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Response of mouse skin and the C3HBA mammary carcinoma of the C3H mouse to X-rays and cyclotron neutrons: Effect of mixed neutron—Photon fractionation schemes
Europ. J. Cancer Vol. 11, pp. 891-901. Pergamon Press 1975. Printed in Great Britain
Response of Mouse Skin and the C3HBA Mammary Carcinoma of the C3H Mouse to X-rays and Cyclotron Neutrons: Effect of Mixed Neutron-Photon Fractionation Schemes* JANET S. R. NELSON, RITA E. CARPENTER and ROBERT G. PARKER Division of Radiation Oncology, School of Medicine, University of Washington, Seattle, Washington, 98195, U.S.A. Abstract--The response of mouse foot skin and the C3HBA mammary adenocarcinoma of the C3H mouse to 250 kVp X-rays and cyclotron-produced neutrons (8 MeV mean energy) has been studied. Neutrons or X-rays were given in single fractions (fx); 2 f x 24 hr apart; 2 f x 96 hr apart; and 5 f x in 5 days. Two f x of neutrons + 3 f x of X-rays in 5 days also were given in the sequence n-n-x-x-x or n-x-x-x-n. When neutron only schemes were compared to X-ray only schemes, RBE's for early skin damage and foot deformity at 6 months post-irradiation increased w~th increasing number of fractions. The RBE for 2 f x of neutrons + 3 f x of X-rays (total dose of neutrons + X-rays) relative to 5 f x of X-rays was 1.3 for both mixed fraction sequences. Tumor growth delay following single and fractionated X-rays suggested an hypoxic fraction of cells which undergoes extensive and long lasting reoxygenation, a situation in which fractionated neutrons might not be expected to show an advantage in terms of tumor damage relative to normal tissue injury. Therapeutic gain factors ( TGF = tumor RBE/skin RBE) for single fractions ranged from 1.6 to 1"8 depending on dose level while TGF's for fractionated neutrons varied from 0.73for 2 f x 24 hr apart to 0.96for 5 f x in 5 days. For both the n-n-x-x-x and n-x-x-x-n fractionation schemes a TGF of 1.1 suggests that the mixed fractionation schemes may be slightly better than neutron only schemes in this tumor system.
INTRODUCTION
times per week. This has indicated that the relative importance of the fractionation scheme, as well as radiation quality, in modifying treatment results also needs to be studied and the response of experimental tumors and normal tissue to various neutron only and X-ray only fractionation schemes has been reported [5-8]. This paper describes the response of mouse skin and the C3HBA m a m m a r y adenocarcinoma of the C3H mouse to 250 kVp X-rays and 8 M e V (mean energy) neutrons produced by the University of Washington cyclotron, using single and multiple fractions of X-rays or neutrons as well as mixed neutron-photon fractionated radiation. The tumor chosen, a
NEUTRON THERAPY of human cancer has necessarily been preceded by experiments intended to biologically characterize different neutron beams [1-4]. Limited medical access to cyclotron-produced neutron beams has resulted in patients being treated 3 or only 2 times per week, rather than the conventional 5
Accepted 8 August 1975. *This investigation was supported by Research Grant # CA-12441-04 from the National Cancer Institute, National Institute of Health. 891
892
J. S. R. Nelson, R. E. Carpenter and R. G. Parker
C3H mammary tumor, is one in which neutrons might be expected not to show an advantage because other mammary tumors in this mouse show rapid and extensive reoxygenation, rendering judiciously fractionated X-rays as effective as neutrons [7, 8]. We chose to examine mixed fractionation schemes, utilizing 2 initial neutron fractions followed by 3 fractions of X-rays over a five day period (n-n-x-x-x) or 2 fractions of neutrons on the first and fifth days, with X-rays on the intervening days (n-x-x-x-n). The rationale was that, in a rapidly reoxygenating tumor, one or two initial neutron fractions could efficiently kill hypoxic tumor cells while subsequent fractions of X-rays would be more effective after the initiation of reoxygenation; a mixed scheme with a final neutron fraction (n-x-x-x-n) would also allow more effective killing of any resistent hypoxic foci left at the end of treatment. Such treatment schemes might produce an increase in therapeutic ratio while taking into account limited cyclotron access for patient treatment.
MATERIAL AND METHODS Radiation sources and dosimetry X-irradiations were performed using a General Electric Maxitron 250; the settings were 250 kVp, 30 mA, 0.5 mm Cu + 1-0 mm A1 added filtration, target to specimen distance (TSD) 50 cm, dose rate 100 R/min. Irradiations were performed with a horizontal beam and an 8 x 8 cm field defined by a ¼ in. thick lead shield. Mice were irradiated while taped to a vertical Lucite board placed directly behind the shield. Dosimetry was done with a Victoreen air equivalent exposure meter using a 250 R chamber and a rad/Roentgen conversion factor of 0.95. Measurements were made with the mouse holder in place to include contributions from backscatter. LiF and LiBO4 thermoluminescent dosimenters were also used to verify absorbed dose in several experiments. These dosimeters were taped to the feet of mice, in the case of skin irradiations, or placed under the skin adjacent to the irradiated tumors. Neutron irradiations were performed at the University of Washington Cyclotron. The neutron beam was produced by bombarding a 1.5 mm thick beryllium target with 21 M e V deuterons, yielding a neutron beam with mean energy of 8 M e V and an energy spread ot 0-25 MeV. Dosimetry was performed with tissue equivalent (TE) ionization chambers (Shonka type A 150 plastic) filled with TE gas [4]. The doses are expressed as total rads of
neutrons plus gammas; gamma contamination was 8% in air at the mouse positions. The neutron beam was defined by a Cu A1 bronze collimator in the initial skin experiment and animals were irradiated at a TSD of 70 cm and a dose rate of 100-125 rad/min. Subsequent experiments employed a borated waterextended polyester (B/WEP) collimator which defined an 18 x 18 cm field at a TSD of 108 cm; the dose rate was 40-50 rads/min. Mice were irradiated while taped to a vertical Lucite board which was placed 2 cm from the collimator exit. Skin irradiations Female BALB/c mice (Jackson Laboratories or Charles River) were anesthetized with Nembutal (sodium pentobarbital, 60/lg/gm body weight) and one rear foot was irradiated with the animals' bodies shielded by the lead shield in the case of X-irradiation and by the bronze or B/WEP collimator in the case of neutron irradiations. Six to nine mice were irradiated at each dose and the feet were immersed in tissue equivalent fluid (C5H4o O18N) [9] to provide for secondary charged particle equilibrium in the neutron irradiation. No significant difference in skin response to single doses of neutrons was seen with the two collimators. C 3 H / H e J male mice, 6-10 weeks old (Jackson Laboratories), were also used in single dose experiments to verify that the two strains of mice had similar skin responses to X-rays and neutrons and yielded the same neutron RBE. Thereafter, skin response RBE's were determined using BALB/C mice and were compared to tumor response RBE's from C3H mice to calculate Therapeutic Gain Factors (TGF = tumor RBE/skin RBE). Early skin response following single fractions was scored over the period of 7-35 days postirradiation, using a scale modified from Fowler et al. [10]. For experiments using multiple fractions of radiation, the ascending curve for skin response vs. days postirradiation was superimposed over similar curves for single fractions to determine which postirradiation day was equivalent to day 7. In all cases, day 8 or 9 was used as the starting point for skin scoring in multiple fraction experiments, and observations were continued until day 36 or 37. Foot deformity at 6 months post-exposure was scored using the scale of Field et al. [11]. Tumor irradiations C 3 H / H e J male mice, 6-10 weeks old (Jackson Laboratories), were housed in the University of Washington Vivarium at least
Response of Mouse Skin and the C3HBA Mammary Carcinoma of the C3H Mouse one week "after arrival before C3HBA mammary adenocarcinomas were transplanted by placing small (1-2 mm dia) pieces of non-necrotic tumor under the skin of the upper back. The C3HBA tumor is a transplantable mammary neoplasm which occurred as a spontaneous tumor in a C3H/An mouse in 1946 and has been carried as a transplantable tumor in C3H/HeJ mice by Jackson Laboratories, Bar Harbor, Maine, since 1949. At 12-13 days posttransplant, tumors of average diameter, 2"7 mm to 5.7 mm were chosen for irradiation. Six to eight mice anesthetized with Nembutal (60 pg/g wt.) were irradiated at each dose. The animals were positioned at the corners of the field with their bodies shielded by the lead shield in the case of X-irradiation or the B/WEP collimator in the case of neutron irradiations.
893
volume was confirmed by comparing calculated volumes to weights and saline displacement volumes of excised tumors. The volume of each tumor on the first day of irradiation was taken as 1.0, and volumes on succeeding days were normalized to this value. The endpoint for tumor radiation response was growth delay, defined as: (no. of days for irradiated tumor to reach 5x starting volume) minus (no. of days for control tumor to each 5x starting volume.) Neutron RBE's were determined by dividing the X-ray dose by the neutron dose which produced equivalent growth delay. RBE's for
Table 1 nud:~o~x
m
Tmm~s Dome/~
1 fx X-za,j,s
x . . . .
2 fx
x-~ca!m
x x
- -
2 fx
X-z'al,'s
x -
- -
5 fx
X--z:m!m
x x
x x
~
dese
Z~ese/fx 1010 - 4540
dome
~
1000 - 4500
1000 - 4000
-
908 - 2270
181.6 - 4 5 4 0
x
1025 - 2465
2050 - 4930
1010 -
2220
2020 - 4440
x
605 - 1260
:3025 - 6 3 0 0
505 -
980
252S - 4540
400 -
2000
400 - 2000
640 - 1810
1010 - 4540 ]280
- 3620
1 fx s'm~cms
n . . . .
