Applicability of animal tumor data to cancer therapy in humans

lnt J. Radiation Oncology Biol. Phys., Vol. 14, pp. 913-927 Printed in the U.S.A. All rights reserved.

0360-3016/88 $3.00 + .00 Copyright © 1988 Pergamon Press pie

• Original Contribution APPLICABILITY

OF ANIMAL TUMOR DATA THERAPY IN HUMANS

TO

CANCER

J O H N E. M O U L D E R , P H . D . , 1 J E A N D U T R E I X , M . D . , P H . D . , 2 SARA R O C K W E L L , P H . D . 3 A N D D I E T M A R W . SIEMANN, P H . D . 4 IDepartment of Radiation Oncology, Medical College of Wisconsin, Milwaukee, WI, U.S.A.; 2Department of Radiation Therapy, Institut Gustave-Roussy, Villejuif, France; 3Department of Therapeutic Radiology, Yale Medical School, New Haven, CT, U.S.A.; and 4Department of Radiation Oncology, University of Rochester Medical Center, Rochester, NY, U.S.A. The problem of applying experimental tumor studies to clinical cancer therapy is a complex one. The radiotherapy literature contains many examples of premature efforts to apply laboratory observations to the clinic, and many examples of failures to adequately consider animal tumor observations in the design of clinical studies. This review covers three areas: tumor hypoxia, where clinical trials based on animal tumor data have been conducted with radiosensitizers, hyperbaric oxygen, and systemic oxygen carriers; dose fractionation, where current trials of hyperfractionation are based in part on animal tumor studies; and chemo-radiotherapy, where clinical trials are only beginning to exploit concepts developed in animal tumor systems. The use of animal tumor systems extends past the screening of new agents. Animal tumor models can be used in biological, physiological, and pharmacological studies to elucidate the biological factors influencing the efficacy of therapeutic agents. Tumor studies can be combined with studies of normal tissues to predict the toxicities to be anticipated in clinical trials, and to assess the potential for therapeutic gain. Animal studies can also provide data which are useful in designing optimal clinical trials of new agents and maximizing the potential for successful clinical application of new approaches. In general, it is not possible to apply specific laboratory data directly to man. To translate, rather than transpose, information from the laboratory to the clinic, the model studies must be directed at evaluating principles, rather than merely quantifying results. Only through studies of mechanisms, by designing experiments to test or refute a hypothesis, will it be possible to apply model studies to man. Tumors, Oxygenation, Radiotherapy, Radiobiology, Fractionation, Animals, Heterogeneity.

INTRODUCTION The problem of applying experimental t u m o r studies to clinical cancer therapy is a complex one which has been the subject of entire conferences. 54 The cancer therapy literature contains numerous examples of premature and inappropriate attempts to apply laboratory animal findings directly to the clinic, without rigorous consideration of the limitations and implications of the experimental studies. The other extreme is represented by those who believe that studies with experimental animals are essentially irrelevant to h u m a n cancer and cannot contribute to the i m p r o v e m e n t of cancer therapy. 87 The clinical trials with misonidazole a4'26'39"4°,5°provide an example of the problems associated with applying animal t u m o r data to clinical trials. When the misonidazole

trials were originally planned, sensitizer enhancement ratios (SER's) of 1.2-1.6 were predicted, based principally on the in v i t r o results obtained by Adams et al. ~ However, single fraction t u m o r control experiments predicted SER's of 1.1-1.4 for the clinical trials, 14 and multifraction tumor control experiments predicted SER's below 1.1571 (Fig. 1). Based on the latter results, the general lack of success of the clinical trials is not unexpected. Depending on how the laboratory and clinical history is viewed, the misonidazole trials can be seen as a failure of animal t u m o r data to apply to humans, as an example of the inappropriate application of experimental data to clinic therapy, or as an example of appropriate animal studies accurately predicting the results of clinical trials. This review covers three areas in which animal t u m o r data are being, or have been, applied to clinical radio-

This work was presented as a panel at the 28th Annual Meeting of the American Society for Therapeutic Radiology and Oncology, Los Angeles, Calif, November 6, 1986. Work in the authors laboratories was supported by grants CA36858 (D.W.S.), CA38637 (D.W.S.), CAll051 (D.W.S.), CA24652 (J.E.M.) and CA35215 (S.R.) from the U.S. National

Cancer Institute, and grant PDT145 (S.R.) from the American Cancer Society. Reprint requests to: John E. Moulder, Radiation Oncology, MCMC Box 165, Medical College of Wisconsin, 8700 W. Wisconsin Avenue, Milwaukee, WI 53226, U.S.A. Accepted for publication 2 December 1987. 913

914

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Fig. I. Misonidazole enhancement ratios projected for clinical misonidazole trials on the basis of: in vitro radiosensitization of hypoxic V-79 cells (A)~; in vivo sensitization of EMT6/St, R I / LBL, C3H/Tif, FSa and BAI 112 tumors with single dose irradiation (E])J4'7~; and in vivo sensitization of BAI 112 sarcomas with fractionated irradiation (1). 7J The 95% confidence limits are shown only for the fractionated data; limits are of similar sizes for the other in vivo and in vitro data. Misonidazole concentrations are in #g/ml for the in vitro data and in #g/g tumor for the in vivo data. Tumor concentrations achieved by the doses administered in clinical trials are shown at the topJ 4

therapy: tumor hypoxia, where clinical trials based on animal t u m o r data have been conducted with radiosensitizers, 14'26'39'5° hyperbaric oxygen, 25 and systemic oxygen carriers34'95; dosefractionation, where current trials ofhyperfractionations are based in part on animal t u m o r studies27,37,120,131; and chemo-radiotherapy, where clinical trials are only beginning to exploit concepts developed in animal t u m o r systems. TUMOR

