Effects of chemotherapy and irradiation on normal tissues.

Meyer JL, Vaeth JM (eds): Radiotherapy/Chemotherapy Interactions in Cancer Therapy. Front Radiat Ther Oncol. Basel, Karger, 1992, vol 26, pp 45-54

Effects of Chemotherapy and Irradiation on Normal Tissues1 Theodore L. Phillips Department of Radiation Oncology, University of California, San Francisco, Calif., USA

The initial observations that a combination of actinomycin and radiotherapy led to enhanced radiation pneumonitis and increased skin reactions including the so-called `recall' reactions led to intense interest in chemoradiation effects in normal tissues [ 1]. It also led to conclusions by many that simultaneous radiation and chemotherapy should be avoided and that they should be administered remotely in time [2]. More recently available laboratory data and the results of clinical trials suggest that although interactions are enhanced, they occur more often in acutely responding tissues which can heal and are less marked in late-responding tissues. This presentation will review the results of a series of laboratory studies and briefly review some of the available clinical data. Although the original idea behind chemoradiation was spatial cooperation [2], with its goal of reducing metastases, it is now clear that gains in many tumors are being achieved by simultaneous radiation and chemotherapy and/or rapid alternation of radiation with chemotherapy. In order to quantitate the effects of radiation and chemotherapy, it is necessary to define a term. Since many of the interactions observed cannot be absolutely classified as subadditive, additive, or supra-additive, the term enhancement is best used to describe an increased effect noted in normal tissues when the two agents are used in combination. The term dose effect factor (DEF) has proven quite useful. This term is the ratio of the radiation dose required for a given effect without chemotherapy divided by the radiation dose for the same level of the effect with chemotherapy [3]. One can go further and determine the therapeutic ratio by dividing the DEF for tumor by the DEF for normal tissues.

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Supported by US Public Health Research Emphasis Grant CREG No. CA-20529.

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Table 1. Evaluated drugs — LAFI mice: maximum tolerated doses, mg/m2 Actinomycin Adriamycin BCNU Bleomycin Cis-platinum

1 24 75 300 40

Cyclophosphamide 5-Fluorouracil Melphalan Methotrexate Nitrogen mustard Vincristine

250 300 30 2,100 9 9

Animal Studies


We have conducted a series of trials in four mouse tissues representing both acute and delayed effect type organs. Studies have been carried out using the LD50 endpoint for the pulmonary lethality, esophageal lethality and renal lethality. All of these were performed with drug administered at the maximum tolerated dose of 2 h before irradiation [4, 5]. A large series of experiments has been carried out with intestinal crypt cells using the crypt regeneration assay of Withers and Elkind [6] with drug delivered at numerous time intervals before and after irradiation, drug delivered 2 h before the determination of a complete crypt survival curve, with drug delivered in increasing doses to determine drug survival curves and with drug given before 2 split doses of irradiation to quantitate the effects on sublethal damage repair. In these experiments the mice were injected intraperitoneally with the drug at various fractions of the previously determined maximum tolerated dose, i.e. that killing less than 1 % of animals with drug alone, and then radiation was delivered at various time intervals relative to that time of intraperitoneal injection. Five days later, the animals were euthanized and the intestine removed and sectioned with enumeration of the numbers of regenerated crypts and conversion of this data to survival curves. These studies were carried out with a total of 11 drugs which are listed in table 1, with their maximum tolerated doses (MTD) in the LAFI mouse, in which these experiments were conducted. The initial series of experiments evaluated the 11 drugs given 2 h prior to graded radiation doses, generating survival curves. The survival curves determined 2 h after drug administration were shifted toward the left or to lower doses for a given level of survival, but the curves remained parallel, in all but a few cases, to the radiation only survival curve. This result suggests that the effect is primarily either additive cell kill or partial removal of the shoulder in the dose-response curve. The former interpretation is preferred.