700 -
2000
700 - 2000
2 fx ntm~.,~,~
n n
- -
-
500 -
1000
1000 - 2000
7 5 - 1Q00
2 /~x n m . , t x m s
n -
-
-
n
.500 " - 1 1 0 0
1000 - 2200
400 - 1250
800 - 2500
5 fx n~;.L~m
n n
n n
n
260 -
1300 - 2500
100 -
500 -
2 f x ~ + :3 ~ X ~
nx
xx
n
50-625 (tO 958 o~" 1261 (x)
29"74 - 50,33 (n ÷ x)
100400 (n) 908 (x)
2 ~ x nt~L,.,~-., + 3 £'x X - . m ~
n n
2974 - 5033 (n ÷ x)
50 - 4 0 0 ( n ) 908 (x)
~: x
x
500
100 - 625 (n) 958 (z 1261 (x)
For each fractionation scheme, mice from the same transplant group were irradiated concurrently with neutrons or X-rays and their responses were used to determine RBE's. However, repeat experiments showed tumor response to a given dose and fractionation scheme was constant enough to allow data from separate experiments employing different transplant groups to be combined without changing RBE values. Tumors were measured in 3 dimensions 3-5 days/week during and after treatment and tumor volumes were calculated, using the simplified formula: length x width x depth
which closely approximates the volume of an ellipsoid. Measurements were corrected for skin thickness. This method of determining tumor
425
100 -
2000
2125
2924 - 3524 2824 -
3524
mixed neutron-photon fractionation schemes were determined by dividing total X-ray dose by total neutron + X-ray dose. Therapeutic gain factors (TGF), defined as tumor RBE/skin RBE for a specified dose and fractionation scheme, were determined.
RESULTS The fractionation schemes, total doses, and dose per fraction used in skin and tumor response experiments are outlined in Table 1. One fraction of neutrons, two fractions 24 or 96 hr apart, and 5 fractions in 5 days were compared to X-rays given in identical fractionation schemes. In the mixed fractionation schemes, 2 fractions of neutrons plus 3 fractions of X-rays were given in 5 days, in the sequence n-n-x-x-x or n-x-x-x-n, and the responses were compared to those obtained with 5 fractions of X-rays in 5 days. In both the all X-ray and mixed neutron-
894
J . S. R . Nelson, R . E . Carpenter and R . G. Parker
photon options in these experiments, the X-ray dose per fraction was held constant while the neutron dose per fraction was varied. Within the context of such an experiment, an RBE could be determined by seeing which dose of neutrons + X - r a y s was equivalent to the constant total X-ray dose in that experiment (for example, see Fig. 2 and 7). MOUSEFOOTRESPONSE
:¢
e~ 1.5 ¢c ! ~
| o.s
A
y
S E] I fx NEUTRONS
i
i
.
I000
3000
5000
(Fig. 3). Neutron RBE's for early and late damage were read directly from smooth curves fitted to the points on these plots and similar ones for all other fractionation schemes, and are summarized in Tables 2-4.
Tumor response O f the mice irradiated for tumor growth delay, 4% died from anesthetic overdose, 4% died within the first 10 days after irradiation, due mostly to radiation effect on the intestine, and 4% died later than 10 days but too soon for their tumors to be included in the growth delay analysis. Tumor growth following graded single doses of X-rays or neutrons is shown in Fig. 4(a and b). Tumor response to radiation was assessed as tumor growth delay, determined from volume change against time for control and irradiated 5.0-
Tolol dole Ruds Fig. 1. Average early skin response vs dose in rads for single fractions of X-rays or neutrons. Each point is the mean +_ standard error of the average skin response over the period 7-35 days postirradiation for the mice in each dosage group.
i~n-X-X-X • X-IK~-I-X
A
o
~0
2.0
J 1.5
~
H-I-l-fl • x-ll-x-x-x 0
B
a5 I
.
L'O00
!
f 4OOO
~
2,0-
1,03obo Total dose, rods
LO
~0,~
3.0-
,o~o
i.s
I.¢
4.0-
i
I 2OOO
I
I
'l
Fig. 3. Average foot deformity at 6 months post treatment vs dose for single fractions of neutrons or X-rays. Each point is the mean +_ standard errorfor all animals in one dosage group. Points without error flags have standard error smaller than the size of the point.
4OOO
ToIOlDOSe, rods Fig. 2. Averageearly skin response vs total dosefor 5 f x of X-rays in 5 days (5 x 958 fads/fx) and for 2 f x of neutrons (variable dose/fx)+ 3 f x of X-rays (958 rads/fx). Each point is the mean +_ standard error for early skin response (7-35 days postirradiation) for all mice in a dosage group. A. 5 f x X-rays in 5 days vs n-n-x-x-x Neutron doses/fx were 100, 200, 325 and 425 fads. B. 5 f x X-rays in 5 days vs n-x-x-x-n Neutron doses/fx were 50, 200, 325 and 475 rads. Skin response
Early skin response to single doses of neutrons or X-rays is shown in Fig. 1, and response to mixed fractionation schemes is shown in Fig. 2(a and b). Each value is the mean + standard error of the average skin response over the period 7-35 days postirradiation for all mice in one dosage group. Foot deformity at 6 months after single doses of X-rays or neutrons is also shown
tumors. Unirradiated control tumors had a doubling time of 4.0 + 0"2 days starting at 1213 days post-transplant (mean _+standard error for 52 tumors from 8 experiments3. In these experiments 5/434 irradiated mice died or were terminated at 69-114 days post-treatment with no measurable tumor. Other mice in the same dosage group who lived longer all had recurrent tumors and some mice dying without a measurable tumor were found upon autopsy to have a small tumor at the primary site. Since recurrences were found to occur up to 150 days after treatment, these mice had not survived long enough to be considered cured and were eliminated from the analysis. Growth delay in reaching 5x starting volume as a function of total dose of neutrons vs total dose X-rays is shown in Figs. 5 and 6. Growth delay as a function of total dose of X-rays vs total dose of neutrons+X-rays in the mixed
Response of Mouse Skin and the C3HBA Mammary Carcinoma of the C3H Mouse fractionation scheme is shown in Fig. 7 (a and b). Each value is the mean + standard error of delay in days for all tumors in a particular dosage group. The neutron and mixed neutronphoton RBE's for tumor growth delay are summarized in Tables 5 and 6.
specified X-ray dose were read directly from the curves. The maximum and minimum X-ray and neutron doses likely to produce the same level of damage as the specified X-ray dose were determined from the envelopes of the standard error bars. Dividing the maximum X-ray dose by the minimum neutron dose, and vice-versa, gives a range of RBE's, which are included in parenthesis below the RBE values listed in Tables 2-6. For sample sizes used here this approximates a 70% confidence limit [12]. The RBE range was expressed as per cent of the RBE value read directly from the curves, and these per cent ranges were used to calculate the ranges of T G F values in Table 6.
Estimation of errors In the absence of an established statistical test for variation in RBE's for skin damage and tumor growth delay read from the graphs, the following method was used. Envelopes of the standard bars were drawn on each curve of early skin damage, foot deformity, or tumor growth delay vs dose. RBE's relative to a
Table 2.
NeutronRBE' sfor early skin damage (7-35 dayspostirradiation) andfoot deformity at 6 mos Skin rmpm'~e
?racttonation schme
2 fx 24 hrs. apart
2 fx 96 hrs. apart
"l~__,~s~
x-r~ dom/fx, r~s
Total X-ray din.e, ~
1 fx
*
RE
ran~
2500
1390 (1290 - 1450}
1.8 (1.6 - 1.9)*
4000
4000
1940 (1880 - 2000)
2.1 (1.8 - 2.6)
1900 (1750 - 1980)
2.1 (1.9 - 2.4)
2500
1250
575 (545 - 580)
2.2 (2.2 - 2.3)
605 (500 - 650)
2.1 (1.9 - 2.5)
4000
2000
915 (865 - 935)
2.2 (2.2 - 2.3)
925 (850 - 960)
2.2 ( 1 . 8 - 2.4)
2500
1250
515 (515 - 540)
2.4 (2.3 - 2.4)
525
2.4 (2.2 - 2.7)
4000
2000
890 (850 - 910)
2.2 ( 2 . 1 - 2,4)"
9~5 (955 - 1005)
2.1 ( 1 . 8 - 2,'~)
are w e t ~ , ~ m t e l y
*
~ h ' ~ n t o sc,mOm-d a ~ . ~
70~ c c ~ f i d e n c e l i m i t .
1260
b a r s on p l o t s o f
See t e x t f o r e ~ l a n a ~ L n n
Neutron and mixed neutron-photon RBE's for early skin damage Bqu~xr~lellt*
2 f x n eut o0ns + 3 fx X-ZayS in 5 d a y s (n-x-x-x-,n) vs. 5 fx X-rays in 5 days ***
2.0 (--~)
2500
d~e/fx
2 fx rls.q:~ns + 3 f x X-~llye i n 5 .~ym ( n - n - x - x - x ) v s . 5 fx X-rays in 5 days***
~tv~e,~ ~/fx
FEE'*
fnp~m~Nsuw~rm~d~nau~bm/fxareth~m',~ofdcws~m
Table 3.
5 fx x-~ vs. 5 f x 9~T.torn
Deformity
~,,u~t* neutron da~e/~x
~iv~l.~t off~-t u dm:m:~sd fro. ~welo~s skin z'Npm~ or defraY.t7 vs. d~se. **
RBE**
dom/£x
Total
EqulV~t
doae
trot radlatton
I~* ~
r~ges
do~
800
276 (258 - 290)
2.9 (2.6 - 3.2)
4000
1380
2.9 (2.6 - 3.2)
950
346 (340 - 382)
2.7 (2.3 - 2.9)
4750
1760
2.7 (2.3 - 2.9)
958
430 (390 - 455)
2.2
4790
3734
1.3 ---
1261
555 (510 - 600)
2.3
6305
4893
1.3
958
320 (285 - 345)
3.0
4790
3514
1.3
1261
500 (475 - 540)
2.5
6305
4783
1.3 ---
~e .uml~n in t ~ e ~ s u . ~ e r eao~ r e . i r o n d o ~ / f x ate b'~ r m g e o~ ~ pmeoctr~ e q u L v s l e ~ e g f o c t a s d e t e n u t n ~ f ~ s n e n v e l o p ~ drmm t o s ~ e ~ m ' d e r r ~ bra's cf~ p l o t s o f ~ d n resp0nse ~ . ~ .
** ~
895
are a~pz~J~ately
709 ccmftdence l h ~ t s .
See t e x t f o r e x ~ w ~ i o n .
**~ mL1d f r a c t t o n a t l o n s d ' e m ~ employ a o o ~ t o n t X - r a y ~ / f x (958 o r 1261 r ~ / f x ) w h i l e the ~ x , on ~ a ~ m ~ c t o n s a t e ~wn, ~w=~d. Thin a a r l y Jkin r e f e r e e (7-35 aays p ~ t irr~1~Ao,) ~ I~, t h e m t t m t ~ n t s Is ~ to ~ ~ by 5 x 958 o r 5 x 1261 ~ X - r a y s givsn in 5 d a y s .
of
I
896
J . S. R. Nelson, R. E. Carpenter and R. G. Parker
Table 4.