HYPOXIA

May 1988, Volume 14, Number 5 and dose rates. 9'43'82'98Recent studies using high-precision plating techniques have shown that 02 also enhances the radiation response of m a m m a l i a n cells at low (0.1-1.0 Gy) doses, although the OER's at low doses are lower than those at high doses. 82,129 In vitro systems have been used to establish the dependence of the radiosensitization on 02 concentration, to show that O2 is required during irradiation for sensitization, and to examine the radiochemistry of the oxygen e f f e c t . 43'44'86"107"129 In vitro systems have also been used to examine the biological effects of hypoxia on m a m m a l i a n cells. M a m malian cells can survive for long periods in hypoxia; survival times vary with the cell line and degree of hypoxia, and also with the pH, glucose concentration, and nutritional environment. 9,11,59'98,1°8,122 Prolonged exposure to hypoxia has been shown to alter cell proliferation patterns, energy metabolism, protein synthesis, gene expression, cell m e m b r a n e properties, intra-cellular pH regulation, enzyme levels and activities, drug metabolism, and the ability of cells to repair radiation- and drug-induced damage.9,11,48,59,89,98,107,108,122 These studies of the biological and radiobiological effects of oxygen could only be performed using in vitro systems, as they require well-defined cell populations, precise environmental control, or levels of experimental precision which cannot be obtained in vivo. In vitro studies are currently yielding data which may offer new insights into the implications of hypoxia for cancer therapy. However, it is clear that the effective use of these data will

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The therapeutic implications of hypoxia have been studied for decades, using subcellular systems, microorganisms, cells in vitro, tumors in animals, and clinical trials. Each of these approaches is valuable, as each system can provide unique and important information. It is clear that the nature and effects of t u m o r hypoxia are more complex than was realized only a few y e a r s ago. 52"75'77 Critical evaluation and integration of data obtained from in vitro, in vivo, and clinical studies will be necessary if cancer treatment is to be improved through the manipulation of t u m o r hypoxia.

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Fig. 2. Radiation cell survival curves for EMT6 mouse mammary tumor ceils irradiated in vitro or in vivo under acute severe hypoxia (e); irradiated in vitro under fully oxic conditions (A); and irradiated as tumors in unanesthetized, air-breathing mice (0). Redrawn with permission from Moulder and Rockwell. 75

Applicability of animal tumor data • J. E. MOULDERet al. require more information on the characteristics of hypoxic cells in solid tumors.

Many investigators have shown that hypoxic cells limit the curability of rodent tumors by single doses of radiation. 75'77 Manipulations which increase the n u m b e r of hypoxic t u m o r cells increase the radiation dose needed to cure the tumors (the TCDso), whereas, manipulations which improve t u m o r oxygenation or sensitize hypoxic cells decrease the TCDso. These results, and similar studies using t u m o r growth delay endpoints, show that hypoxic cells determine the response of tumors remaining in situ after treatment, and are not merely an artifact associated with the use of an in vitro cell survival assay. Data comparing survival curves, TCDso'S, or t u m o r growth in normally-aerated tumors and in tumors made acutely hypoxic can be used to calculate effective hypoxic fractions for the tumors. These data have been reviewed in a series of papers by Moulder and Rockwell75'76'77; the latest review considers 1 17 hypoxic fraction determinations in 46 t u m o r systems. O f the 46 rodent t u m o r lines which have been examined at macroscopic sizes, 41 have been shown to contain significant numbers of hypoxic cells. 77 Data available on the remaining 5 tumors (which include 2 leukemias) are statistically compatible with hypoxic fractions as large as 5.5%, but are also compatible with completely aerobic radiation responses. 75'77 The hypoxic fraction data have been analyzed to examine factors which m a y affect extrapolations from animal data to cancer in humans. In most animal t u m o r systems, hypoxic fraction increases with t u m o r size (Fig. 3). Notably, even microscopic tumors do contain hypoxic cells, implying that hypoxia may be a factor in subclinical, as well as bulky, disease. No t u m o r characteristics have yet been identified which predict hypoxic fractions. 75'77 It had been hypothesized that slowly-growing tumors might have lower hypoxic fractions, because the vascular bed would be better able to "keep up" with the growing tumor;

Hypoxic fractions of rodent tumors The existence of hypoxic areas in solid tumors has been suspected since M o t t r a m ' s histologic studies of irradiated tar warts in 1936. 69 In 1964, Powers and Tolmach s5 showed that cells irradiated in solid lymphosarcomas had a two-component survival curve which indicated the existence of a small, radioresistant t u m o r cell subpopulation with a Do similar to that of hypoxic cells. Since these classic studies, m a n y other animal t u m o r systems have been examined. In general, fully-oxygenated or fully hypoxic t u m o r cell populations irradiated in vivo have survival curves similar to those observed for populations irradiated in vitro under the same conditions of oxygenation. In some cases in vitro and in vivo survival curves show differences reflecting differences in cell population structures or reflecting the effects of intercellular contacts on repair or radiosensitivity. 2,29,56,77 Survival curves for cells irradiated in solid tumors are complex, with an initial radiosensitive region reflecting the survival of the well-oxygenated cell population and a more resistant region reflecting the hypoxic subpopulation (Fig. 2). The dose at which a t u m o r cell survival curve differs from that of a fully oxic cell population varies considerably. In tumors with a small hypoxic fraction (e.g., Power's and Tolmach's survival curve for lymphosarcoma 85) this m a y occur at a dose of greater than 15 Gy. In tumors with large hypoxic fractions, the survival curves may begin to separate at a dose of less than 5 Gy (e.g., Fig. 2). In some tumors moderately hypoxic cells influence the shoulders of the survival curves and affect the response of the tumors to radiation doses in the range used in conventionally fractionated radiotherapy. 88,~27

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Tumor Diameter (mm) Fig. 3. Effect of tumor size on the hypoxic fraction of 7 transplanted rodent tumors. Redrawn from Moulder and Rockwell 77 with the addition of EMT6 and SSC-VII data from Shibamoto et al. 97 Solid symbols are for subcutaneous and intramuscular tumors, open symbols are for pulmonary nodules; all hypoxic fractions are shown with 95% confidence intervals.

