Failure of hyperfractionated radiotherapy to reduce bone growth arrest in rats

Inl J Rudurron Onrolo~~~ Bd Phyc Vol. 26, pp. 427-43 I Pnnted in the U S.A All nghts reserved

0360.3016193 $6.00 + .oO Copyright F 1993 Pergamon Press Ltd.

??Biology Original Contribution

FAILURE

OF HYPERFRACTIONATED RADIOTHERAPY GROWTH ARREST IN RATS

K. WECHSLER-JENTZSCH,

‘George Washington

TO REDUCE

M.D.,’ H. HUEPFEL,

M.D.,2 W. SCHMIDT, PH.D.,* AND B. KAHN, M.D.’

University, Division of Radiation Oncology and Biophysics, Klinik und lnstitut fuer Strahlenbiologie der Universittiet

BONE

E. WANDL,

M.D.2

Washington, DC; and 2Strah1entherapeutische Wien, Vienna, Austria

Purpose: Radiotherapy of the craniospinal axis is causing an age dependent growth arrest in children. The purpose ofpaper was to examine in an animal model, whether hyperfractionated radiotherapy, given with twice daily fractions in conventional overall treatment time, would cause less growth arrest of the spinal column than a regular treatment schedule. Methods and Materials: The time-dose-fraction schedule for the treatment of the craniospinal axis of children with medullohlastomas was used as model for the treatment of the spine in rats. The entire spine of weanling rats received either 3570 cCy in 21 daily fractions of the 170 cCy, 5 times per week over 27 days, or 3630 cGy in 33 fractions of 110 cGy, given twice daily with 6-hr intervals over 21 days. Results: Both fraction schedules were isoeffective and caused a growth inhibition of 9.5%. The growth arrest was complete after 1870-2420 cGy. The alpha/beta ratio for the growing rat vertebrae was 3400 cGy. This result contrasts with the growth sparing effect observed with hyperfractionation of accelerated treatment schedules. Conclusion: Growing bone is a fast proliferating tissue. Hyperfractionation with 110 cGy BID compared to 170 cGy given once a day, has no sparing effect on bone growth in rats if given in conventional overall treatment time. Radiation therapy, Vertebral growth, Hyperfractionation,

Rats.

INTRODUCTION

to the conventional

clinical schedule, which requires 2025 days for the same total dose. The purpose of this experiment was to compare in an animal simulation model the effect on growth inhibition of two clinically used fraction schedules and to examine whether the hyperfractionated schedule could reduce the growth retardation of the spinal axis in rats. One group of rats received the conventional treatment consisting of 3570 cGy given in 21 single doses of 170 cGy over 27 days. The second group received 3630 cGy in 33 twice daily fractions of 110 cGy, 6 hr apart over 2 1 days.

Postoperative radiotherapy to the crania-spinal axis is an integral part of the treatment of children with medulloblastomas and other brain tumors. Given at a young age, this treatment results in stunted vertebral growth. The degree of the growth arrest is age and dose dependent (9, 10, 12, 13). Experimental and clinical evidence suggest that smaller single doses are less damaging for slow proliferating tissues than higher single doses (5, 7). Treatment regimens with multiple small fractions per day are successfully used for radiation of brain stem tumors in children (6, 14). Hyperfractionated treatment with small single doses appears to have also a protective effect on growing bone of rats (4,8). This observation is now under investigation in clinical trials (2). The treatment regimens used in experiments and in the clinic differ considerably with regard to overall treatment time and fraction size (2, 4, 8). In animal experiments hyperfractionated, accelerated schedules with overall treatment times of 5-9 days were used, as opposed

METHODS

AND MATERIALS

Outline of the experiment Weanling female rats weighing 55-60 grams were divided in 4 groups of 12 animals. Group I served as control, Group II received anesthesia only two times a day, 6 hr apart in order to evaluate for a possible growth inhibition through repeated anesthesia. Group III received 2 1 daily fractions of 170 cGy, to a total dose of 3570 cGy. Group

Acknowledgement-The

Reprint requests to: K. Wechsler-Jentzsch, M.D., Division of Radiation Oncology and Biophysics, The George Washington University Medical Center, 901 23rd St., N.W., Washington, DC 20037.

authors would like to thank Jack F. Fowler Ph.D. for his helpful and stimulating suggestions. Accepted for publication 3 1 December 1992.