Fractionation Scheme
Neutron and mixed neutron-photon R B E ' s f o r late foot deformity
X-ray c~>se/fx
5 fx X-rays vs. 5 fx nmutrons
Equivalent*
ItP_~**
Total
~i.',,r'~ent:
neuzzon c~ee/fx
/ x dcee/fx ~ ~ /
x-ray dose
total dose: test radiation
I~E** / total Xlra~ ~ 1 ~ total d O N te~t ) radiatlon
800
300 (270 - 346)
2.7 (2.2 - 3.4)
4000
1500
2.7 (2.2 - 3.4)
950
340 (322 - 390)
2.7 {2.2 - 3.1)
4750
1740
2.7 (2.2 - 3.1)
1250
490 (450 - 530)
2.6 (2.3 - 2.8)
6250
2450
2.6 {2.3 - 2.8)
958
370 (340 - 405)
2.6
4790
3614
1.3 ---
1261
520 (455 - 580)
2.4
6305
4823
1.3 ---
958
330 (220 - 400)
2.9
4790
3534
1.4 ---
1261
550 (510 - 605)
2.3
6305
4883
1.3 ---
inS~
5 fx X-ray~ in 5 day~ vs. n-n-x-x-X* *~
5 fx X-rays in 5 days vs. n-x.x-x-n ~**
*
The n u e t ~ r s ~ p a r e ~ e s e s ~ L i v a l e n t effect_ ~ ~
**
~
under each n e u ~ dose/f× ~ from ~ v e l o p e s t o ~
ranges are a~proximately 70% confidence limits.
the r a n g e o f ~o s e s p _ ~ d u c ~ e r r o r hail; On p l o t s o f f o o t d e f o ~ .
See text for e~planatlo~.
*** The ~ fractionation schemes employ a constant X-ray dcse/fx, (958 c~ 1261 rads/fx) ~ / l e the n e u t n m dose/fx, o~ those days when neutrons a r e g i w m , is varied. ~ e late foot deformity p r o d u ~ by these tr~a~ma~ts is compared to d a ~ a ~ produced by 5 ~ 958 or 5 x 1261 rads of X-rays give~, in
5 days.
Ioo.--
Single Fraction, X-Ray,
~0
•
0
o
0 • • • o
°
~
o
0
0 I0-
~
Controls IOlO rods 3 0 2 5 rods 4 0 3 5 rods 4 5 4 0 rods
iOO --
~)t ¢
e • o o
I
ti
¢¢
@O •
o,{.
°~
lO' a
I..t° t °t
it .I. '
E I.C
t~ ° I'"
"
0.4 i i
I ~0
Days Post
i
i 50
,
i TO
irradiation
a B
Fig. 4. Normalized C3HBA tumor volume vs time after irradiation; each point is the mean normalized tumor volume on a specific day for all mice in a dosage group. Standard errors were calculated on all days but are drawn on only every third point for clarity. (A) Normalized tumor volume vs days postirradiation for single doses of X-rays.
•
* 8 0 0 r~ls • 1200 r.ds • 1600 reds m ZOO0 rads
m
O, 08 i
IO
i
i
30
i
i
5O
I
I
70
i
I
gO
Days Post I,ra~atlan
(B) Normalized tumor volume vs single doses of neutrons.
Response o f Mouse Skin and the C 3 H B A Mammary Carcinoma o f the C 3 H Mouse
DISCUSSION
C3HBA Tumor, Growth Delay Follo~f~l Irradiation
X-rays
lOCI,
I froction 2 froctiom 24 hrs ap~t Jo 2 fractions [ . 96 h ~ oport
e I& Ne~m:~ [0
80,
[ fraction 2 froctio~s 24 hrs ot~rt 2 fractions
1 9 6 hrs ~ r t
.20
ToIol dose, rods
Fig. 5. Growth delay in days, for the C3HBA tumor to reach 5x the volume at the beginning of irradiation vs dose of neutrons or X-rays, for single fractions, 2 f x 24 hr apart, and 2 fractions 96 hours apart. Each point is the mean growth delay in days +_ standard error for all mice in one dosage group.
O 5 Freclkm, Ne~r~ • 5 Fr(Ictl~l, X - I k y l
80
g~
40
.
i
~ 1200)
3000
5000
16O01
11000)
Total INxle, rods ~10eee/fx., rods]
Fig. 6. C3HBA tumour growth delay in days, to reach 5x starting volume vs dose for 5 f x X-rays or 5 f x neutrons in 5 days. Each point is the mean growth delay +_ standard error for all tumors in a single dosage group.
It-11-X*X-X • X-X-X- X-R 0 6O
£ oj
2O i 2000
I
n-H-H x-x-x-x-s
• o
I
I
6C
/
40
a
A
4C
20 I
I
I
4OOO
4000 ToloI DON, rads
Fig. 7. C3HBA tumor growth delay, in days, to reach 5x starting volume vs total dose for 5 f x of X-rays in 5 days (5 x 908 fads) and for 2 f x of neutrons (variable dose/fx) + 3 f x of X-rays (908 rads/fx). (A) 5 f x X-rays in 5 days vs n-n-x-x-x Neutron doses/fx were 50, 100, 200, 300 and 400 fads. (B) 5 f x X-rays in 5 days vs n-x-x-x-n Neutron doses/fx were 50, 100, 200, 300 and 400
fads. E*
897
The neutron RBE's for early mouse skin response and foot deformity at 6 months after irradiation with the University of Washington neutron beam (8 MeV mean energy) increase with increasing number of fractions and decreasing dose/fx, as reported previously [7, 11, 13]. The skin RBE's calculated for total dose of 2 fx of neutrons + 3 fx of X-rays, given in the mixed fractionation scheme and compared to 5 fx of X-rays in 5 days, are the same for both mixed schemes, n-n-x-x-x and n-x-x-x-n: 1.3 (Table 3). The deformity RBE for total dose of neutrons + X-rays in the mixed scheme, relative to 5 fx of X-rays in 5 days, is 1.3 for the n-n-x-xx scheme and 1.3 or 1.4 for the n-x-x-x-n scheme. The response of the C3HBA tumor to single doses of X-rays (Fig. 5) shows that growth delay increased quite rapidly with increasing dose up to 2000 rads and then shows less rapid change between 2000 and 4500 rads. The mean values for delay at each dose define a plateau in response between 2000 and 4000 fads, with increasing response as a function of dose above 4000 fads. This is similar to the response of the RIB-5 tumor of the rat to single doses of X-rays [11] and this refractoriness between 2000 and 4000 rads indicates that this C3H m a m m a r y tumor contains an hypoxic fraction of cells which render it resistant to further growth delay over an intermediate dose range. A plateau region in the growth delay vs dose curve is not seen for single fractions of neutrons; this is expected even if hypoxic cells are present because they provide relatively less protection against high LET radiation [14]. Between 2000 and 4500 rads, two fractions of X-rays given 24 or 9 6 h r apart are more effective than the same dose given in a single fraction (Fig. 5). Neutrons given in 2 fractions 24 or 96 hr apart are slightly less effective in producing tumor growth delay than the same total dose given in one fraction (Fig. 5). The net effect is a reduced neutron RBE for 2 fx 24 or 96 hr apart, relative to a single fraction (Table 5). Field et al. suggested [5] that this elimination of a plateau region in the growth delay response curve with fractionated X-irradiation is due to reoxygenation. Fowler et al. [7] and Suit and Schiavone [15] showed that, following large single doses of X-rays rapid reoxygenation occurred in primary and early generation explants of C3H mouse m a m m a r y tumors, and Howes [16] showed that the minimum proportion of hypoxic cells occurred at 96 hr after
J. S. R. Nelson, R. E. Carpenter and R. G. Parker
898
Table 5. Fractlonaticm ~chmm
X-ray d~m/~ fads
Total X-ray dram, ~ d ~
1 fx
Neutron RBE's for tumor growth delay
2000
2000
2500
2500
Equi~d~mt neuCztm* do~/fx 980 (910 - 1130)
4000
2000
i000
2500
1250
3500
2 f x - 96 h r s .
1750
2000
I000
apart
*
Thl m l ~ e r s in ~ as ds~-ed f~
3.8
~
600
1.7 (1.6 - 1.9)
--
790
1.6
(760 - 835)
(1.5 - 1.7)
940 (860 - 940)
1.9 (1.7 - 2.1)
---
1.6/2.2 s 0.73(0.65-0.9)
--
445
2.2
(430 - 450)
(2.0 - 2.5) 2.1 (1.8 - 2.4)
1250
600 (585 - 650)
4400
2200
1100 (1010 - 1 1 2 5 )
2.1/2.4 - 0.88(0.7-1.0)
2.2 (1.7
-
2.8)
under eada nmatz~n c~¢m/fx are the nmcj~ of ¢]o~s p ~ u C l r q e c ~ v a l e n t e m w k ~ s d r a m ~o s t m d a r d ~ ~zs c~ plo~ of ~mr gn~h delay w. ~ .
.
Table 6.
5 fx in 5 days
3.8/2.1 s 1.8(1.2-2.1)
(2.5 - 4.5)
(550 - 630)
2500
** RB~ rmcjes arts ~ v ~ x i ~ t o l y
~
2.5/1.6 m 1.6(0.90-3.1)
(1.4 - 4.0)
1050
a[oert
--
2.5
(950 - 1200)
2 f X - 24 hrs.
2.0 (1.2 - 4.6)
1000 (860 - 1180)
4000
TGF . tu~or ~B~ ** ~ RB~*
70~ co~fJ~ea'~e IL~Lts, and are used to calculate ~
of T ~
eff~-"t
values.
Neutron and mixed neutron-photon RBE's for tumor growth delay
706
240 (210 - 272)
2.9 (2.2 - 3.6)
3530
1200
2.9 (2.2 - 3.6)
807
295 (266 - 312)
2.7 (2.4 - 3.2)
4035
1475
2.7 (2.4 - 3.2)
2.7/2.9 - 0.93(0.0-1.1}
350
2.6 (2.2 - 3.5)
4540
1750
2.6 (2.2 - 3.5)
2.6/2.7 - 0.96(0,81-1.3)
900
(306 - 356}
~---
908
250 (235 - 270)
3.6
4540
3224
1.4
1.4/1.3 = I.I
2 fx reutzo~ + 908 3 f x X-rays i n 5 day~ ( n - x - x - x - n ) vs. 5 f x X-rays i n 5 day~ * * *
250 (215 - 275)
3.6
4540
3224
1.4
1.4/1.3 - 1.1
2 fx n e u t x ~ m + 3 fx X-rm/l in 5 days ( , - c ~ x - x - x ) v s . 5 f x X-rays i n 5 da~ ***
*
The numbers in p a x ~ t h e ~ detatsdned f ~ emmlop~
**
R~E r ~ are ~ . d l m t e l y explh~at Lon.
under each n m C r o n d m m / e x a ~ the range of ~ producing equlvalent effect as d r m m to standazd error bars on plots of t u m ~ g r o ~ h delay vs. dome. 709 c o n f i ~ K ~
IL~[ts and are used to calculate limits f ~ T~P'S.