916

I.J. Radiation Oncology• Biology• Physics

however, no correlation has been found between the hypoxic fraction and the t u m o r volume doubling time. 77 Neither the site of t u m o r growth, the degree of differentiation, nor the histologic classification were correlated with the hypoxic fraction, except that the few l y m p h o m a s and leukemias studied appear to be well-oxygenated. 77 The failure to find correlations between t u m o r characteristics and hypoxic fraction may be due, in part, to the high degree of uniformity of the animal tumors that have been studied to date (e.g., mouse m a m m a r y tumors, undifferentiated tumors and rapidly growing tumors are overrepresented in the literature). 75'77 These analyses also examined the hypothesis that hypoxic cells might be an artifact of t u m o r transplantation. Because there are inbred mouse strains in which a large proportion of the females develop m a m m a r y carcinomas, m a n y studies have been performed with primary and transplanted mouse m a m m a r y tumors. Radiobiological data on mouse m a m m a r y tumors can be used to estimate hypoxic fractions for primary tumors in their hosts of origin, for tumors transplanted once, a few times, m a n y times, or hundreds of times, and for culture-adapted tum o r lines. As Figure 4 shows, these data provide no evidence that the hypoxic fractions of mouse m a m m a r y carcinomas vary with the duration of transplantation or that transplanted tumors differ from primary tumors. Several groups have investigated the radiation responses of h u m a n t u m o r cell lines xenografted into nude mice. The hypoxic fractions of these xenografts are similar to the hypoxic fractions of transplanted rodent tumors analyzed under similar conditions. 77 Xenografts may be limited as models for oxygenation in h u m a n tumors, as the vascular bed supporting the t u m o r is of mouse origin, 93 and as 02 transport to the t u m o r is dependent upon the

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Fig. 4. Hypoxic fractions of mouse mammary carcinomas as a function of transplant history. Closed symbols are for excision assays, open symbols are for in situ assays. Hypoxic fractions are shown for autochthonous tumors (×), 1st generation tumors (O), 2nd through 5th generation tumors (~), 10th through 100th generation tumors (V, ~'), tumors transplanted for more than 100 generations or more than 10 years (D, II), and cultureadapted lines (A, A). Reprinted with permission from Moulder and Rockwell. 77

May 1988, Volume 14, Number 5 hematologic system of the mouse, which has characteristics very different from that of humans. However, because h u m a n t u m o r xenografts produce tumors with hypoxic fractions similar to those of rodent tumors, several potential bases for postulating systematic differences between the oxygenation of h u m a n and rodent tumors are eliminated.

Characteristics of hypoxic cells in tumors The biological characteristics of the naturally occurring hypoxic cells in solid h u m a n cancers are essentially undefined; little information is even available on the characteristics of hypoxic cells in solid rodent tumors. There is evidence that some tumors contain chronically hypoxic ce118,19,42,52,117j18.121,124 whereas some contain cells which are transiently hypoxic. 13'18'79'115 Chronically and transiently hypoxic cells would result from different mechanisms, have different characteristics, and have different implications for therapy, v7 Several groups have begun to develop probes for chronically and transiently hypoxic t u m o r cells. 17,~8,19.42. 80,83,103,104 The hypoxic cell radiosensitizer misonidazole undergoes reductive bioactivation to an alkylating species more rapidly in hypoxic cells than in aerobic cells. The pattern of drug binding can be used to identify chronically hypoxic cells in diffusion-limited cell culture systems and in tumors. 19,42,124Because the radiolabeled nitroimidazoles have long biological half-lives and their diffusion and metabolism are slow, these drugs may prove useful as probes for measuring and identifying chronically hypoxic t u m o r cells. Durand, Olive, Chaplin, and their collaborators have used tracer dyes combined with histologic and flow cytometric techniques to examine hypoxia and blood flow in tumors. 17,18,80These studies provide the most convincing evidence to date that variations in perfusion through individual blood vessels produce transient local hypoxia in some tumors.~8 These techniques also allow the identification and isolation of chronically and transiently hypoxic t u m o r cells, and thereby allow a detailed examination of their biological characteristics and response to therapy (as described below). Nuclear magnetic resonance spectroscopy and imaging may likewise prove useful in the detection, measurement, and characterization ofhypoxic t u m o r cells, as may more invasive studies with 02 and pH electrodes and with centrifugal elutriation techniques. 77

Reoxygenation It is generally assumed that reoxygenation occurs between treatments in fractionated radiotherapy and minimizes the importance of hypoxic t u m o r cells in determining the outcome of therapy. Unfortunately, data on reoxygenation in animal tumors are limited and variable. 52'53'6°'92'96 Figure 5 compares reoxygenation patterns for six transplanted rodent tumors after large single doses of radiation; it is obvious from these data that reoxygenation is not necessarily rapid or complete, and that dif-

