Fraction size-dependent acute skin reaction of mice after multiple twice-a-day doses

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??Original Contribution

FRACTION SIZE-DEPENDENT ACUTE SKIN REACTION OF MICE AFTER MULTIPLE TWICE-A-DAY DOSES YOSHINAO

ABE,

M.D.

AND MUNEYASU

URANO,

M.D.

Edwin L. !jteele Laboratory, Department of Radiation Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02 114 To study the effect of multiple daily fractions on acute murine foot reaction, the left feet of C3Hf/Sed

mice were irradiated by a Cs-137 irradiator at intervals of 6 and 18 hr, alternately. Fraction sizes ranged from 1 to 12 Gy, and various numbers of fractions were given. At the end of fractionation, a top-up dose of 20 Gy was administered. The overall treatment time ranged from 2 to 21 days. The average skin reaction from 10 to 35 days after completion of irradiations was determined. The skin reactions started from 8 to 27 days following the completion of irradiations. The skin reaction after fraction sizes of 3.5 and 5 Gy was dependent on the total dose. The skin reaction after fraction sizes of 1.5 and 2 Gy showed a biphasic response; at lower total doses, the skin reaction was proportional to the total dose, but at higher total dose, the skin reaction reached a plateau. The average skin reaction at the plateau with fraction sizes of 1, 1.5, and 2 Gy were 0.79 * 0.03, 1.12 f 0.13, and 1.37 f 0.06, respectively. The (Y/B ratio for acute skin reaction following the twice a day scheme was 9.09 Gy (at dose/fraction 2 3.5 Gy). The plateau may be attributed to the effect of accelerated proliferation and a balance between cell depletion and proliferation during twice-a-day irradiations. It is also suggested that twice-a-day irradiations induce more proliferation in skin than once a day treatments. Fraction size, Proliferation during irradiations, Skin reactions, Multiple fractionation, cr/j3 ratio.

sponding tissues and tumors is very likely related to both fraction size and treatment interval. To study the effects of multiple daily fractions on acute murine foot reactions, we have designed a series of experiments using a fixed time interval-twice a day-and a various range of fraction sizes-l to 12 Gy with a final single “top-up” dose of 20 Gy (1, 10).

INTR.ODUCTION

An introduction of the a/P ratio to radiotherapy has contributed to clarifying the differences between acute and late responding normal tissues after multiple fractions (2, 17) and supports the rationale for multiple daily fractions (7, 16,22). Numerous a//3 ratios have been obtained from animal studies. A common procedure for the determination of the a//3 ratio uses multiple fractions at an interfractional interval which is short enough to minimize the effect of proliferation but long enough to allow maximum repair from sublethal and potentially lethal damage. In clinical practice, however, proliferation of cancer cells during conventional fractionation radiotherapy may be significant ( 16,23). This may be true for acute responding normal tissues. Although some studies regarding proliferation during and following irradiations have been reported in mouse and rat skin (4, 9, 13, 21) using 5 times a week and/or 3 times a week treatment schemes, data regarding proliferation during multiple daily fractions are still scanty. The extent of proliferation in the acute re-

METHODS

AND

MATERIALS

Male C3Hf/Sed mice were used throughout the experiments. They were produced and maintained in our defined flora mouse colony. At the start of irradiations, the mice were lo- 12 weeks old. Irradiations were performed using a cesium irradiator with two portals. Dose rate was -7.5 Gy/min. The left foot was hooked by a wire and fixed on a brass plate to be placed in an irradiation field (3 cm in diameter). Prior to irradiation, mice were anesthetized by an intraperitoneal injection of sodium pentobarbital of 0.05 mg/g body weight.

Acknowledgement-The authors wish to thank Drs. E. L. Travis, S. L. Tucker and H. D. Thames for their advice and discussions. This study was partially supported by NC1 grant CA-26350 awarded by the National Cancer Institute, Department of Health and Human Services. Accepted for publication 15 June 1989.

Presented at the American Society for Therapeutic Radiology and Oncology meeting, New Orleans, Louisiana, October 9- 14, 1988. Reprint requests to: Muneyasu Urano, M.D., Dept. of Radiation Medicine, University of Kentucky Medical Ctr., 800 Rose St., Lexington, KY 40536. 359

Fraction size dependent acute skin reaction 0 Y. ABE AND M. URANO

nificant differences were observed among these various methods (data not shown). Figures 2A and 2B show the average skin score plotted against the total dose. The single-fraction experiment showed a good correlation between the skin score and the radiation dose. Within the range of test dose, the average skin score did not reach the upper limit of scoring. The average skin score obtained from 2 fractions with a topup dose also showed a good correlation with total dose. The slope of the 2 fraction response was not as steep as that of the single dose response. The skin scores after fraction sizes greater than 2 Gy were proportional to the total dose. However, after fraction sizes of 2 and 1.5 Gy, the skin scores showed a brphasic response. At lower dose, the average skin scores were proportional to total dose.