*** The ~ f ~ o n schemes employ a ccmst~mt x-ray ~ / f x days a w a . m u t ~ m ~ ~'~ g i ~ . , i~ ~ a r i ~ . ~e t u m r ~ l delay produced by 5 x 908 racEl of X-rays g i v ~ in 5 days.
exposure, although, more importantly, the minim u m number occurred at 48 to 72 hr. Like primary and early explant C3H mammary tumors, the C3HBA m a m m a r y adenocarcinoma probably undergoes rapid reoxygenation, rendering 2 fx of X-rays at 24 or 96 hr intervals more effective than the same dose delivered as a single fraction. To accomplish this reoxygenation must outweigh the effect of repair between fractions in the case of the 24 hr interval and additionaly the effect of repopulation during the 96 hr interval. When 5 fx of neutrons in 5 days are compared to 5 fx of X-rays in 5 days, RBE's are
a
See text for
(908 rads) while the neutrom ~ / f x , on y ~ - a ~ ~ ~ ~T~. ~to
increased relative to two fractions, presumably because the accumulated effect of less repair between fractions of neutrons tends to raise the RBE, although differences in the pattern of reoxygenation during the 5 fx X-ray treatment relative to the 2 fx treatments cannot be ruled out (Table 6). The RBE for neutrons plus X-rays in the mixed fractionation schemes (2 fx of neutrons + 3 fx of X-rays in 5 days in the sequence n-n-xx-x or n-x-x-x-n) relative to 5 fx X-rays in 5 days, is 1.4 for both schemes (Table 6). The RBE's here are lower than those calculated when neutron only schemes are compared to
Response of Mouse Skin and the C3HBA Mammary Carcinoma of the C3H Mouse X-ray only schemes and reflect the relatively large contribution of the 3 X-ray fractions to the total dose. TGF's calculated by dividing tumor RBE by skin RBE for a particular dose and fractionation scheme are close to 1.0 for all treatment regimens except single fractions of neutrons (Table 5, 6). The T G F for both mixed fractionation schemes is 1.1, which is marginally better, although the confidence limits for T G F values for some neutron only schemes overlap this value (Table 6). The T G F values of less than 1.0 can be explained in terms of the rapid and extensive reoxygenation in C3H m a m m a r y tumors such that fractioned X-irradiation can be highly effective. Fowler and others [7, 8, 17] have studied the response of first and second generation explants of C3H mouse m a m m a r y tumors to neutrons and X-rays and have shown that the appropriate fractionation of X-rays, to take advantage of the known reoxygenation pattern of those tumors, makes photon treatment as effective as neutrons when treatment effectiveness is expressed in terms of tumor growth delay or cure probability relative to skin damage. Neutrons m a y give more reliable results, being less dependent on fractionation scheme [18], but not necessarily better results. These experiments described here are consistent with these findings. The TGF's for all schemes other than single fx are close to 1.0. Also, the sequence of neutron fractions in the mixed irradiations does not alter the RBE. The RBE for skin damage (1.3) is the same whether neutrons are given on the first and second days or the first and fifth days of a 5 day treatment schedule; the tumor growth delay RBE of 1"4 is the same for both sequences. The hypothesis to be tested was that an advantage might be seen with neutrons in a rapidly reoxygenating tumor if neutrons were given as the first one or two fractions, when hypoxic cells were present in the largest numbers, followed by X-ray fractions to kill increasingly better reoxygenated cells. The hypothesis is not borne out; the TGF's of 1.1 for both mixed fractionation schemes are not significantly different from TGF's for 5 fx of neutrons in 5 days. At any rate, the mixed schemes are no worse than and may be slightly better than 5 fx of neutrons in 5 days. The C3HBA tumor's response to 2 fx of Xrays vs single doses strongly suggests that reoxygenation occurs in this tumor as in primary C3H m a m m a r y tumors and early generation explants. These latter tumors reoxygenate after large single doses of X-rays such that the proportion of hypoxic cells 24 to 96 hrs after
899
irradiation is considerably less than in the unirradiated tumor [16]. Such extensive reoxygenation is not seen in other experimental tumors including the K H T mouse sarcoma [18, 19], a mouse osteosarcoma [20] and the RIB-5 tumor of the rat [21], where at best the proportion of hypoxic cells in irradiated tumors returns to levels close to those in untreated controls. Thus C3H m a m m a r y tumors are probably the system least likely to demonstrate an advantage for neutron therapy. The theoretical basis for using one or two initial fractions of neutrons followed by fractionated photons may be valid in other tumors. The practice of expressing neutron RBE in terms of dose/fx rather than total dose has been widely used and has yielded useful insight into the variation of RBE with increasing number of fractions and decreasing dose per fraction in cell systems which can repair sublethal damage [13]. In the mixed fractionation schemes, a variable neutron dose/fx was coupled with a fixed X-ray dose/fx, the latter being given on 3 of the 5 days. The response to the mixed radiation was compared to 5 fx of X-rays using the same X-ray dose per fraction as in the mixed scheme. Neutron dose/fx relative to X-ray dose/fx can be used to calculate an RBE here if one wishes to focus on neutron dose/fx equally effective as a fixed X-ray dose in the context of these experiments. In neutron only vs X-ray only schemes, RBE's are the same, whether one uses dose/fx or total dose in the calculations while in the mixed schemes, an elevated RBE is obtained if one uses dose/fx rather than total dose (Tables 3, 4, 6). The dose/fx approach to calculating RBE's ignores the 3 fx of X-rays common to the mixed scheme and to 5 fx of X-rays in 5 days. The definition of RBE is: (dose of X-rays necessary to produce a specific effect)/(dose of test radiation to produce the same effect). No effect was examined which resulted from only the neutron component of the mixed irradiation. All endpoints were the result of the total treatment, and consequently RBE's calculated from (total X-ray dose)/(total dose of (neutrons + X-rays)) should be used to calculate TGF's.
Clinical correlation The conventional pattern of irradiation with external beams, 5 consecutive daily increments per week, developed empirically. Although there are factors of custom and convenience, this so-called "Coutard method" is based on clinically useful therapeutic ratios resulting from preservation of the integrity of normal tissues concurrent with tumor cell killing. This
900
J. S. R. Nelson, R. E. Carpenter and R. G. Parker
single p a t t e r n of t r e a t m e n t is unlikely to be the best possible for all, or even many, clinical situations. However, at this time, there is no scientific basis for tailoring the p a t t e r n of treatm e n t to maximize the therapeutic ratio for each specific clinical problem. W h e n e v e r a new r a d i 0 t h e r a p e u t i c modality is to be tested, the p a t t e r n of application is likely to be conventional, based on custom, or expedient, based on a variety o f extraneous factors. Restricted patterns of application m a y be based on the limited availability of the n e u t r o n generators r a t h e r t h a n on a n y biological evidence. Therefore, it is essential that a wide spectrum o f t r e a t m e n t patterns be studied prior to a n y c o m m i t m e n t for definitive clinical trials.
M i x e d n e u t r o n - p h o t o n fractionation schemes would facilitate the selected t r e a t m e n t o f a larger n u m b e r o f patients with each expensive n e u t r o n generator, and are attractive on this basis alone. However, a favorable therapeutic ratio must be shown. In this relatively unfavorable t u m o r system, this m o d e of t r e a t m e n t is at least as effective as a n y fractionated n e u t r o n only scheme and deserves further consideration in other t u m o r systems.
Acimowledgements--We gratefully acknowledge the support of Dr. Juri Eenmaa, Dr. Keith Weaver, and Dr. David Williams of the Division of Medical Radiation Physics for performing the neutron dosimetry and providing other services at the University of Washington Cyclotron.
REFERENCES 1.
2.
3. 4.
5.
6. 7.
8.
9. 10. 11. 12. 13. 14.
J . F . FOWLER and R. L. MORGAN, Pre-therapeutic experiments with the fast neutron beam from the Medical Research Council cyclotron. VIII. General review. Brit. J. Radiol. 36, 115 (1963). J . L . MONTOUR,J. D. WILSON, C. C. ROGERS,R. B. THEUS and F. H. ATTIX, Biological characterization of a high energy neutron beam for radiation therapy. Cancer (Philad.) 34, 54 (1974). P. TODD, C. B. SCHROY, F. H. ATTIX and R. B. THEUS, Spatial distribution of human cell survival in a neutron beam designed for therapy. Cancer (Philad.) 34, 33 (1974). P. WOOTTON, K. ALVAR, H. BICHSEL,J. EENMAA,J. S. R. NELSON, R. G. PARKER, K. A. WEAVER, D. L. WILLIAMSand W. G. WYCKOFF, Fast neutron beam radiotherapy at the University of Washington. or. Canad. Assoc. Radiol. 26, 44 (1975.) 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. Fractionation, recovery and reoxygenation. Brit. J. Radiol. 41, 597 (1968). J. DENEKAMP,E. W. EMERY and S. B. FIELD, Response of mouse epidermal cells to single and divided doses of fast neutrons. Brit. or. Radiol. 45, 80 (1971). J . F . FOWLER,J. DENEKAMP,A. L. PAGE,A. C. BEGG, S. B. FIELD and K. BUTLER, Fractionation with X-rays and neutrons in mice: response of skin and C3H mammary tumors. Brit. or. Radiol. 45, 237 (1972). J . F . FOWLER, S. B. FIELD and J. DENEKAMP, The effect of fractionated doses of neutrons on C3H mouse mammary tumours. Europ. J. Cancer 10, 281 (1974). N.A. FRIGERIO,R. F. COLEYand M. J. SAMPSON,Depth dose determinations. I. Tissue equivalent liquids for standard man and muscle. Physics Med. Biol. 17, 792 (1972). J . F . FOWLER, R. J. BERRY, R. E. ELLIS, K. KRAGT and P . J . LINDOP, The effect of divided doses of 15 MeV electrons on the skin response of mice. Int. J. Radiat. Biol. 9, 241 (1965). S . B . FIELD, T. JONES and R. H. TROMLINSON, The relative effects of fast neutrons and X-rays on tumour and normal tissue in the rat. I. Single doses. Brit. J. Radiol. 40, 834 (1967). A. GOLDSTEIN,Biostatistics: An Introductory Text, Macmillan, New York (1964). S.B. FIELD and S. HORNSEY, RBE values for cyclotron neutrons for effects on normal tissues and tumors as a function of dose and dose fractionation. Europ. J. Cancer 7, 161 (1971). G . W . BARENDSEN, Cellular responses determining the effectiveness of fast neutrons relative to X-rays for effects on experimental turnouts. Europ. J. Cancer 7, 181 (1971).