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Fig. 5. Patterns ofreoxygenation for 6 transplanted rodent tumors after large single doses of X rays. Data are shown for C3H mouse mammary carcinomas (O), RIB5 rat sarcomas (1), mouse C22LR osteosarcomas (A), mouse RIF-I fibrosarcomas (t), mouse SSK31 fibrosarcomas (i), and EMT6 mouse mammary tumors (~). Redrawn from Rockwell and Moulder, 92 with additional data from Kummermehr et al. 6° and Shibamoto et al. 96

ferent tumors behave differently. Moreover, for a specific tumor, the rate and extent of reoxygenation can depend on the radiation dose and fractionation pattern. 46"53'92 There is also evidence that hypoxic cells can influence the response of rodent tumors irradiated with chronic low dose rate irradiation, as well as with acute irradiation. 5~ Animal data on reoxygenation are too fragmentary to allow meaningful extrapolation to h u m a n cancers. However, the existing rodent t u m o r data provide a warning that we cannot expect all h u m a n tumors to reoxygenate to the same extent, or at the same time, and that we should expect hypoxic cells to influence the outcome of radiotherapy in some h u m a n tumors, even with fractionated radiotherapy regimens. Approaches to improving radiotherapy

The data on experimental rodent tumors support the hypothesis that solid tumors contain hypoxic cells which influence the outcome of cancer therapy. Approaches to minimizing the therapeutic resistance induced by hypoxia can be based on: increasing the relative radiosensitivity of the hypoxic cells (e.g., using chemical radiosensitizers k26'39'5°'9°or high LET radiation132), increasing t u m o r oxygenation (e.g., with hyperbaric oxygen 25'34 or perflurochemical emulsions34'73'91'95"i19), or killing the hypoxic cells (e.g., with hyperthermia or bioreductive alkylating agents58'9°). The unspectacular overall results of the clinical radiosensitizer trials have discouraged m a n y cliniciansY '39'7~ Many radiobiologists would argue, however, that the low levels of radiosensitization achieved in rodent tumors with clinically relevant drug and radiation schedules (Fig. 1)

917

should have warned us that only modest clinical gains could be expected, 14's°'71"9° and that clinical trials with small numbers of patients or variable clinical material (e.g., multiple histologies, sites, or stages) would be doomed, by statistics, to failure. 14'9° Adjuvant therapy with agents toxic to hypoxic cells has theoretical advantages over the use of radiosensitizers, as it is less dependent on the radiation dose per fraction and on the achievement of high drug levels during irradiation. 58'9° Some agents of this class look extremely promising in the laboratory and in preliminary clinical trials/s'9° Recent reevaluations of laboratory data and clinical hyperbaric oxygen studies suggest that it is appropriate to reconsider the use of hyperbaric oxygen and to consider other approaches to modulating t u m o r oxygenation during therapy (e.g., by increasing arterial pO2, increasing 02 transport, altering t u m o r blood flow, or decreasing 02 utilization by tumor cells). 25'34'36'39'52'1°5 For example, data from several laboratories have shown that perfluorochemical emulsions plus 02 breathing can improve the responses of solid tumors to large single doses of radiation and to fractionated regimens using doses as low as 2.5 Gy per fraction. 34'9k~]9 Experiments with B A l l l 2 rhabdomyosarcomas 73 treated with three fractions per week show that treatment with a perfluorochemical emulsion plus 02 only once per week significantly lowers the TCDso; this change in the TCDso is significantly larger than that produced by misonidazole at toxic doses. Survival curves for tumors treated with both hyperbaric oxygen and perfluorochemical emulsions show that even greater effects can be achieved with the combination of these two agents. 34'66 Clinical trials of perfluorochemical emulsions are now in progress. 34'95 RADIATION FRACTIONATION Animal studies of the relationship between the dose per fraction and the radiation tolerance of normal tissues have made major contributions to clinical radiotherapy. A large body of experimental and clinical data has demonstrated the similarity in response to fractionation of animal and human normal tissues. The variation of the tolerance dose as a function of dose per fraction is the same for early skin reaction in mice, 28'41 in rats, v° and in man. 31 The results obtained for late skin reaction 1°,~33 in mice also agree with h u m a n data. 7'35 Experiments on rat spinal cord, 125'13° mouse lung, 3°'33'128 and mouse ll° and rat 74,92 kidney provide a satisfactory explanation for clinical observations of the sparing effect of fractionation on these tissues. The contribution of experimental studies to an understanding of the relationship between the dose per fraction and t u m o r control has been far more modest, due to specific features of the animal t u m o r models and to technical problems in the experiments. Transplantable animal tumors differ in m a n y respects from spontaneous h u m a n tumors. The clonogenicity of animal tumors is higher,

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I.J. Radiation Oncology • Biology • Physics

their cell loss rates are greater, 64'~°9 and they grow faster than h u m a n tumors. They are smaller in absolute size than h u m a n tumors, but are larger in proportion to body weight. The immunogenicity of animal tumors is variable, and can be very high. 49'112 These features may influence the t u m o r radiation response, particularly in fractionated studies.

Dose per fractionation studies with rodent tumors. The influence of the dose per fraction is usually expressed by the a/fl coefficient of the formula:

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D(c~/fl + d) = constant where D is the dose required to produce an effect with a dose per fraction ofd. 5"28'38"123'134This formula was derived from the linear-quadratic expression of the cell survival curve. This derivation is based on a n u m b e r of assumptions. It is assumed that cell survival can be represented by a linear-quadratic expression over the range of doses per fraction used, and that cell kill is the same for each fraction in a fractionated course. It is also assumed that the same biological effect is observed with different fractionation schedules producing the same final cell survival. Finally, it is presumed that overall treatment time is either the same in all schedules, or is so short that t u m o r proliferation is not a factor. The a/~ relationship can also be viewed as an empirical curve-fitting formula. The graphical method introduced by Douglas and Fowler 2s consists of plotting the reciprocal of the total dose D (i.e., l/D) as a function of the dose per fraction (d). The experimental data should fall on a straight line which intercepts the abscissa at a dose of -'~/~.

