal fractions, E is a constant for the chosen level of injury, and CIand p are constants for a particular tissue assay and type of radiation. For the full course fractionation data (Fig. 1) where 1-16 fractions were given (i.e. 12= 1, 2, 4, 8 or 16) total doses were read from the dose-response curves at an isoeffect level of skin reaction equal to 1.5, for each value of n. For the low dose per fraction study (Fig. 2) where top-up doses of d(4)-Be neutrons were given, the top-up doses at skin reaction = 1.5 were read from the curves for each small dose per fraction. These top-up doses were then used to calculate an Equivalent Total Dose and Equivalent Number of Fractions, which would be the values if the whole treatment had been given as the small dose fractions [22]. These equivalent total dose and fraction number data were then grouped together with the total doses for the full course fractionation (1-16 fractions) and all the data fitted to Eqn. (Al) using a weighted non-linear least squares regression of total dose against number of fractions as described by Tucker [28] and van Putten [29]. d(4)-Be neutrons were given as 16 small doses plus top-up as well as full course fractionation, and since the top-up radiation is identical to the radiation given in the 16 small doses, the method of Joiner [ZO]was used to derive the LQ parameters in this case. In Table III, values of a/E in Eqn. (Al) are nor-

(B) Fitting the RBE versus dose relationships directly

This technique has two potential advantages over the method of fitting the LQ model to isoeffect doses. First, all the data from both the full course fractionation study (Fig. 1) and the top-up study (Fig. 2) may be included together directly in one analysis, without the need for calculating Equivalent Total Doses from the top-up data. Only the RBE values are used, which are measured off as simple dose ratios from Figs. 1 and 3. Second, confidence limits on the LQ parameter estimates should be generally reduced because the RBEs used in the analysis are mean values derived from all levels of effect in Figs, 1 and 2, not just from a single set of isoeffect doses. Using X-rays as a “reference”, we calculated the RBE of each neutron beam as a function of X-ray dose per fraction. These three RBE relationships are shown in Fig. 5 and were fitted simultaneously with non-linear least squares regression, using the reciprocal square of the standard error on each RBE value to weight each point (The SYSNLIN procedure, SAS/ETS Version 5, 1984, SAS Institute Inc., Cary, N.C., U.S.A.). The equation used was K + JKz + 4Kd(l + d/u/C RBE = 2(1 + d/v)

(A2)

where d is the X-ray dose per fraction, K is rxneu_ trons/Olx_rays, C is or/p for neutrons, v is a//3 for Xrays.