427

I. J. Radiation

428

Oncology 0 Biology 0 Physics

IV received 33 fractions of 110 cGy, twice daily, 6 hr apart to a total dose of 3630 cGy. The treatments were given at the same time of the day on 5 consecutive days, followed by 2 days of rest. Weight and length were recorded from day 1 through day 300. During the treatments and up to 3 months after completion of the experiment, the animals were weighed 4-5 times per week. This was mainly done in order to monitor the condition of the animals during the treatment period. The length was recorded at least once a week. The rats were measured from the tip of the nose to the end of the hairline of the body.

Experimental conditions For the first 10 days the animals were anesthetized with Pentobarbital sodium, 0.03 mg/g i.p.* Nine animals were lost between day 7 and day 10 of the experiment, three of the once a day, 6 of the twice a day radiation group. This was attributed to the long acting anesthetic in conjunction with stress from the treatment. The pentobarbital sodium was replaced by diethyl either, which was better tolerated. The rats were placed in the prone position on a Plexiglas board with a small wedge for elevation of the upper thoracic spine, in order to bring the spine to a depth of approximately 1 cm from the surface. The animals were secured with an aquaplast mould, which was fixed to the board. The treatment field of 6 X 1 cm2 was outlined on the simulator and marked on the cast. It covered the thoracic and lumbar spine and the sacrum, and was kept constant throughout the experiment. The correct position was palpated prior to each treatment and was checked multiple times on the simulator. A 250 KV x-ray tube+ was used for the treatments: conditions were 200 kV x rays, 20 mA, 1 mm Cu, FSD 37 cm, dose rate 92 cGy/min. Depth dose distribution was measured in a polystyrene phantom using a Simplex 0.66 cm3 PTW chamber. The dose was calculated to 1 cm depth or the 89% isodose line. The depth of the spine varied from 0.5- 1 cm over the length of the field, resulting in 10% depth dose variation. The dose distribution was confirmed with TLD measurements placed in a rat phantom with removable spine. The field was outlined with the block collimator system of the x-ray tube which allowed a sharp delineation of the field margins and adequate shielding of the body of the animal. The dose homogeneity and the dose outside of the field were measured with film dosimetry, using a Plexiglas phantom.

Data analysis The groups of the Group

loss of animals for evaluation. treatment were: III: 9/ 12, Group

* Phentobarbital Animale,

Paris.

during the treatment left unequal The number of animals at the end Group I: 12/ 12, Group II: 11/ 12, IV: 6/ 12. Standard deviations were

sodium

(Nembutal),

Ceva

Sanoti,

Sante

Volume 26, Number 3. 1993

calculated for all measuring points. The level of significance between groups I and II, groups III and IV and groups II and IV was calculated using the Student’s t-test. Statistical comparisons of group I with group II and group III with group IV found that the differences between the compared groups are not significant. Similar comparisons between group IV and group II respectively group II and group I found the differences significant, both for length (p = < 0,015) and for weight (p = < 0,0025) data, independent of the variation in the frequency of group III (nine animals) and group IV (six animals). The fraction schedules used in group III and group IV were isoeffective for growing bone. The alpha/beta ratio was calculated using the biologically effective dose concept (1, 5) (Fowler, J. F. written communication, March 199 1):

where:

D = total dose in Gy, d = fraction dose, cu/p = ratio between

E a!

- = biologically

the linear and quadratic

coefficient,

effective dose in Gy.

RESULTS

Effkct on spinal growth (Fig. 1) Anesthesia qfect: comparison between group I and II. The animals in Group II, who received twice a day anesthesia remained 1.5% (range O-5%) shorter than the untreated controls in Group I. This difference was first noted on day 9 and persisted through day 42. It was not significant (p > 0.3).