Attempts to determine the c~/fi ratio for tumors has been confounded because most tumors are a mix of radiosensitive aerobic cells and radioresistant hypoxic cells. To circumvent this problem, most dose per fraction studies have used tumors made uniformly radiosensitive with hyperbaric oxygen or radiosensitizers, or tumors made uniformly anoxic by clamping the blood supply to the t u m o r or asphyxiating the animal with nitrogen. If we assume that oxygen acts as a pure dose-modifying factor, the same effect is produced on oxic cells by a dose D, as is produced on anoxic cells by this dose D multiplied by the OER. Thus the od/3 values obtained for anoxic tumors can be compared with those for oxic tumors by dividing the values of anoxic cells by an assumed OER.

Tumor dose per fraction studies in situ Barendsen and Broerse 6 used a t u m o r growth delay assay to study the effects of dose fractionation in a rat rhabdomyosarcoma. Growth delay was found to correlate with the total dose, not the fraction size; the difference between the growth delays for single-fraction and multi-fraction irradiation was minimal (Fig. 6). The lack of a significant change in growth delay when the fraction size was reduced from 4 to 2 Gy was unexpected: a 30% dose increase was

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tionated irradiation delivered in a single dose (ff]), or at 2 (A), 3 (m) or 4 (O) Gy per fraction. Redrawn with permission from Barendsen and Broerse. 6 anticipated from the a/13 value of 4.9 G y measured in vitro with cultured cells of this tumorS; an additional increase was anticipated from repopulation during the longer irradiation time required for the smaller fractions. The t u m o r control dose increased from 78 Gy to 98 Gy when the fraction size was decreased from 4 to 2 Gy; this is as expected from the a/13 value of 4.9 Gy. Suit and Wette 1~3assessed tumor control doses (TCDso) for a mouse m a m m a r y carcinoma rendered anoxic by clamping. For a single dose, the TCDso was 46 Gy. For a single fraction of 9.9 Gy, plus a top-up dose, the TCDso was 51 Gy (i.e., repair equivalent to 5 Gy occurred when a single dose was replaced by two fractions). For 9 fractions of 6.75 Gy, plus a top-up dose, the TCDs0 was 84.0 Gy (i.e., repair equivalent to 38 Gy occurred when a single dose was replaced by nine fractions). This experiment demonstrates that increased repair occurs when the number of fractions is increased and the dose per fraction is decreased. However, it does not assess the effect of increasing the number of fractions (decreasing the dose per fraction) in a schedule where all fractions are the same size. That computation would necessitate knowing the shape of the dose-response relationship for the 250 kV X rays used for the top-up dose. Subsequently, Suit et al. ~' carried out a study using an equal dose per fraction for all treatments (Fig. 7). An a/fl value of 35 Gy can be derived from the data for clamped tumors if the single dose (71 Gy) point is excluded; assuming an OER of 3, the a/fl ratio for oxic cells would be 12 Gy. For animals breathing hyperbaric oxygen, the data for 5 and 10 fractions also fit an a/fl value of 12 Gy; for a smaller n u m b e r of fractions the projected a/13 is larger, as would be expected if hyperbaric oxygen does not eliminate all hypoxic cells. For air-breathing animals the a/fl ratio was between that for hyperbaric oxygen and that for clamped tumors, and was closer to the latter than

Applicability of animal tumor data • J. E. MOULDERet al. I

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Table 1. a/13 values derived from clinical studies

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Dose per Fraction(Gy) Fig. 7. Reciprocal of the tumor control dose plotted against the dose per fraction for the response of a mouse mammary carcinoma to fractionation. Tumors were irradiated in normal airbreathing animals (V1), in animals breathing hyperbaric oxygen (O) or in animals where the blood supply to the tumor had been clamped off (11). Data from Suit et al. ~ to the former, as expected for a tumor with a large hypoxic fraction. Williams et al. TM reviewed studies in which misonidazole or clamping was used to achieve a uniform t u m o r radiosensitivity (Fig. 8). Larger a/t3 ratios were found for misonidazole treated animals than for clamped tumors. Fowler 37 considered this apparent difference insignificant because of the wide spread of confidence limits; an alternative explanation is that misonidazole did not eliminate all hypoxic radioresistance. Data derived from clinical studies are scarce (Table 1), and the influence of reoxygenation and proliferation necessitates some assumptions for the calculation of a/fl ratios from clinical results obtained with different fractionation schedules.