361

At higher dose, the average skin scores became independent of the total dose. The skin reaction reached a plateau around the total dose of 45 and 50 Gy at fraction sizes of 2 and 1.5 Gy, respectively. At the fraction size of 1 Gy the dose response curve showed a plateau between 52 and 60 Gy. The average skin scores at plateaus observed following the fraction sizes of 1, 1.5, and 2 Gy were 0.79 + 0.03, 1.14 f 0.13, and 1.37 + 0.06, respectively. This means that these plateau values depend on the fraction size. Figure 3 illustrates the average skin score plotted against the overall treatment time. Again, average skin scores obtained from fraction size greater than 2 Gy were proportional to the overall treatment time, although skin scores obtained for fraction size up to 2 Gy showed a biphasic

W 3

bi

30

40

I

I

50

GO

Total Dose

(Gy)

Fig. 2. The avera,ge skin reactions plotted against the total dose. Single dose and 2 fractions and fraction sizes of l-5 Gy (B). Mean f standard error.

with top-up dose (A)

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February 1990, Volume 18, Number 2

OVERALL

Fig. 3. The average skin reactions

TREATMENT

plotted against the overall treatment

response. The skin reaction reached a plateau around 7 days and 11 days at the fraction size of 2 and 1.5 Gy, respectively. The plateau obtained from a fraction size of 1 Gy lay between 17 and 2 1 days. Using the data obtained from single-fraction, 2 fractions and multiple fractions with 3.5 Gy and 5 Gy each, the a)/ /3 ratio was calculated at the skin reaction level of 1.5. These schedules with a large size of fractions and a small number of fractions were chosen to minimize the influence of proliferation. The total dose which induced a skin reaction score of 1.5 was determined by using logit analysis for each fraction size and the a/P ratio was obtained according to the method of Joiner (10) and Tucker ( 18). The (Y/P value was 9.09 Gy with a 95% confidence interval from 5.09 to 13.1 Gy. The error obtained from predicted isoeffect dose and observed isoeffect dose ranged from -0.55 to 0.44 Gy. This value is well coincident with earlier studies ( 15). DISCUSSION The skin reaction obtained following twice a day fractions showed several significant features which related to both fraction size and number of fractions. At higher fraction sizes (23.5 Gy), the skin scores were proportional to both overall treatment time and total dose. At small fraction sizes, the skin reaction appeared to reach a plateau value; namely, at small size of doses, skin reaction increased with increasing total dose and at large doses skin reaction became independent of total dose. The level of skin reaction at each plateau was dependent on the fraction size. The larger the fraction size the more severe the plateau skin reaction was. Six and 18 hr intervals of irradiations are long enough to allow repair from sublethal damage (6, 21). The con-

TIME-

DlYS

time. Mean k standard

error.

stant repair capacity of skin cells following multiple fractions up to 20 fractions has been reported ( 11). The plateau phase of skin reaction is neither due to the upper limit of the skin scoring system nor to the complete repair of sublethal damage. It is very likely that cell proliferation was accelerated during irradiations. It has been demonstrated that accelerated proliferation occurs in skin following and during multiple fractions. Denekamp described the accelerated proliferation of mouse skin following 14 fractions of 3 Gy given 5 days per week (4). She also reported that this proliferation was not due to an increase in the growth fraction but due to a shortening of the cell cycle time (5). Moulder and Fischer ( 13) showed the accelerated proliferation of rat skin during multiple fractionation. These two studies indicate that the daily increment dose corresponds to 1.25-l .3 Gy during the most accelerated proliferation period. Accordingly, the plateau of skin reaction observed in the present study is likely attributed to the accelerated proliferation induced by fractionation and to the balance between cell depletion and proliferation. The plateau skin reactions were also observed by van Rongen and Kal(2 1) and by Howes and Brown (9). The former showed the plateau of rat skin reaction when the total dose was more than 80 Gy (5-6 weeks) of 3 Gy daily fractions. The latter investigators found that a mouse skin score for 20 fractions of daily 4 and 4.5 Gy was equal to that of 30 fractions of the same daily dose. In other words, acute skin reaction reached a plateau at the total dose of 80-90 Gy of 4-4.5 Gy daily fractions. They also demonstrated that the magnitude of the plateau obtained by daily doses of 4.5 Gy each was more severe than that obtained by daily doses of 4 Gy each. Their data together with ours suggest that the proliferation of rodent skin stem cells was able to compensate for the cell damage caused