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Table 2. Intestinal crypt cell DEF values Cyclophosphamide Bleomycin BCNU Cis-platinum Cyclophosphamide Actinomycin D Adriamycin Fluorouracil Nitrogen mustard Methotrexate Vincristine

1.05 2.4 1.25 1.4 1.05 1.1 1.2 1.3 1.4 1.8 1.1

The dose-effect factor for intestinal crypt cells was calculated by dividing the radiation dose required to reduce the number of crypt cells per circumference to 50 with radiation alone by the dose required when radiation was delivered 2 h after drug treatment.


Examples of these dose-response curves are shown in figure 1 for four different drugs [7-10]. The calculated dose effect factors at a survival level of 50 crypts is shown in table 2 for 10 drugs. It can be seen that the maximum enhancement occurred with bleomycin and that little or no enhancement occurred with cyclophosphamide. The next series of experiments was performed with a fixed radiation dose given 2 h after drug or 3 h before drug administration. In this way the experiments could determine whether there was an effect on radiation repair from the drug given before and could determine a drug dose response curve. Results for the four different drugs shown in figure 1 are shown for this assay in figure 2. It can be seen that there is generally a dose response with decreasing crypt survival with increasing drug dose. Generally, there is only a small difference in the results obtained when drug is given before versus after, with the exception of bleomycin and BCNU where there is a much more marked effect when it is given before, suggesting an interaction between the repair mechanisms of drug and radiation injury. The estimated intestinal crypt cell survival at the drug MTD is summarized in table 3. In all cases this was determined from the experiments in which drug was given after irradiation to eliminate the effects of repair interaction and determine the true cell kill by the drug alone. The next series of experiments evaluated 10 drugs in terms of the effect of time between drug and radiation administration which varied from —48 to +48 h relative to the time of irradiation. There was a wide range of effects of time between drug and radiation with very large effects seen with bleomycin and BCNU when given 18-2 h before irradiation and little

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Phillips

Cyclophosphamíde 1,000 500 -

500 -

σ 75 mg/kg • 250 mg/kg

o 137Cs

50 -

10-

10-

5-

5-

0.5 -

05-

1

1,000500 -

1

1

1

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0.1 -

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1,400 1,800 Dose, rad

σ 3 U/kg • 100 U/kg

100-

50 -

0 1,000

2 h before írradiatιοn

1,000-

ο137 Cs

100-

Crypt cells per circumference

Bleomycin

2 h before irradiahon

2,200

BCNU 2 h before irradiation

Ο137 Cs

1,200 1,600 Dose, rad

800

Adriamycin 1,000-

2 h before irradiation

500 -

~

137Cs σ 8 mg/kg

σ 8 mg/kg

100-

• 25 mg/kg

2,000

• 15 mg/kg

100 -

50 -

50 -

10 -

10-

5-

5-

0.5 -

0.5 -

0.17' ι ι ι 0 800 1,200 1,600 Dose, rad

0.1 • i 0 600

• i

i i i ι 1,000 1,400 1,800 Dose, rad


Fig. 1. Examples of intestinal crypt cell radiation survival curves measured 2 h after drug delivery at MTD. From [7-10], with permission.


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Bleomycin

200 -

iiiiiiiiiiiiiiiiiiii

100 -

1370s

control, 1,100

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50 -

Cyclophosphamide 137 Cs control, 1,100 rad

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Crypt cells per circumference

5-

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62.5 125.0 187.5 250.0 Dose, mg/kg 200 100 -

BCNU

25

Adriamycin

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// ~

50 75 Dose, units/kg

100

13705

control,

1,100

rad

50 -

10 6.25

12.50 18 75 Dose, mg/kg

25.00

2.0

4.0 6.0 Dose, mg/kg

Fig. 2. Survival of intestinal crypt cells with increasing chemotherapy dose given 2 h before (•) or 3h after (0) irradiation. From [7-10], with permission.