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Dose per fraction studies in vitro Survival curves of some established animal tumor cell lines can also be assessed by in vitro cloning. Under these conditions, a/fl ratios range from 4.1 Gy to 14.5 Gy. 4 The cell survival curves obtained in 40 human tumor cell lines were reviewed by Fertil and Malaise. 32 The distribution o f a~/3 values is shown in Table 2. Deacon et al. 2° reviewed published data on 51 human cell lines, and concluded that the radiosensitivity of the cell lines was correlated with clinical radiosensitivity. Malaise et al. 65 reviewed published data on 49 h u m a n tumor cell lines (Table 3), and reached conclusions similar to those of Deacon et al., 2° even though the two groups did not analyze all of the same cell lines, and although they used different criteria for assessing both in vivo and in vitro radiosensitivity. Both analyses 2°'65 emphasized radiosensitivity at low doses, which is mainly related to parameter a. Problems associated with derivation o f t u m o r a/13 values The different t u m o r response assays used in the animal studies have different technical constraints and raise difTable 2. a/fl ratios derived from cell survival curves of 40 human tumor cell lines*

>

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Dose per fraction studies by excision assay In excision assays small radiation doses can be used without causing the problems encountered for in situ experiments, and in some studies doses of less than 3 Gy have been used. Williams et al. ~3~derived a/fl values from the results of 26 excision assays (Fig. 8). For 19 sets of data a/fl is less than 12 Gy; the mean value is 7.7 Gy. The 7 other a / ~ values are larger than 20 Gy. The cases for which a / ~ is larger than 20 Gy are mainly observed for naturally oxic tumors (air breathing animals) or misonidazole treated animals; only two high a/fl values were observed among the 14 experiments on anoxic tumors. The a/fl ratio is smaller for fibrosarcomas and rhabdomyosarcomas than for the other types, but the small number of tumors studied for most histologic types does not permit any further conclusions.

0

10 a/0 Value (Gy)

100

Fig. 8. Cumulative distribution of a/fl values for in situ assays of misonidazole sensitized tumors (..... ), in situ assays of anoxic tumors ( . . . . . ), and excision assays ( ). Data from Williams et al. TM

a/fl (Gy)

Number of cell lines

Distribution (%)

2-4 4-6 6-10 10-20 20+

8 7 8 7 10

20 17 20 17 25

* Adapted from Fertil and Malaise. 32

920

I.J. Radiation Oncology• Biology• Physics Table 3. Survival curve parameters for histologic groups of human tumor cell lines*

Tumor type

Number of cell lines

c~ (Gy l)

/3 (Gy -2)

c~/fl(Gy)

Glioblastoma Melanoma Squamous cell ca. Adenocarcinomas Lymphomas Oat cell ca.

5 19 6 6 7 6

0.24 0.26 0.27 0.31 0.45 0.65

0.029 0.053 0.045 0.055 0.051 0.081

8.3 4.8 6.1 5.6 8.8 8.0

* Adapted from Malaise el al. 65 ferent biological problems. 21'22'76 In the studies of Barendsen and Broerse 6 the fractionation effect is less for growth delay than for t u m o r control. The same trend is seen in the data analyzed by Williams et al. TM The two t u m o r response assays may not assess the same cell population; tumor regression assesses the response of the bulk of the t u m o r cell population, whereas the t u m o r control is determined by the most radioresistant clonogenic cells. I n situ experiments require repeated irradiations to achieve an observable gross reaction. When a large number of fractions is used, the overall time may not be negligible with respect to the t u m o r doubling time and repopulation during the irradiation may partially compensate for the cell kill. The role of overall time has been studied by Suit et al. ~ml for a m a m m a r y carcinoma with a volume doubling time of 2.5 days. The TCDs0 was not dependent on the interval between fractions until some critical time was reached; this critical time depended on the dose per fraction. Thus, for a constant overall time, the increase of the TCDs0 with increasing fraction number m a y not be determined solely by the dose per fraction. The effect of the overall time can be minimized by keeping the interval between fractions as short as possible. However, to allow for complete repair of sublethal damage between fractions, the fractionation cannot safely exceed three fractions per day. The m a x i m u m n u m b e r of fractions is therefore limited when the overall time is kept short enough to make repopulation negligible. Because of these constraints, most o~//3values determined in situ have been based on relatively large doses. Smaller doses can be used in excision assays than in in situ assays, and several technical and biological problems arising with in situ assays disappear with excision assays. Proliferation, redistribution, completion of sublethal damage repair, for example, do not interfere with the effect of the single doses used in excision assays. However, repair of potentially lethal damage may be less than it is in situ. Malaise et al. 65 have compared cell survival curves for the t u m o r cells irradiated in vitro with those for solid tumors irradiated as xenografts (the contribution of the hypoxic fraction was excluded to compare the effect on presumably oxygenated cells). They found higher survivals for in vivo irradiations than for in vitro for all dose levels. Whether

May 1988, Volume 14, Number 5 this difference in radiosensitivity is associated with a difference in response to fractionation is not known. Hypoxia poses a serious problem when tumors are irradiated in situ. For a mixed oxic-hypoxic population the cell survival curve is biphasic (Fig. 2). The a/{3 value for oxygenated cells must be assessed on the initial portion of the cell survival curve. When data for small fractions are not available, the variation of the t u m o r response with the dose per fraction is underestimated, and a/t3 is overestimated. An alternative to using small doses per fraction is to achieve uniform sensitivity in the entire t u m o r cell population. Misonidazole has been used in an attempt to render the hypoxic cells equally sensitive to the oxygenated cells. However, even with the largest misonidazole dose that can be used, the sensitizer enhancement ratio is still less than the O E R . 4° Incomplete sensitization of hypoxic cells results in an overestimate of the a/~3 ratio; this may be responsible for the large c~//3 values obtained in misonidazole experiments (Fig. 8). An alternate approach to radiosensitization is to use a vascular clamp, or nitrogen asphyxiation of the host, to render the entire t u m o r uniformly hypoxic. If uniform hypoxia is not achieved, or if the trauma caused by clamp produces some additional cell kill, the a/t3 ratio will again be overestimated. The a/13 values obtained with uniform hypoxia are much smaller than those obtained with misonidazole (Fig. 8). This suggests that misonidazole is not a reliable method of achieving uniform radiosensitivity. Even with uniform oxygenation, the validity at low radiation doses o f a~13 values measured from high dose data is questionable. 5 At large doses m a n y cell survival curves become exponential, rather than following a strict o~//3 model. As a result, the cell survivals for small numbers of fractions (i.e., large dose per fraction) are larger than the values predicted by a linear-quadratic fitted to the initial part of the survival curve. Thus, a higher a//3 value is obtained when large doses are used. For example, from the TCD50 data of Suit, 1~ an o~//3 value of 19 Gy is obtained if all the points are used, but a value of 12 Gy is obtained if the single-dose point is excluded (Fig. 7). Derivation of the c~//3 value for oxic t u m o r cells from the data obtained in hypoxic cells poses a different set of problems. This derivation presumes that oxygen acts as a pure dose-modifying agent. In fact, some experiments show that the OER may be smaller for low doses than for large doses, that is, parameter c~ is less influenced by hypoxia than parameter/L 67's2'~29The assumption of a constant OER may lead to an overestimation of the a/13 value of oxic cells. The problems of uniform oxygenation and fraction size disappear for cell survival curves obtained in vitro. Cell culture allows direct assessment of the parameters a and /3 in the dose range of interest. The relevance of the values obtained in vitro to the results of fractionated irradiation is questionable, however, even when the roles of repopulation and reoxygenation are ignored. Some experiments