Efect of.fiaction schedule: comparison between group III and group IV. A growth inhibition was noticeable in both groups on day 9, after 1020 cGy in Group III and 1320 cGy in group IV. The maximum growth delay was reached in groups III and IV on day 16 with 9.5% and 1 l%, after 1870 cGy and 2420 cGy, respectively. The difference in growth delay of 1.5% between group III and IV was not significant (p > 0.3). A slight difference between both groups remained until day 75. EJect of radiation: comparison between group II and IV. The effect on growth of twice daily anesthesia on the control animals was of the same magnitude as the difference between the two treatment arms and most likely represents the effect of anesthesia. Therefore, for further analysis we are comparing only Group II with Group IV. The growth inhibition in the twice daily treated animals

+ Stabilan

1, Tube TR 250 f, Siemens,

Germany.

RT on growing

bone 0 K.

WECHSLER-JENTZSCH

Average

429

al.

et

Length

24 23 2221 E

20-

g

19-

p

ta-

--t

Group1

I? -

-

Group2

ia -

--c

Grcup3

15 -

-o-

Group4

f

14 1 Average

Standard

Deviation

13,

I

12!1,,11,1111 0

5

I

III

I

III

I/h

101520253035404550556065707560659095100

300

Days

Fig. 1. Effect of anesthesia and hyperfractionated radiotherapy on the spinal growth of weanling rats. Group lControls, untreated; Group 2-Twice daily anesthesia; Group 3-I X 170 cGy/day, to 3570 cGy in 27 days; Group 4-2 X 110 cGy/day, given in 6-hr intervals, 33 fractions, to 3630 cGy in 21 days.

Efect offraction schedule: comparison between group III and IV. The twice daily treated animals gained an

compared with their control group was 9.5% (range 613%). A growth retardation was already noticeable on day 9, after 1320 cGy. It had reached its maximum on day 16 after 2420 cGy. The difference of 9.5% persisted through the entire observation period. It was statistically significant (p < 0.0 15).

average of 4% less weight than the once daily treated group. The difference was not significant (p > 0.3).

Efect of radiation: comparison between group II and group IV The weight of the animals in the treatment group was 19% lower than in the control group. This weight gap was already apparent on day 5 and persisted until the end of the experiment on day 300. The difference was significant (p = < 0.0025).

Efect on weight (Fig. 2) The pattern of weight change for the control and treatment groups paralleled that of the spinal growth.

Anesthesia efect: comparison between group I and group II. The anesthesia only animals gained an average

Calculation of the alpha/beta ratio for growing bone

of 2% less weight than the untreated was not significant (p > 0.2).

The dose of 3630 cGy given in 33 fractions of 110 cGy twice daily was isoeffective with 3570 cGy given in 21

group. The difference

Average

0

5

10

15

20

25

30

35

40

45

50

Weight

55

60

65

-

Group1

-

Group2

-

Group3

-O-

Group4

70

75

60

65

90

95 100

300

Davs

Fig. 2. Effect of anesthesia and hyperfractionated radiotherapy on the weight of weanling rats. Group I-Controls, untreated; Group 2-Twice daily anesthesia; Group 3-l X 170 cGy/day, to 3570 cGy in 27 days; Group 4-2 X 110 cGy/day, given in 6-hr intervals, 33 fractions, to 3630 cGy in 21 days.

430

I. J. Radiation Oncology 0 Biology 0 Physics Table 1. Radiation

Exp. no. la lb 2a 2b 2c 2d 3a 3b

Fraction-size/&y 500 250 180 150 125 100 170 110

Number fract 5 10 14 17 20 25 21 33

Volume 26, Number 3, I993

effect on growth in weanling

rats

Treat.

Total

days

dose/cGy

% growth inhibit.

Treat. area

Ref.

5 5 9 8 8 9 27 21

2500 2500 2500 2500 2500 2500 3570 3630

35 22 16 13 10 10 9.5 11.0

Leg Leg Spine Spine Spine Spine Spine Spine

4 4 8 8 8 8 Present Present

fractions of 170 cGy once a day. Using the biologically effective dose concept ( 1, 5), the calculation of the alpha/ beta ratio gave a value of approximately 3400 cGy.