Response of Mouse Skin and the C3HBA Mammary Carcinoma of the C3H Mouse 15.
16.
17. 18.
19.
20.
2 I.
H . D . SUIT and J. V. SCI-IIAVONE,Effect of a single dose of radiation on proportion of hypoxic cells in a C3H mouse mammary carcinoma. Radiology ~ , 325 (1968). A . E . Howls, An estimation of changes in the proportions and absolute numbers of hypoxic cells after irradiation of transplanted C3H mouse mammary tumours. Brit. J. Radiol. '1~, 441 (1969). J . F . FOWL,.R, J. D~NEr~MP and S. B. FIF.LD, RBE values for regrowth of C3H mouse mammary carcinomas after single doses of cyclotron neutrons or X-rays. Europ. or. Cancer 9, 853 (1973). L . M . VAN PUWTENand R. F. KALLMAN, Oxygenation status of a transplantable tumor during fractionated radiotherapy, or. nat. Cancer Inst. 4111, 441 (1968). R. F. KALLMAN,L. J. JARDINE and C. W. JOHNSON, Effects of different schedules of dose fractionation on the oxygenation status of a transplantable mouse sarcoma. J. nat. Cancer Inst. 44, 369 (1970). L . M . VAN PUTTEN, T u m o u r reoxygenation during fractionated radiotherapy: Studies with a transplantable mouse osteosarcoma. Europ. J. Cancer 4, 173 (1968). R . H . THOMLINSON,Changes in oxygenation in tumours in relation to irradiation. Front. Radiat. Ther. Oncol. 3, 109 (1968).
901
Response of Mouse Skin and the C3HBA Mammary Carcinoma of the C3H Mouse to X-rays and Cyclotron Neutrons: Effect of Mixed Neutron-Photon Fractionation Schemes* JANET S. R. NELSON, RITA E. CARPENTER and ROBERT G. PARKER Division of Radiation Oncology, School of Medicine, University of Washington, Seattle, Washington, 98195, U.S.A. Abstract--The response of mouse foot skin and the C3HBA mammary adenocarcinoma of the C3H mouse to 250 kVp X-rays and cyclotron-produced neutrons (8 MeV mean energy) has been studied. Neutrons or X-rays were given in single fractions (fx); 2 f x 24 hr apart; 2 f x 96 hr apart; and 5 f x in 5 days. Two f x of neutrons + 3 f x of X-rays in 5 days also were given in the sequence n-n-x-x-x or n-x-x-x-n. When neutron only schemes were compared to X-ray only schemes, RBE's for early skin damage and foot deformity at 6 months post-irradiation increased w~th increasing number of fractions. The RBE for 2 f x of neutrons + 3 f x of X-rays (total dose of neutrons + X-rays) relative to 5 f x of X-rays was 1.3 for both mixed fraction sequences. Tumor growth delay following single and fractionated X-rays suggested an hypoxic fraction of cells which undergoes extensive and long lasting reoxygenation, a situation in which fractionated neutrons might not be expected to show an advantage in terms of tumor damage relative to normal tissue injury. Therapeutic gain factors ( TGF = tumor RBE/skin RBE) for single fractions ranged from 1.6 to 1"8 depending on dose level while TGF's for fractionated neutrons varied from 0.73for 2 f x 24 hr apart to 0.96for 5 f x in 5 days. For both the n-n-x-x-x and n-x-x-x-n fractionation schemes a TGF of 1.1 suggests that the mixed fractionation schemes may be slightly better than neutron only schemes in this tumor system.
INTRODUCTION
times per week. This has indicated that the relative importance of the fractionation scheme, as well as radiation quality, in modifying treatment results also needs to be studied and the response of experimental tumors and normal tissue to various neutron only and X-ray only fractionation schemes has been reported [5-8]. This paper describes the response of mouse skin and the C3HBA m a m m a r y adenocarcinoma of the C3H mouse to 250 kVp X-rays and 8 M e V (mean energy) neutrons produced by the University of Washington cyclotron, using single and multiple fractions of X-rays or neutrons as well as mixed neutron-photon fractionated radiation. The tumor chosen, a
NEUTRON THERAPY of human cancer has necessarily been preceded by experiments intended to biologically characterize different neutron beams [1-4]. Limited medical access to cyclotron-produced neutron beams has resulted in patients being treated 3 or only 2 times per week, rather than the conventional 5
Accepted 8 August 1975. *This investigation was supported by Research Grant # CA-12441-04 from the National Cancer Institute, National Institute of Health. 891
892
J. S. R. Nelson, R. E. Carpenter and R. G. Parker
C3H mammary tumor, is one in which neutrons might be expected not to show an advantage because other mammary tumors in this mouse show rapid and extensive reoxygenation, rendering judiciously fractionated X-rays as effective as neutrons [7, 8]. We chose to examine mixed fractionation schemes, utilizing 2 initial neutron fractions followed by 3 fractions of X-rays over a five day period (n-n-x-x-x) or 2 fractions of neutrons on the first and fifth days, with X-rays on the intervening days (n-x-x-x-n). The rationale was that, in a rapidly reoxygenating tumor, one or two initial neutron fractions could efficiently kill hypoxic tumor cells while subsequent fractions of X-rays would be more effective after the initiation of reoxygenation; a mixed scheme with a final neutron fraction (n-x-x-x-n) would also allow more effective killing of any resistent hypoxic foci left at the end of treatment. Such treatment schemes might produce an increase in therapeutic ratio while taking into account limited cyclotron access for patient treatment.
MATERIAL AND METHODS Radiation sources and dosimetry X-irradiations were performed using a General Electric Maxitron 250; the settings were 250 kVp, 30 mA, 0.5 mm Cu + 1-0 mm A1 added filtration, target to specimen distance (TSD) 50 cm, dose rate 100 R/min. Irradiations were performed with a horizontal beam and an 8 x 8 cm field defined by a ¼ in. thick lead shield. Mice were irradiated while taped to a vertical Lucite board placed directly behind the shield. Dosimetry was done with a Victoreen air equivalent exposure meter using a 250 R chamber and a rad/Roentgen conversion factor of 0.95. Measurements were made with the mouse holder in place to include contributions from backscatter. LiF and LiBO4 thermoluminescent dosimenters were also used to verify absorbed dose in several experiments. These dosimeters were taped to the feet of mice, in the case of skin irradiations, or placed under the skin adjacent to the irradiated tumors. Neutron irradiations were performed at the University of Washington Cyclotron. The neutron beam was produced by bombarding a 1.5 mm thick beryllium target with 21 M e V deuterons, yielding a neutron beam with mean energy of 8 M e V and an energy spread ot 0-25 MeV. Dosimetry was performed with tissue equivalent (TE) ionization chambers (Shonka type A 150 plastic) filled with TE gas [4]. The doses are expressed as total rads of
neutrons plus gammas; gamma contamination was 8% in air at the mouse positions. The neutron beam was defined by a Cu A1 bronze collimator in the initial skin experiment and animals were irradiated at a TSD of 70 cm and a dose rate of 100-125 rad/min. Subsequent experiments employed a borated waterextended polyester (B/WEP) collimator which defined an 18 x 18 cm field at a TSD of 108 cm; the dose rate was 40-50 rads/min. Mice were irradiated while taped to a vertical Lucite board which was placed 2 cm from the collimator exit. Skin irradiations Female BALB/c mice (Jackson Laboratories or Charles River) were anesthetized with Nembutal (sodium pentobarbital, 60/lg/gm body weight) and one rear foot was irradiated with the animals' bodies shielded by the lead shield in the case of X-irradiation and by the bronze or B/WEP collimator in the case of neutron irradiations. Six to nine mice were irradiated at each dose and the feet were immersed in tissue equivalent fluid (C5H4o O18N) [9] to provide for secondary charged particle equilibrium in the neutron irradiation. No significant difference in skin response to single doses of neutrons was seen with the two collimators. C 3 H / H e J male mice, 6-10 weeks old (Jackson Laboratories), were also used in single dose experiments to verify that the two strains of mice had similar skin responses to X-rays and neutrons and yielded the same neutron RBE. Thereafter, skin response RBE's were determined using BALB/C mice and were compared to tumor response RBE's from C3H mice to calculate Therapeutic Gain Factors (TGF = tumor RBE/skin RBE). Early skin response following single fractions was scored over the period of 7-35 days postirradiation, using a scale modified from Fowler et al. [10]. For experiments using multiple fractions of radiation, the ascending curve for skin response vs. days postirradiation was superimposed over similar curves for single fractions to determine which postirradiation day was equivalent to day 7. In all cases, day 8 or 9 was used as the starting point for skin scoring in multiple fraction experiments, and observations were continued until day 36 or 37. Foot deformity at 6 months post-exposure was scored using the scale of Field et al. [11]. Tumor irradiations C 3 H / H e J male mice, 6-10 weeks old (Jackson Laboratories), were housed in the University of Washington Vivarium at least
Response of Mouse Skin and the C3HBA Mammary Carcinoma of the C3H Mouse one week "after arrival before C3HBA mammary adenocarcinomas were transplanted by placing small (1-2 mm dia) pieces of non-necrotic tumor under the skin of the upper back. The C3HBA tumor is a transplantable mammary neoplasm which occurred as a spontaneous tumor in a C3H/An mouse in 1946 and has been carried as a transplantable tumor in C3H/HeJ mice by Jackson Laboratories, Bar Harbor, Maine, since 1949. At 12-13 days posttransplant, tumors of average diameter, 2"7 mm to 5.7 mm were chosen for irradiation. Six to eight mice anesthetized with Nembutal (60 pg/g wt.) were irradiated at each dose. The animals were positioned at the corners of the field with their bodies shielded by the lead shield in the case of X-irradiation or the B/WEP collimator in the case of neutron irradiations.