Table 3. Estimated intestinal crypt cell survival at drug MTD (% values) Actinomycin Adriamycin BCNU Bleomycin Cis-platinum

40 30 35 40 20

Cyclophosphamide 5-Fluοrοuracil Methotrexate Nitrogen mustard Vincristine

60 10 10 10 70


effect when given after irradiation. Wide swings in survival were seen for two cell cycle active agents, i.e. 5-FU and methotrexate, presumably due to synchronization of cells. Table 4 shows the time of maximum and minimum cell kill in intestinal crypt cells relative to the drug administration time. There is clearly no constant maximum or minimum time of cell kill among the drugs; each is unique in the effect of time between drug and radiation on crypt cell survival.

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Table 4. Time of maximum and minimum cell kill in intestinal crypt cells — drug administration time relative to radiation time Drug Actinomycin Adriamycin BCNU Bleomycin Cis-platinum Cyclophosphamide 5-Fluorouracil Methotrexate Nitrogen mustard Vincristine

Maximum kill, h —6 + 12 —2 —6 0 —18 +24 —12 —12 +48

Minimum kill, h +48 0 +24 +48 +2 + 18 +48 +24 —48 —48

Negative values indicate drug before radiation; positive values indicate drug after radiation.


A final series of experiments carried out with the intestinal crypt cell assay involved delivery of maximum tolerated drug dose 2 h before the first of two radiation doses. The two radiation doses were divided by times from 2 to 24 h. The drugs which caused significant impairment of split dose repair in the intestinal crypt cell system are summarized in table 5. The percent decrease in repair at 4 h ranges from 10 to 100%, depending on the drug type. Because of the synchronizing effect of methotrexate, the studies are probably unreliable in determining its effect on sublethal damage repair, and there was apparent increased repair. The pulmonary lethality endpoint determined at 160 days following irradiation was then applied. Drug was given 2 h before irradiation of the thorax in animals observed for 160 days. Enhanced response was seen with six drugs ranging from a DEF of 1.2-2.1, with cyclophosphamide being the most active agent (table 6). A series of experiments were carried out using the esophageal lethality endpoint in which mice are observed for 28 days for deaths following thoracic irradiation due to esophageal denudation and obstruction. Only two drugs caused a significant enhancement in the dose effect factor for esophageal irradiation, BCNU and cis-platinum at 1.4 and 1.7, respectively. The renal lethality endpoint in which the right kidney is resected and the left allowed to hypertrophy and then palpated through the skin for irradiation was employed. The 1-year LDSos were used and indicated that only two drugs caused significant enhancement of radiation injury, i.e. adriamycin and cyclophosphamide with DEFs of 1.9 and 1.8. Although

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Table 5. Drugs causing impaired split dose repair in intestinal crypt cells Drug Actinomycin Adriamycin BCNU Cis-platinum Methotrexate

Percent decrease at 4 h 50 10 100 100 (2 h) 50 (4 h) -100

Table 6. Drugs yielding significant enhancement Drug Lung Bleomycin Cisplatin Cyclophosphamide Actinomycin D (2.2 mg/m2) Adriamycin Methotrexate Esophagus BCNU Cis-platinum Kidney Cyclophosphamide Adriamycin

Dose effect factor 1.2 1.2 2.1 1.6 1.3 1.6

(1.0-1.4) (1.1-1.4) (1.6-2.7) (1.3-2.3) (1.1-1.6) (1.3-2.0)

1.4 (1.2-1.8) 1.7 (1.3-2.5) 1.8 (1.2-2.7) 1.9 (1.2-3.2)


cis-platinum is known to be renal toxic, it appears that a single administration of the maximum tolerated dose does not cause sufficient injury to interact with the radiation damage or to dominate the effect of radiation damage with the late endpoint. Although acute tubular necrosis may occur, it does not seem to, in this experimental design, change survival. In reviewing this and other animal data, it is clear that there are a number of factors involved in the interactions of chemotherapy and radiotherapy in normal tissues. Additive and supra-additive effects are seen more frequently in acute-reacting tissues such as intestinal crypt cells than in late-reacting tissues such as lung and kidney. Enhanced normal tissue damage is seen primarily with drugs which have specific organ effects of their own and is, therefore, very much drug-type dependent [ 11]. A few agents are seen which caused enhanced response in almost every endpoint, the most notable of this is actinomycin-D, which fortunately is now rarely used in the treatment of adult tumors in chemoradiation protocols.