Applicabilit~ of animal tumor data • J. E. MOULDER el a[.

have shown that in a fractionated regimen the effect of subsequent fractions increases progressively. 68~26"~35 This change has been attributed to cell cycle redistribution, reoxygenation, or a reduction of the repair ability. The consequence is that tumor response is less affected by fractionation than expected from assumption of an equal effect at each fraction. This would suggest a larger c~/~ value in situ than in cell culture. Transplantable rodent tumors clearly provide a model for analyzing the mechanisms which play a role in determining the response of tumors to fractionated irradiation. However, the parameter W/3, commonly used to express the effect of fractionation, varies over a broad range, and artifacts appear to lead to spuriously high c~//3 values and to an underestimation of the fractionation effect. Most experimental tumors studied to date respond to fractionation in a fashion similar to acutely reacting normal tissues; however, some tumors will probably be found to react like late reacting tissues (Fig. 8). Data obtained for human tumors lead to the same suggestions (Tables 13). With a total dose giving an equal tumor effect, reducing the dose per fraction spares the late responding tissue if the tumor response to fractionation is small (high c~/¢3 value) and increases the late reactions if the tumor response to fractionation is large (low c~/fl value). CHEMO-RADIOTHERAPY Applying data obtained from combined modality studies in animals to the clinic is a problematic task. Mechanistic studies performed in tissue culture are clearly important. However, in vitro investigations may not always predict in vivo t u m o r responses. For example, drug pharmacokinetics and metabolism have been shown to affect agents known to be effective in vitro such that they are ineffective in vivo. This failure often is caused by the inability of the drug to penetrate to relevant tumor cells or because of rapid drug deactivation by the host. 12.:2,J14,1~6 The sensitivity and repair capacity of cells derived from tissue culture or solid tumors may not be the same. Figure 9 shows that while human squamous cell carcinoma cells grown in tissue culture demonstrate significant repair of potentially lethal damage, this form of repair is absent when these cells were grown as xenografts in nude mice. 2 The age-response of ceils from tissue culture or solid tumors also may not be the same. When mouse K H T tumor cells were evaluated for their radiation response across the cell cycle, 56 cells in vitro exhibited a fairly conventional response but the cell age response of K H T cells derived from solid tumors was different (Fig. 10). These data demonstrate that the direct extrapolation of in vitro data to the in vivo situation may not always be valid. T u m o r heterogeneity a n d chemo-radiotherapy. The differences between sensitivity and repair capacity of cells in vitro and in vivo may be related, in part, to the heterogeneity of tumors in vivo. As a t u m o r grows the vasculature often cannot keep pace with the rapid neoplastic

921

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Fig. 9. Clonogenic cell survival as a function of time before subculture for HEp3 cells irradiated in plateau-phase monolayers with 9 Gy (m), or as xenografts with 20 Gy (O). Data from A1lalunis-Turner and Siemann. 2 cell proliferation. This can lead to microenvironmental variations within a tumor such that areas of good nutrition (near blood vessels) exist along with areas of impaired nutrition, poor nutrition, and necrosis. Thus, as a consequence of their growth characteristics, tumors are comprised of various cell subpopulations consisting not only of nutritionally deficient cells, but also cells in various phases of the cell cycle, quiescent cells and non-neoplastic host cells. The existence of tumor cell heterogeneity can influence the response of tumors to single agent or combined modality therapies. For example, the presence of hypoxic cells limits the success of radiotherapy in animal tumor models and clinical evidence exists which implicates these cells as a cause for local failures in at least some human c a n c e r s . 15"L6'46"77"124 Hypoxic cells also may be preferentially spared by some commonly used chemotherapeutic agents because of their location in poorly vascularized areas of tumors, or by their cell cycle state. 58'9°'94"99 Similarly quiescent tumor cells may demonstrate intrinsically different sensitivities to drugs or radiation compared to their rapidly proliferating counterparts, s.84 More recently it has been suggested that the presence of cell subpopulations possessing high intracellular glutathione levels may be a mechanism for tumor resistance to certain anticancer agents. 45'47'62 Thus, the existence of various tumor cell subpopulations may lead to resistance to treatment with single agents. Alternatively, the existence of tumor cell subpopulations with different properties may allow the development of therapeutic strategies aimed at exploiting the cooperation or interaction between radiation and drugs against different cell subpopulations.