893
volume was confirmed by comparing calculated volumes to weights and saline displacement volumes of excised tumors. The volume of each tumor on the first day of irradiation was taken as 1.0, and volumes on succeeding days were normalized to this value. The endpoint for tumor radiation response was growth delay, defined as: (no. of days for irradiated tumor to reach 5x starting volume) minus (no. of days for control tumor to each 5x starting volume.) Neutron RBE's were determined by dividing the X-ray dose by the neutron dose which produced equivalent growth delay. RBE's for
Table 1 nud:~o~x
m
Tmm~s Dome/~
1 fx X-za,j,s
x . . . .
2 fx
x-~ca!m
x x
- -
2 fx
X-z'al,'s
x -
- -
5 fx
X--z:m!m
x x
x x
~
dese
Z~ese/fx 1010 - 4540
dome
~
1000 - 4500
1000 - 4000
-
908 - 2270
181.6 - 4 5 4 0
x
1025 - 2465
2050 - 4930
1010 -
2220
2020 - 4440
x
605 - 1260
:3025 - 6 3 0 0
505 -
980
252S - 4540
400 -
2000
400 - 2000
640 - 1810
1010 - 4540 ]280
- 3620
1 fx s'm~cms
n . . . .
700 -
2000
700 - 2000
2 fx ntm~.,~,~
n n
- -
-
500 -
1000
1000 - 2000
7 5 - 1Q00
2 /~x n m . , t x m s
n -
-
-
n
.500 " - 1 1 0 0
1000 - 2200
400 - 1250
800 - 2500
5 fx n~;.L~m
n n
n n
n
260 -
1300 - 2500
100 -
500 -
2 f x ~ + :3 ~ X ~
nx
xx
n
50-625 (tO 958 o~" 1261 (x)
29"74 - 50,33 (n ÷ x)
100400 (n) 908 (x)
2 ~ x nt~L,.,~-., + 3 £'x X - . m ~
n n
2974 - 5033 (n ÷ x)
50 - 4 0 0 ( n ) 908 (x)
~: x
x
500
100 - 625 (n) 958 (z 1261 (x)
For each fractionation scheme, mice from the same transplant group were irradiated concurrently with neutrons or X-rays and their responses were used to determine RBE's. However, repeat experiments showed tumor response to a given dose and fractionation scheme was constant enough to allow data from separate experiments employing different transplant groups to be combined without changing RBE values. Tumors were measured in 3 dimensions 3-5 days/week during and after treatment and tumor volumes were calculated, using the simplified formula: length x width x depth
which closely approximates the volume of an ellipsoid. Measurements were corrected for skin thickness. This method of determining tumor
425
100 -
2000
2125
2924 - 3524 2824 -
3524
mixed neutron-photon fractionation schemes were determined by dividing total X-ray dose by total neutron + X-ray dose. Therapeutic gain factors (TGF), defined as tumor RBE/skin RBE for a specified dose and fractionation scheme, were determined.
RESULTS The fractionation schemes, total doses, and dose per fraction used in skin and tumor response experiments are outlined in Table 1. One fraction of neutrons, two fractions 24 or 96 hr apart, and 5 fractions in 5 days were compared to X-rays given in identical fractionation schemes. In the mixed fractionation schemes, 2 fractions of neutrons plus 3 fractions of X-rays were given in 5 days, in the sequence n-n-x-x-x or n-x-x-x-n, and the responses were compared to those obtained with 5 fractions of X-rays in 5 days. In both the all X-ray and mixed neutron-
894
J . S. R . Nelson, R . E . Carpenter and R . G. Parker
photon options in these experiments, the X-ray dose per fraction was held constant while the neutron dose per fraction was varied. Within the context of such an experiment, an RBE could be determined by seeing which dose of neutrons + X - r a y s was equivalent to the constant total X-ray dose in that experiment (for example, see Fig. 2 and 7). MOUSEFOOTRESPONSE
:¢
e~ 1.5 ¢c ! ~
| o.s
A
y
S E] I fx NEUTRONS
i
i
.
I000
3000
5000
(Fig. 3). Neutron RBE's for early and late damage were read directly from smooth curves fitted to the points on these plots and similar ones for all other fractionation schemes, and are summarized in Tables 2-4.
Tumor response O f the mice irradiated for tumor growth delay, 4% died from anesthetic overdose, 4% died within the first 10 days after irradiation, due mostly to radiation effect on the intestine, and 4% died later than 10 days but too soon for their tumors to be included in the growth delay analysis. Tumor growth following graded single doses of X-rays or neutrons is shown in Fig. 4(a and b). Tumor response to radiation was assessed as tumor growth delay, determined from volume change against time for control and irradiated 5.0-
Tolol dole Ruds Fig. 1. Average early skin response vs dose in rads for single fractions of X-rays or neutrons. Each point is the mean +_ standard error of the average skin response over the period 7-35 days postirradiation for the mice in each dosage group.
i~n-X-X-X • X-IK~-I-X
A
o
~0
2.0
J 1.5
~
H-I-l-fl • x-ll-x-x-x 0
B
a5 I
.
L'O00
!
f 4OOO
~
2,0-
1,03obo Total dose, rods
LO
~0,~
3.0-
,o~o
i.s
I.¢
4.0-
i
I 2OOO
I
I
'l
Fig. 3. Average foot deformity at 6 months post treatment vs dose for single fractions of neutrons or X-rays. Each point is the mean +_ standard errorfor all animals in one dosage group. Points without error flags have standard error smaller than the size of the point.
4OOO
ToIOlDOSe, rods Fig. 2. Averageearly skin response vs total dosefor 5 f x of X-rays in 5 days (5 x 958 fads/fx) and for 2 f x of neutrons (variable dose/fx)+ 3 f x of X-rays (958 rads/fx). Each point is the mean +_ standard error for early skin response (7-35 days postirradiation) for all mice in a dosage group. A. 5 f x X-rays in 5 days vs n-n-x-x-x Neutron doses/fx were 100, 200, 325 and 425 fads. B. 5 f x X-rays in 5 days vs n-x-x-x-n Neutron doses/fx were 50, 200, 325 and 475 rads. Skin response
Early skin response to single doses of neutrons or X-rays is shown in Fig. 1, and response to mixed fractionation schemes is shown in Fig. 2(a and b). Each value is the mean + standard error of the average skin response over the period 7-35 days postirradiation for all mice in one dosage group. Foot deformity at 6 months after single doses of X-rays or neutrons is also shown
tumors. Unirradiated control tumors had a doubling time of 4.0 + 0"2 days starting at 1213 days post-transplant (mean _+standard error for 52 tumors from 8 experiments3. In these experiments 5/434 irradiated mice died or were terminated at 69-114 days post-treatment with no measurable tumor. Other mice in the same dosage group who lived longer all had recurrent tumors and some mice dying without a measurable tumor were found upon autopsy to have a small tumor at the primary site. Since recurrences were found to occur up to 150 days after treatment, these mice had not survived long enough to be considered cured and were eliminated from the analysis. Growth delay in reaching 5x starting volume as a function of total dose of neutrons vs total dose X-rays is shown in Figs. 5 and 6. Growth delay as a function of total dose of X-rays vs total dose of neutrons+X-rays in the mixed
Response of Mouse Skin and the C3HBA Mammary Carcinoma of the C3H Mouse fractionation scheme is shown in Fig. 7 (a and b). Each value is the mean + standard error of delay in days for all tumors in a particular dosage group. The neutron and mixed neutronphoton RBE's for tumor growth delay are summarized in Tables 5 and 6.
specified X-ray dose were read directly from the curves. The maximum and minimum X-ray and neutron doses likely to produce the same level of damage as the specified X-ray dose were determined from the envelopes of the standard error bars. Dividing the maximum X-ray dose by the minimum neutron dose, and vice-versa, gives a range of RBE's, which are included in parenthesis below the RBE values listed in Tables 2-6. For sample sizes used here this approximates a 70% confidence limit [12]. The RBE range was expressed as per cent of the RBE value read directly from the curves, and these per cent ranges were used to calculate the ranges of T G F values in Table 6.
Estimation of errors In the absence of an established statistical test for variation in RBE's for skin damage and tumor growth delay read from the graphs, the following method was used. Envelopes of the standard bars were drawn on each curve of early skin damage, foot deformity, or tumor growth delay vs dose. RBE's relative to a
Table 2.
NeutronRBE' sfor early skin damage (7-35 dayspostirradiation) andfoot deformity at 6 mos Skin rmpm'~e
?racttonation schme
2 fx 24 hrs. apart
2 fx 96 hrs. apart
"l~__,~s~
x-r~ dom/fx, r~s
Total X-ray din.e, ~
1 fx
*
RE
ran~
2500
1390 (1290 - 1450}
1.8 (1.6 - 1.9)*
4000
4000
1940 (1880 - 2000)
2.1 (1.8 - 2.6)
1900 (1750 - 1980)
2.1 (1.9 - 2.4)
2500
1250
575 (545 - 580)
2.2 (2.2 - 2.3)
605 (500 - 650)
2.1 (1.9 - 2.5)
4000
2000
915 (865 - 935)
2.2 (2.2 - 2.3)
925 (850 - 960)
2.2 ( 1 . 8 - 2.4)
2500
1250
515 (515 - 540)
2.4 (2.3 - 2.4)
525
2.4 (2.2 - 2.7)
4000
2000
890 (850 - 910)
2.2 ( 2 . 1 - 2,4)"
9~5 (955 - 1005)
2.1 ( 1 . 8 - 2,'~)
are w e t ~ , ~ m t e l y
*
~ h ' ~ n t o sc,mOm-d a ~ . ~
70~ c c ~ f i d e n c e l i m i t .
1260
b a r s on p l o t s o f
See t e x t f o r e ~ l a n a ~ L n n
Neutron and mixed neutron-photon RBE's for early skin damage Bqu~xr~lellt*
2 f x n eut o0ns + 3 fx X-ZayS in 5 d a y s (n-x-x-x-,n) vs. 5 fx X-rays in 5 days ***
2.0 (--~)
2500
d~e/fx
2 fx rls.q:~ns + 3 f x X-~llye i n 5 .~ym ( n - n - x - x - x ) v s . 5 fx X-rays in 5 days***
~tv~e,~ ~/fx
FEE'*
fnp~m~Nsuw~rm~d~nau~bm/fxareth~m',~ofdcws~m
Table 3.