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Fraction size dependent acute skin reaction 0 Y. ABE AND M. URANO

by daily fractions of 4-4.5 Gy or twice-a-day doses of 2 Gy each. Our present data indicated that the total dose to reach the plateau following twice-a-day irradiation was much less than that obtained following daily doses. This difference may be attributed to the difference in the treatment intervals (twice-a-day vs. daily). Twice-a-day irradiations appeared to induce more proliferation in skin than once-a-day irradiations. A similar phenomenon has been observed in vitro and in vivo using continuous irradiations. Mitchel et al. (12) observed in vitro that survival curves of 3 out of 7 mammalian cells bent upward continuously as a function of the total dose and finally reached a plateau during very low dose rate (0.1 Gy/hr) irradiations. This expresses the accelerated proliferation and the balance between cell depletion and rapid cell proliferation. An in vivo study reported by Quastel ( 14) also showed that such proliferation took place in the small intestine of rats during continuous irradiations. Quastel’s experiments suggest that the plateau occurs 2 days following initiation of 4.15 Gy/day continuous exposure. Since LQ (linear-quaidratic) model is additive (3) the following equation is obtained:

d b / d

OQ’

9

20

1

25

30

Single Dose or Equivalent Dose (Gy)

Deq(Deq + a/p) = C nidi(di + a/P), where Deq is an equivalent dose to a single dose, ni and di are a number of fractions and a fraction size at ith treatment, respectively, and cu/p is the a/P ratio. This equation allows the conversion of the total dose of multiple daily fractionation schedule into the equivalent dose. Results of this analysis are shown in Figure 4. The curve in the figure is based on the results of single fraction. The equivalent doses estima.ted from 2 fractions, and fraction sizes of 3.5 Gy and 5 Gy each are located closely to the single dose. This result suggests that cell proliferation does not occur during fractionation treatments. The equivalent doses estimated from h-action sizes of 1, 1.5 and 2 Gy also show plateaus. Overkilling of stem cells is also suggested for mice irradiatled with doses of 2 Gy each before they reach the plateau. This overkilling may be attributed to the effect of redistribution because this cannot be explained by repair kinetics. It is not easy to answeer the question of why the plateau level of skin reaction depends on fraction size. As quoted before, accelerated proliferation may be attributed to a shorter cell cycle time. A large radiation dose can kill more cells than a small dose. During the plateau, the cell cycle time may decrease with increasing fractional dose and thus a balance is maintained between cell depletion and proliferation. Figulre 5 illustrates a schematic model of the accelerated proliferation during fractionated irradiations. Since the flexure dose of the skin obtained from our data is estimated to be between 0.45 and 1.36 Gy (8, 19) slopes of dose response curves following multiple doses of 1, 1.5, and 2 Gy each are different and expected to become steeper with increasing fraction size. Initially,

Fig. 4. The average skin reactions plotted against the single dose (open triangles) or equivalent dose as described in text. Symbols indicate the following fraction sizes: closed triangle-l Gy; closed Gy; open circles-3.5 Gy; circles-l.5 Gy; closed squares-2

open squares-5

Gy. Open triangles include 2 fractions.

there is no rapid cell proliferation. At the damage level ‘a’in Figure 5, accelerated proliferation begins and compensates the cell depletion of the smallest fraction size of Total Dose -

1.5 Gy

2 GY

Fig. 5. The schematic model to interpret pendent skin reactions due to accelerated multiple fractions. See details in text.

the fraction proliferation

size deduring

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1 Gy. At ‘b’ in the figure, cell depletion caused by 1.5 Gy is compensated by accelerated proliferation. The same phenomenon may be observed for the highest dose of 2 Gy at level ‘c’. Thus, the fraction-size dependent skin reaction is observed. A critical dose may exist over which cell depletion cannot be compensated and under which

February 1990, Volume 18, Number 2

cell depletion is compensated since the shortening of the cell cycle time may be limited. The critical dose is also related to the interval of irradiations. It is important to understand this critical dose among various acute responding tissues to prevent severe acute reactions following multiple fractions.

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