DISCUSSION Young children receiving radiotherapy to the craniospinal axis will develop a short stature ( 10, 12, 13). Treatment with 2700-3600 cGy in approximately 150- 180 cGy fractions over 22-27 days at age one, 5 and 10 years caused a growth retardation of 9.0, 7.0 and 5.5 cm, or 19%, 11% and 8%, respectively (12). Our study was designed to follow closely the treatment regimen used clinically to evaluate for a possible bone sparing effect of hyperfractionated treatment in a clinical setting. We found a spinal growth inhibition of 9.5% for the conventional treatment arm and of 11% for the hyperfractionation arm of the experiment. The difference of 1.5% between both treatment arms was not significant and is attributed to the effect of twice daily anesthesia. This result differs with the observations of other investigators, who observed the opposite: less growth arrest with smaller single doses given in multiple fractions per day (Table 1) (4, 8). The major difference in the three studies listed in Table 1 is the overall treatment time used: Eifel (4), as well as Hartsell et al., (8) used accelerated fraction schedules as base line experiments. Accelerated treatment used in experiments # 1a and #2a caused a growth arrest of 35% and 16% compared to only 9.5% with the conventional treatment schedule 3a. A reduction of the single dose from 500 cGy to 125 cGy, helped to reduce the bone growth retardation from 35% to 10% (Exp. #la-2c, Table 1). This sparing effect of the smaller fraction size was limited to the hyperfractionated, accelerated treatment regimes. In contrast, if used with a regular treatment time, hyperfractionation with 110 cGy twice a day did not provide any further sparing of bone growth. Incomplete repair of sublethal damage between fractions could be a possible explanation for the different results in the otherwise very similar experiments #2 and #3 (Table 1) (3, 11). Hartsell et al. (8)

used 4 to 6-hr intervals between their fractions. In the present study this interval was not less than 6 hr. The growing spine of rats responded early to the effect of radiation: with protracted treatments (Fig. l), growth inhibition was already noticeable on day 9 after 10201 1320 cGy, the maximal effect was reached on day 16 at 1870/2420 cGy, continued treatment to 3570/3630 cGy did not result in further growth delay. The hyperfractionated, accelerated treatment with single doses of 125 and 100 cGy (Table 2, Exp. #2c, 2d) caused the same degree of growth inhibition with 2500 cGy as the regular fraction schedule using 170 cGy single dose up to 3630 cGy (8). The high value of 3400 cGy for the alpha/beta ratio of growing bone is characteristic for fast proliferating tissues and in agreement with the rapid growth observed in weanling rats (5). The alpha/beta ratio, as well as the early manifestation of a radiation induced growth delay rank the growing bone of rats with the fast proliferating, early responding tissues. This could explain why substituting of 1 X 170 cGy by 2 X 110 cGy did not result in an improved normal tissue sparing. The degree of growth inhibition observed in young children and in weanling rats (Table 1) ( 12) for similar fraction schedules is within the same order of magnitude. This supports the extrapolation that growing bone of children belongs as well to the fast proliferating, early responding tissues and observations made with the rat model can be of relevance for the treatment of children.

CONCLUSION There appears to be a lower limit to the fraction size which still can spare bone growth. For the present study it was 170 cGy. Hyperfractionated treatment used with conventional treatment times as currently applied in Children’s Cancer Study Group Protocols (2) had no sparing effect on bone growth of rats. But hyperfractionation allowed accelerated treatments without further deterioration of bone growth arrest beyond the level that would be induced by conventional fractionation (8). This could improve the therapeutic ratio for the treatment of pediatric malignancies without added toxicity for growing bone.

-fvI:('lddnS)z1

'[ox10

'~e!psa

‘9861:PPl '1 'IUI .('Jlsqv) dnoJ8

iCpn~sJa3ue3s,uaJpl!y3 pu~Ja~ua3q3JeasaJJorun~u!eJqaql JO iCpnlslo[!d v :seuro!fiuralsu!eJq JOJ uau@aJ Mau

v

'a ‘oysods I.d ‘JaApS t's 'x ‘zeg

‘UlXUp!aH

‘[OXQ

‘layJ_

waruieaJ~

:.w 'M

‘le!peX

uo 13aBa uogwpe~

'm

'Jay]o!pea wyau!y

-Ja snopalalap aql sasvaJ3ap UO!lWJO!lX?J~Jad~H -y+IpUaH

f'~'a‘Olt?JalUO~i~~ 'M‘UOSUt?H

1.3 'm‘uaqo3

‘p~oJuvs :.r‘Jaqx~~

'a ‘J ‘uos

f'd 'M‘I[aSlJt?H

‘8

'n

:'a ~3'uemaaJ~

‘9

'1 'Ipa .iCdl?JaqlO!pEJ paleUO!lX?J~ U! SSaJ%OJd pul!