5 fx x-~ vs. 5 f x 9~T.torn
Deformity
~,,u~t* neutron da~e/~x
~iv~l.~t off~-t u dm:m:~sd fro. ~welo~s skin z'Npm~ or defraY.t7 vs. d~se. **
RBE**
dom/£x
Total
EqulV~t
doae
trot radlatton
I~* ~
r~ges
do~
800
276 (258 - 290)
2.9 (2.6 - 3.2)
4000
1380
2.9 (2.6 - 3.2)
950
346 (340 - 382)
2.7 (2.3 - 2.9)
4750
1760
2.7 (2.3 - 2.9)
958
430 (390 - 455)
2.2
4790
3734
1.3 ---
1261
555 (510 - 600)
2.3
6305
4893
1.3
958
320 (285 - 345)
3.0
4790
3514
1.3
1261
500 (475 - 540)
2.5
6305
4783
1.3 ---
~e .uml~n in t ~ e ~ s u . ~ e r eao~ r e . i r o n d o ~ / f x ate b'~ r m g e o~ ~ pmeoctr~ e q u L v s l e ~ e g f o c t a s d e t e n u t n ~ f ~ s n e n v e l o p ~ drmm t o s ~ e ~ m ' d e r r ~ bra's cf~ p l o t s o f ~ d n resp0nse ~ . ~ .
** ~
895
are a~pz~J~ately
709 ccmftdence l h ~ t s .
See t e x t f o r e x ~ w ~ i o n .
**~ mL1d f r a c t t o n a t l o n s d ' e m ~ employ a o o ~ t o n t X - r a y ~ / f x (958 o r 1261 r ~ / f x ) w h i l e the ~ x , on ~ a ~ m ~ c t o n s a t e ~wn, ~w=~d. Thin a a r l y Jkin r e f e r e e (7-35 aays p ~ t irr~1~Ao,) ~ I~, t h e m t t m t ~ n t s Is ~ to ~ ~ by 5 x 958 o r 5 x 1261 ~ X - r a y s givsn in 5 d a y s .
of
I
896
J . S. R. Nelson, R. E. Carpenter and R. G. Parker
Table 4.
Fractionation Scheme
Neutron and mixed neutron-photon R B E ' s f o r late foot deformity
X-ray c~>se/fx
5 fx X-rays vs. 5 fx nmutrons
Equivalent*
ItP_~**
Total
~i.',,r'~ent:
neuzzon c~ee/fx
/ x dcee/fx ~ ~ /
x-ray dose
total dose: test radiation
I~E** / total Xlra~ ~ 1 ~ total d O N te~t ) radiatlon
800
300 (270 - 346)
2.7 (2.2 - 3.4)
4000
1500
2.7 (2.2 - 3.4)
950
340 (322 - 390)
2.7 {2.2 - 3.1)
4750
1740
2.7 (2.2 - 3.1)
1250
490 (450 - 530)
2.6 (2.3 - 2.8)
6250
2450
2.6 {2.3 - 2.8)
958
370 (340 - 405)
2.6
4790
3614
1.3 ---
1261
520 (455 - 580)
2.4
6305
4823
1.3 ---
958
330 (220 - 400)
2.9
4790
3534
1.4 ---
1261
550 (510 - 605)
2.3
6305
4883
1.3 ---
inS~
5 fx X-ray~ in 5 day~ vs. n-n-x-x-X* *~
5 fx X-rays in 5 days vs. n-x.x-x-n ~**
*
The n u e t ~ r s ~ p a r e ~ e s e s ~ L i v a l e n t effect_ ~ ~
**
~
under each n e u ~ dose/f× ~ from ~ v e l o p e s t o ~
ranges are a~proximately 70% confidence limits.
the r a n g e o f ~o s e s p _ ~ d u c ~ e r r o r hail; On p l o t s o f f o o t d e f o ~ .
See text for e~planatlo~.
*** The ~ fractionation schemes employ a constant X-ray dcse/fx, (958 c~ 1261 rads/fx) ~ / l e the n e u t n m dose/fx, o~ those days when neutrons a r e g i w m , is varied. ~ e late foot deformity p r o d u ~ by these tr~a~ma~ts is compared to d a ~ a ~ produced by 5 ~ 958 or 5 x 1261 rads of X-rays give~, in
5 days.
Ioo.--
Single Fraction, X-Ray,
~0
•
0
o
0 • • • o
°
~
o
0
0 I0-
~
Controls IOlO rods 3 0 2 5 rods 4 0 3 5 rods 4 5 4 0 rods
iOO --
~)t ¢
e • o o
I
ti
¢¢
@O •
o,{.
°~
lO' a
I..t° t °t
it .I. '
E I.C
t~ ° I'"
"
0.4 i i
I ~0
Days Post
i
i 50
,
i TO
irradiation
a B
Fig. 4. Normalized C3HBA tumor volume vs time after irradiation; each point is the mean normalized tumor volume on a specific day for all mice in a dosage group. Standard errors were calculated on all days but are drawn on only every third point for clarity. (A) Normalized tumor volume vs days postirradiation for single doses of X-rays.
•
* 8 0 0 r~ls • 1200 r.ds • 1600 reds m ZOO0 rads
m
O, 08 i
IO
i
i
30
i
i
5O
I
I
70
i
I
gO
Days Post I,ra~atlan
(B) Normalized tumor volume vs single doses of neutrons.
Response o f Mouse Skin and the C 3 H B A Mammary Carcinoma o f the C 3 H Mouse
DISCUSSION
C3HBA Tumor, Growth Delay Follo~f~l Irradiation
X-rays
lOCI,
I froction 2 froctiom 24 hrs ap~t Jo 2 fractions [ . 96 h ~ oport
e I& Ne~m:~ [0
80,
[ fraction 2 froctio~s 24 hrs ot~rt 2 fractions
1 9 6 hrs ~ r t
.20
ToIol dose, rods
Fig. 5. Growth delay in days, for the C3HBA tumor to reach 5x the volume at the beginning of irradiation vs dose of neutrons or X-rays, for single fractions, 2 f x 24 hr apart, and 2 fractions 96 hours apart. Each point is the mean growth delay in days +_ standard error for all mice in one dosage group.
O 5 Freclkm, Ne~r~ • 5 Fr(Ictl~l, X - I k y l
80
g~
40
.
i
~ 1200)
3000
5000
16O01
11000)
Total INxle, rods ~10eee/fx., rods]
Fig. 6. C3HBA tumour growth delay in days, to reach 5x starting volume vs dose for 5 f x X-rays or 5 f x neutrons in 5 days. Each point is the mean growth delay +_ standard error for all tumors in a single dosage group.
It-11-X*X-X • X-X-X- X-R 0 6O
£ oj
2O i 2000
I
n-H-H x-x-x-x-s
• o
I
I
6C
/
40
a
A
4C
20 I
I
I
4OOO
4000 ToloI DON, rads
Fig. 7. C3HBA tumor growth delay, in days, to reach 5x starting volume vs total dose for 5 f x of X-rays in 5 days (5 x 908 fads) and for 2 f x of neutrons (variable dose/fx) + 3 f x of X-rays (908 rads/fx). (A) 5 f x X-rays in 5 days vs n-n-x-x-x Neutron doses/fx were 50, 100, 200, 300 and 400 fads. (B) 5 f x X-rays in 5 days vs n-x-x-x-n Neutron doses/fx were 50, 100, 200, 300 and 400
fads. E*
897
The neutron RBE's for early mouse skin response and foot deformity at 6 months after irradiation with the University of Washington neutron beam (8 MeV mean energy) increase with increasing number of fractions and decreasing dose/fx, as reported previously [7, 11, 13]. The skin RBE's calculated for total dose of 2 fx of neutrons + 3 fx of X-rays, given in the mixed fractionation scheme and compared to 5 fx of X-rays in 5 days, are the same for both mixed schemes, n-n-x-x-x and n-x-x-x-n: 1.3 (Table 3). The deformity RBE for total dose of neutrons + X-rays in the mixed scheme, relative to 5 fx of X-rays in 5 days, is 1.3 for the n-n-x-xx scheme and 1.3 or 1.4 for the n-x-x-x-n scheme. The response of the C3HBA tumor to single doses of X-rays (Fig. 5) shows that growth delay increased quite rapidly with increasing dose up to 2000 rads and then shows less rapid change between 2000 and 4500 rads. The mean values for delay at each dose define a plateau in response between 2000 and 4000 fads, with increasing response as a function of dose above 4000 fads. This is similar to the response of the RIB-5 tumor of the rat to single doses of X-rays [11] and this refractoriness between 2000 and 4000 rads indicates that this C3H m a m m a r y tumor contains an hypoxic fraction of cells which render it resistant to further growth delay over an intermediate dose range. A plateau region in the growth delay vs dose curve is not seen for single fractions of neutrons; this is expected even if hypoxic cells are present because they provide relatively less protection against high LET radiation [14]. Between 2000 and 4500 rads, two fractions of X-rays given 24 or 9 6 h r apart are more effective than the same dose given in a single fraction (Fig. 5). Neutrons given in 2 fractions 24 or 96 hr apart are slightly less effective in producing tumor growth delay than the same total dose given in one fraction (Fig. 5). The net effect is a reduced neutron RBE for 2 fx 24 or 96 hr apart, relative to a single fraction (Table 5). Field et al. suggested [5] that this elimination of a plateau region in the growth delay response curve with fractionated X-irradiation is due to reoxygenation. Fowler et al. [7] and Suit and Schiavone [15] showed that, following large single doses of X-rays rapid reoxygenation occurred in primary and early generation explants of C3H mouse m a m m a r y tumors, and Howes [16] showed that the minimum proportion of hypoxic cells occurred at 96 hr after
J. S. R. Nelson, R. E. Carpenter and R. G. Parker
898
Table 5. Fractlonaticm ~chmm
X-ray d~m/~ fads
Total X-ray dram, ~ d ~
1 fx
Neutron RBE's for tumor growth delay
2000
2000
2500
2500
Equi~d~mt neuCztm* do~/fx 980 (910 - 1130)
4000
2000
i000
2500
1250
3500
2 f x - 96 h r s .