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Clinical Data


It is not appropriate at this point to go through an exhaustive review of all of the recent clinical trials from which one might determine the relative enhancement that is seen in acute and late damage. A few interesting examples, however, can be presented. Earlier in the study of chemoradiation excess normal tissue damage was frequently observed. Reports were published on the interaction of methotrexate and radiation in the central nervous system and the enhancement of lung damage by radiation and actinomycin. Subsequently, extensive data was collected and reported on the interaction of radiation and adriamycin in the heart, indicating that significant adriamycin dose reductions were required for patients who had had cardiac irradiation. In the Northern California Oncology Group study 7Η61 [ 12], acute reactions were clearly increased by the administration of 5 units of bleomycin twice weekly in conjunction with conventional radiotherapy at 1.8 Gy per fraction. Grade 3 or 4 reactions occurred in 26% of radiationonly patients and 71 % of radiation plus chemotherapy patients. On the other hand, interruptions in therapy were not longer in the combined modality group than in the radiation alone group. Analysis of late effects, including fibrosis and necrosis in this group, by an actuarial method indicated that the rate at 4 years was approximately 20% for radiation alone and 38% for the combination, although this difference had not yet reached statistical significance. This study had shown a very marked improvement in local-regional control and disease-free survival, suggesting that the gain in tumor effect was greater than the increase in late tissue damage. Quantitative information on the dose effect factor in humans is very limited, although some is available for skin reactions suggesting that it may be as high as 3 for actinomycin-D and between 1.6 and 1.8 for methotrexate. Clinical dose effect factors in the lung for actinomycin have been in the 1.3 range. Data on the more popular regimens containing 5-FU, 5-FU plus platinum or mitomycin, and platinum alone are less quantitative but suggest that although increases in pulmonary toxicity, acute mucositis and skin reactions are clearly seen, increases in late effects are less prominent. We await the final reports on a number of these exciting new studies to be able to determine the dose effect factor for acute and late damage, relative to that for tumor. Table 7 shows that the various critical organs in those drugs which have been found to enhance radiation response as well as the timing for maximum response. Clearly, concurrent administration yields the highest number of organs in which interactions have been observed.


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Table 7. Clinical examples of increased normal tissue response with systemic single drugs and radiation Tissue

Drug

Timing for maximum response

CIS/PIS Lung Heart Intestine Liver Kidney Skin/mucosa Botte/soft tissue Esophagus Bladder Eye

MTX, CisPlat ActD, CTX, Adria, Blei, CisPlat Adria, Mito, 5FU ActD, Adria, 5FU ActD, VCR ActD, Adria, CisPlat ActD, Adria, MTX, Bleo, 5FU 5FU, Blei ActD, Adria, CisPlat CTX 5FU

B, C, A B, C B, C C C B, C B, C C C C C

B = Before; C = concurrent; A = after.

Conclusion Α few drugs have been found to cause supra-additive effects in almost all normal tissues, i.e. actinomycin and perhaps, adriamycin. Other drugs such as vincristine, vinblastine and cyclophosphamide seldom show enhancement of radiation injury. Enhanced normal tissue damage is seen primarily with drugs which have specific organ effects of their own if one excludes the adriamycin and actinomycin compounds. It is clear that quantitative dose effect factors are needed for normal tissues with newer regimens which primarily emphasize 5-fluoruracil and cis-platinum. Data should be available from ongoing and recently completed clinical trials to provide this information and determine clearly whether the therapeutic ratio has been enhanced. References


1 Wara WI, Phillips TL, Margolis LW, Smith V: Radiation pneumonitis: A new approach to the derivation of time-dose factors. Cancer 1973; 32:547-552. 2 Steel GG, Peckham MJ: Exploitable mechanisms in combined radiotherapy-chemotherapy: The concept of additivity. Int J Radiat Oncol Biol Phys 1979;5:85-91. 3 Phillips TL, Fu KK: The interaction of drug and radiation effects on normal tissues. Int J Radiat Oncol Biol Phys 1978;4:59-64.