E~nUJJOJ 3!lt?Jpt?nb-JRaU!1 aqL

.a[D$It?Ma!Aax

‘8861fSPI-II7141

‘ltXPl?x ‘1

'1UI 'hp

S~EJ %ugueaM

Jad SUO!lXJ~

u! isa*le qwoJ?l

‘686IfSOf-fOf:PI 3~~1”

'A 'r ‘Ja[MOd

‘5

‘SkCqd‘IO!H ‘103UO UO!WpL?J

auoq

a[d!qnuI ql!M

paseaJ3aa

‘IO=‘0 ‘JWo!PW

'r 'd ‘laJ!g ‘P

‘(6SSOO O_LLtI)

aql o$sa!pnls 3y?q LUOJ~ :sa$eJJ!Edaa 'r ‘dweyauaa

‘f

‘6

.iCpn$Slol!d-AI iCpnlSEUIO3Jl?SOkUOpqE~J

‘01

'r ‘uaqo3

:'a '8 '3 ‘JasneqnaN

:yy

‘6861:P69-6L9:Z9

J!edaJ uo

'3 'I/V‘Jayor 1'~ ‘sefo~ ‘II

ayL

‘3Joquau!M

'8~~33861 tiio3urdd!7 '8 'r :vd ‘E!qdIaPl?l!qd 'UO!l!pa pJf ‘@O[O!p&?J aql JOJ k~O[O!qO!pQ '1 '3 ‘[[EH ‘L -n] LLIalSU!EJq U' kdt?Jaql UO!lE?!peJpaWJO!lXJ~Jad/(H

‘l?J\?M ‘PI

'El

:P9P

'[O!pQ

's!a 'y3Jv 'ql~oJ8 uo uoge!p~JJ! leu!dsJo

.[osug aDuanuu!

JagU’G

1.~ 'g ‘Jayled f.3 '[ QaqoJd

ua8luaoJ JO s)gaga uope!ptxq

yeu~~ue

Ja3UE!3 'SJOLU

‘~~JeqM~'I‘Jay~OJ3:'3'3‘U~adleH~.~‘Un~~'~'d‘Ja~Jn8

‘lU’JJ,I ‘i#aL

‘L861 'ply3

‘6861:9~~-6Z~:~l Jad asopjo

‘utqdex

-LNI-DN :6888-f)(3d : 1889-933 ‘AI kP”lS ‘=UO~J~Od~OP -‘MJ dUOBJalU1 ‘L I 10-1NL-13N :OS 16-SOd i 1069~!D>33 ‘Z ‘Z861:L661-186118 ‘SA’Id ‘IO!%I‘V’UO ~03Sd!ySUO~lqaJ 13aga 'M 'E) ‘uaspuaJea .I

7.$$[1()99-Lf9:65 d8OIO!pl?~ .au!ds %U!MOJ% KdeJaql

f.z ‘3 ‘utxu_raa :'a 'm

Ja3ue3

‘[66[:18P-PLP:89

I.x ‘UnSIgJn :'a

f'v '12 ‘U!Aa7 :'a 'S 'm ‘SpJEMpa 'CL6 1:11E-685:9 pue auoq %U~MOI%

'ai%qyw

-19v:Z9

i3aBa 'a ‘uosJead :'x ‘IIapu!Ms fy ‘uosq!g f.w 's ‘iap2ys ‘ZI uo!peg

‘fL61t6f9-Pf9:Zf

.au!ds ay$Jo uo!le!ptx~!a%lloAe8aur Jagt? uaJpl!yD u! uopep -Je]aJ QMOJI;) 's g

aql uo

'6861f~~9z-.3~z:f9

IvJqal_raAuo uo~~wxo~~3eJ~ uoy3peJ

dnOJ&alUI

7600

'lv~pe~I'r'lu1~sasuodsaJanss~lpxuJou

u! ql~oJS

-OS! pue

leuo!luaAuo3 JO si3aJ

alx2Jasop ‘uoy3uo~p~~3

asoa

IEP