1750
2000
I000
apart
*
Thl m l ~ e r s in ~ as ds~-ed f~
3.8
~
600
1.7 (1.6 - 1.9)
--
790
1.6
(760 - 835)
(1.5 - 1.7)
940 (860 - 940)
1.9 (1.7 - 2.1)
---
1.6/2.2 s 0.73(0.65-0.9)
--
445
2.2
(430 - 450)
(2.0 - 2.5) 2.1 (1.8 - 2.4)
1250
600 (585 - 650)
4400
2200
1100 (1010 - 1 1 2 5 )
2.1/2.4 - 0.88(0.7-1.0)
2.2 (1.7
-
2.8)
under eada nmatz~n c~¢m/fx are the nmcj~ of ¢]o~s p ~ u C l r q e c ~ v a l e n t e m w k ~ s d r a m ~o s t m d a r d ~ ~zs c~ plo~ of ~mr gn~h delay w. ~ .
.
Table 6.
5 fx in 5 days
3.8/2.1 s 1.8(1.2-2.1)
(2.5 - 4.5)
(550 - 630)
2500
** RB~ rmcjes arts ~ v ~ x i ~ t o l y
~
2.5/1.6 m 1.6(0.90-3.1)
(1.4 - 4.0)
1050
a[oert
--
2.5
(950 - 1200)
2 f X - 24 hrs.
2.0 (1.2 - 4.6)
1000 (860 - 1180)
4000
TGF . tu~or ~B~ ** ~ RB~*
70~ co~fJ~ea'~e IL~Lts, and are used to calculate ~
of T ~
eff~-"t
values.
Neutron and mixed neutron-photon RBE's for tumor growth delay
706
240 (210 - 272)
2.9 (2.2 - 3.6)
3530
1200
2.9 (2.2 - 3.6)
807
295 (266 - 312)
2.7 (2.4 - 3.2)
4035
1475
2.7 (2.4 - 3.2)
2.7/2.9 - 0.93(0.0-1.1}
350
2.6 (2.2 - 3.5)
4540
1750
2.6 (2.2 - 3.5)
2.6/2.7 - 0.96(0,81-1.3)
900
(306 - 356}
~---
908
250 (235 - 270)
3.6
4540
3224
1.4
1.4/1.3 = I.I
2 fx reutzo~ + 908 3 f x X-rays i n 5 day~ ( n - x - x - x - n ) vs. 5 f x X-rays i n 5 day~ * * *
250 (215 - 275)
3.6
4540
3224
1.4
1.4/1.3 - 1.1
2 fx n e u t x ~ m + 3 fx X-rm/l in 5 days ( , - c ~ x - x - x ) v s . 5 f x X-rays i n 5 da~ ***
*
The numbers in p a x ~ t h e ~ detatsdned f ~ emmlop~
**
R~E r ~ are ~ . d l m t e l y explh~at Lon.
under each n m C r o n d m m / e x a ~ the range of ~ producing equlvalent effect as d r m m to standazd error bars on plots of t u m ~ g r o ~ h delay vs. dome. 709 c o n f i ~ K ~
IL~[ts and are used to calculate limits f ~ T~P'S.
*** The ~ f ~ o n schemes employ a ccmst~mt x-ray ~ / f x days a w a . m u t ~ m ~ ~'~ g i ~ . , i~ ~ a r i ~ . ~e t u m r ~ l delay produced by 5 x 908 racEl of X-rays g i v ~ in 5 days.
exposure, although, more importantly, the minim u m number occurred at 48 to 72 hr. Like primary and early explant C3H mammary tumors, the C3HBA m a m m a r y adenocarcinoma probably undergoes rapid reoxygenation, rendering 2 fx of X-rays at 24 or 96 hr intervals more effective than the same dose delivered as a single fraction. To accomplish this reoxygenation must outweigh the effect of repair between fractions in the case of the 24 hr interval and additionaly the effect of repopulation during the 96 hr interval. When 5 fx of neutrons in 5 days are compared to 5 fx of X-rays in 5 days, RBE's are
a
See text for
(908 rads) while the neutrom ~ / f x , on y ~ - a ~ ~ ~ ~T~. ~to
increased relative to two fractions, presumably because the accumulated effect of less repair between fractions of neutrons tends to raise the RBE, although differences in the pattern of reoxygenation during the 5 fx X-ray treatment relative to the 2 fx treatments cannot be ruled out (Table 6). The RBE for neutrons plus X-rays in the mixed fractionation schemes (2 fx of neutrons + 3 fx of X-rays in 5 days in the sequence n-n-xx-x or n-x-x-x-n) relative to 5 fx X-rays in 5 days, is 1.4 for both schemes (Table 6). The RBE's here are lower than those calculated when neutron only schemes are compared to
Response of Mouse Skin and the C3HBA Mammary Carcinoma of the C3H Mouse X-ray only schemes and reflect the relatively large contribution of the 3 X-ray fractions to the total dose. TGF's calculated by dividing tumor RBE by skin RBE for a particular dose and fractionation scheme are close to 1.0 for all treatment regimens except single fractions of neutrons (Table 5, 6). The T G F for both mixed fractionation schemes is 1.1, which is marginally better, although the confidence limits for T G F values for some neutron only schemes overlap this value (Table 6). The T G F values of less than 1.0 can be explained in terms of the rapid and extensive reoxygenation in C3H m a m m a r y tumors such that fractioned X-irradiation can be highly effective. Fowler and others [7, 8, 17] have studied the response of first and second generation explants of C3H mouse m a m m a r y tumors to neutrons and X-rays and have shown that the appropriate fractionation of X-rays, to take advantage of the known reoxygenation pattern of those tumors, makes photon treatment as effective as neutrons when treatment effectiveness is expressed in terms of tumor growth delay or cure probability relative to skin damage. Neutrons m a y give more reliable results, being less dependent on fractionation scheme [18], but not necessarily better results. These experiments described here are consistent with these findings. The TGF's for all schemes other than single fx are close to 1.0. Also, the sequence of neutron fractions in the mixed irradiations does not alter the RBE. The RBE for skin damage (1.3) is the same whether neutrons are given on the first and second days or the first and fifth days of a 5 day treatment schedule; the tumor growth delay RBE of 1"4 is the same for both sequences. The hypothesis to be tested was that an advantage might be seen with neutrons in a rapidly reoxygenating tumor if neutrons were given as the first one or two fractions, when hypoxic cells were present in the largest numbers, followed by X-ray fractions to kill increasingly better reoxygenated cells. The hypothesis is not borne out; the TGF's of 1.1 for both mixed fractionation schemes are not significantly different from TGF's for 5 fx of neutrons in 5 days. At any rate, the mixed schemes are no worse than and may be slightly better than 5 fx of neutrons in 5 days. The C3HBA tumor's response to 2 fx of Xrays vs single doses strongly suggests that reoxygenation occurs in this tumor as in primary C3H m a m m a r y tumors and early generation explants. These latter tumors reoxygenate after large single doses of X-rays such that the proportion of hypoxic cells 24 to 96 hrs after
899
irradiation is considerably less than in the unirradiated tumor [16]. Such extensive reoxygenation is not seen in other experimental tumors including the K H T mouse sarcoma [18, 19], a mouse osteosarcoma [20] and the RIB-5 tumor of the rat [21], where at best the proportion of hypoxic cells in irradiated tumors returns to levels close to those in untreated controls. Thus C3H m a m m a r y tumors are probably the system least likely to demonstrate an advantage for neutron therapy. The theoretical basis for using one or two initial fractions of neutrons followed by fractionated photons may be valid in other tumors. The practice of expressing neutron RBE in terms of dose/fx rather than total dose has been widely used and has yielded useful insight into the variation of RBE with increasing number of fractions and decreasing dose per fraction in cell systems which can repair sublethal damage [13]. In the mixed fractionation schemes, a variable neutron dose/fx was coupled with a fixed X-ray dose/fx, the latter being given on 3 of the 5 days. The response to the mixed radiation was compared to 5 fx of X-rays using the same X-ray dose per fraction as in the mixed scheme. Neutron dose/fx relative to X-ray dose/fx can be used to calculate an RBE here if one wishes to focus on neutron dose/fx equally effective as a fixed X-ray dose in the context of these experiments. In neutron only vs X-ray only schemes, RBE's are the same, whether one uses dose/fx or total dose in the calculations while in the mixed schemes, an elevated RBE is obtained if one uses dose/fx rather than total dose (Tables 3, 4, 6). The dose/fx approach to calculating RBE's ignores the 3 fx of X-rays common to the mixed scheme and to 5 fx of X-rays in 5 days. The definition of RBE is: (dose of X-rays necessary to produce a specific effect)/(dose of test radiation to produce the same effect). No effect was examined which resulted from only the neutron component of the mixed irradiation. All endpoints were the result of the total treatment, and consequently RBE's calculated from (total X-ray dose)/(total dose of (neutrons + X-rays)) should be used to calculate TGF's.
Clinical correlation The conventional pattern of irradiation with external beams, 5 consecutive daily increments per week, developed empirically. Although there are factors of custom and convenience, this so-called "Coutard method" is based on clinically useful therapeutic ratios resulting from preservation of the integrity of normal tissues concurrent with tumor cell killing. This
900
J. S. R. Nelson, R. E. Carpenter and R. G. Parker
single p a t t e r n of t r e a t m e n t is unlikely to be the best possible for all, or even many, clinical situations. However, at this time, there is no scientific basis for tailoring the p a t t e r n of treatm e n t to maximize the therapeutic ratio for each specific clinical problem. W h e n e v e r a new r a d i 0 t h e r a p e u t i c modality is to be tested, the p a t t e r n of application is likely to be conventional, based on custom, or expedient, based on a variety o f extraneous factors. Restricted patterns of application m a y be based on the limited availability of the n e u t r o n generators r a t h e r t h a n on a n y biological evidence. Therefore, it is essential that a wide spectrum o f t r e a t m e n t patterns be studied prior to a n y c o m m i t m e n t for definitive clinical trials.
M i x e d n e u t r o n - p h o t o n fractionation schemes would facilitate the selected t r e a t m e n t o f a larger n u m b e r o f patients with each expensive n e u t r o n generator, and are attractive on this basis alone. However, a favorable therapeutic ratio must be shown. In this relatively unfavorable t u m o r system, this m o d e of t r e a t m e n t is at least as effective as a n y fractionated n e u t r o n only scheme and deserves further consideration in other t u m o r systems.
Acimowledgements--We gratefully acknowledge the support of Dr. Juri Eenmaa, Dr. Keith Weaver, and Dr. David Williams of the Division of Medical Radiation Physics for performing the neutron dosimetry and providing other services at the University of Washington Cyclotron.
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