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4 Phillips TL, Margolis L: Radiation pathology and the clinical response of lung and esophagus. Front Radiat Ther Oncol 1972;6:254-273. 5 Phillips TL, Ross G: A quantitative technique for measuring renal damage after irradiation. Radiology 1973;109:457-462. 6 Withers HR, Elkind MM: Microcolony survival assay for cells of mouse intestinal mucosa exposed to radiation. Int J Radiat Oncol Biol Phys 1970;17:261-267. 7 Phillips TL, Ross GY, Goldstein LS, Begg AC: The interaction of radiation and bleomycin in intestinal crypt cells. Int J Radiat Oncol Biol Phys 1979;5:1509-1512. 8 Phillips TL, Ross GY, Goldstein LS: The interaction of radiation and cyclophosphamide in intestinal crypt cells. Int J Radiat Oncol Biol Phys 1979;5:1441-1444. 9 Goldstein LS, Ross GY, Phillips TL: The interaction of irradiation and BCNU in intestinal crypt cells. Int J Radiat Oncol Bul Phys 1979;5:1569-1571. 10 Ross GY, Phillips TL, Goldstein LS: The interaction of irradiation and adriamycin in intestinal crypt cells. Int J Radiat Oncol Biol Phys 1979;5:1313-1315. 11 Phillips TL, Fu ΚΚ: Quantification of combined radiation therapy and chemotherapy effects on critical normal tissues. Cancer 1976;37:1186-1200. 12 Fu KK, Phillips TL, Silverberg IJ, Jacobs C, Gomnet DR, Chun C, Friedman MA, Kohler M, McWhirter K, Carter SK: Combined radiotherapy and chemotherapy with bleomycin and methotrexate for advanced inoperable head and neck cancer: Update of a Northern California Oncology Group randomized trial. J Clin Oncol 1987;5:14101418.


Theodore L. Phillips, MD, Department of Radiation Oncology, University of California, San Francisco, CA 94143-0226 (USA)

Detoxifying enzymes in human ovarian tissues: comparison of normal and tumor tissues and effects of chemotherapy.

The interaction of drug and radiation effects on normal tissues.

Changes in the microcirculation of normal tissues after irradiation.

Irradiation effects in the electron microprobe quantitation of mineralized tissues.

Metabolic profiling of somatic tissues from Monochamus alternatus (Coleoptera: Cerambycidae) reveals effects of irradiation on metabolism.

Magnetic resonance imaging of the acute effects of interstitial neodymium:YAG laser irradiation on tissues.

Early and late effects of accelerated charged particles on normal tissues.

Effects of X-rays on vascular function in transplanted tumours and normal tissues in the mouse.

The effect of laser irradiation on oral tissues.

A study of the effects of photodynamic therapy on the normal tissues of the rabbit jaw.

Effects of intravenous amiodarone on electrical dispersion in normal and ischaemic tissues and on arrhythmia inducibility: monophasic action potential studies.

Effects of chemotherapy on fertility.

Combined radiation and hyperthermia: effects of the number of heat fractions and their interval on normal and tumour tissues.

Effects of hyperbaric oxygen and a perfluorooctylbromide emulsion on the radiation responses of tumors and normal tissues in rodents.

[Diagnosis and prognosis of gonadal damage after chemotherapy and irradiation].

Effects of bleomycin and irradiation on euoxic and hypoxic cells.

The prevalence and severity of late effects in normal rat duodenum following intraoperative irradiation.

O2 levels in normal tissues.

Effects of carbon ion irradiation and X-ray irradiation on the ubiquitylated protein accumulation.

The relative biological effectiveness of fractionated doses of fast neutrons (42 MeVd----Be) for normal tissues in the pig. II. Late effects on cutaneous and subcutaneous tissues.

Tolerance of normal tissue to therapeutic irradiation.

Chemotherapy and radiotherapy effects on the skin.

Comparison of the effects of photon versus carbon ion irradiation when combined with chemotherapy in vitro.

Effects of ultraviolet irradiation on growth and pigmentation in seedlings.