922

I.J. Radiation Oncology • Biology • Physics

May 1988, Volume 14, Number 5

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Fig. 10. Cell age response to 10 Gy for KHT sarcoma cells derived from tissue culture (O) or solid tumors (11). Redrawn with permission from Keng et al. 56 Techniques for studying tumor heterogeneity To use animal t u m o r models to evaluate the therapeutic importance of t u m o r cell heterogeneity, it has been necessary to develop techniques to isolate and characterize the various cell subpopulations comprising tumors, and to establish the role of the cell subpopulations in determining the overall t u m o r response to therapy. Two effective techniques for evaluating different cell populations in solid tumors are centrifugal elutriation and fluorescence activated cell sorting. Centrifugal elutriation separates cells on the basis of size and density, and homogeneous subpopulations with a high degree of cell cycle synchrony can be derived directly from solid tumors. 55'57J°4 Consequently it has been possible to assess the in situ sensitivity of cell subpopulation to radiation and chemotherapeutic agents used alone or in combination. 56'1°1J°3'1°6 Centrifugal elutriation was used to evaluate the response of K H T sarcoma cell subpopulations to combinations of CCNU, misonidazole and radiation (Fig. 1 1). When K H T sarcomas were irradiated under air-breathing conditions, hypoxic G1 cells dominated cell survival. ~°3 In contrast, when C C N U and misonidazole were combined, killing of the hypoxic G l cells was increased, presumably because potentiation of C C N U by sensitizers such as misonidazole requires hypoxic conditions99; and survivors were found primarily in S and G2/M. When C C N U and misonidazole were given 24 hours before irradiation, the survival was uniform throughout the various cell fractions, m6 This probably occurred because the CCNU-misonidazole combination significantly reduced the radiation-resistant subpopulations; and the radiation treatment, in turn, reduced the cell populations preferentially surviving the CCNU-misonidazole treatment. The combination, therefore, reduced the importance of both hypoxia and cell cycle specificity in determining the outcome of the treatment. When the CCNU-misonidazole-radiation combination was assessed for overall t u m o r response, it was found that the three-agent treatment led to supra-additive cell kill in

the tumor 1°6 and to a significantly enhanced tumor control probability.l°° These results demonstrate that the use of centrifugal elutriation in combination with animal t u m o r models may be an important method of predicting which treatment modalities might be successfully used in combination. An alternative to centrifugal elutriation is the use of fluorescence activated cell sorting to isolate cell subpop-

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Applicability of animal tumor data • J. E. MOULDERet al. ulations from tumors. In the past, most investigations sorted cells on the basis of DNA-bound fluorochromes. 3'83 However, stain concentrations required for effective cell sorting often proved toxic, particularly to cells treated with drugs or radiation. 83'1°2 More recently, a cell separation technique based on the diffusion properties of the fluorochrome Hoechst 33342 has been developed which allows cells to be separated by a cell sorter on the basis of their Hoechst 33342 staining intensity.17'79 This technique is based on the fact that after intravenous injection of the dye, cells close to the blood vessels stain more brightly than cells further away. By irradiating tumors after Hoechst injection, dissociating the tumors into single cells, sorting on the basis of fluorescence intensity and determining clonogenic cell survival in the sorted fractions, Chaplin and colleagues 17'79have been able to demonstrate that the brightly staining tumor cells were oxic at the time of irradiation whereas dimly staining cells were hypoxic. Consequently this method of cell separation will allow the direct in situ evaluation of agents known to be activated or selectively toxic under hypoxic conditions. Techniques such as centrifugal elutriation and fluorescence activated cell sorting can be used effectively in animal models to evaluate agents used alone, or in combination, against various tumor cell subpopulations. Such studies of tumor cell heterogeneity can address the mechanisms of interaction in combined modality treatments and may suggest future strategies for optimally combining agents. Normal tissue toxicity in chemo-radiotherapy Although combined modality investigations are usually aimed at achieving an increase in tumor response, it is clear that the evaluation of any combination of agents must also assess normal tissue toxicity. For therapeutic benefit to result from treatment with a combination of chemotherapy and radiation, interactions must occur which lead to greater damage to the tumor cells than to the normal tissues in the radiation field. To complement tumor response assessments, animal model systems can be used to study side-effects of combination therapies. Complete dose response curves for both acute and late reactions following combined modality therapies can be determined in a number of normal tissues. TM However, for information gathered from such investigations to be applicable to man, model investigations should be directed at determining mechanisms of drug-radiation interaction in the tissue(s) of interest, and whenever possible should include fractionated dose regimens. This approach can be illustrated by some studies of cisplatinum (cis-Pt) toxicity. Reports that patients who relapsed after total body irradiation (TBI) and bone marrow transplantation had a low chemotherapy tolerance led to studies of cis-Pt toxicity in rats surviving TBI. Rats surviving TBI had a dose-related decrease in cis-Pt tolerance, which could be observed at radiation doses that alone demonstrated little or no renal toxicity; the mechanism

923

responsible for the increased cis-Pt toxicity was found to be a decrease in drug clearance in the TBI animals. 7~'74 Using crypt survival as the endpoint and different fractionation schedules in combination with cis-Pt, DeWit 23 was able to show that the a/~ value for the combined treatment was larger than that for X rays alone. This implied that cis-Pt not only caused cell killing directly, but also inhibited sublethal radiation damage repair. Clinical evidence also exists24 for enhanced damage to small bowel when cis-Pt is included with radiation. Laboratory findings such as these could have an impact on the future design of protocols combining these agents. CONCLUSIONS Studies with animal tumor models have provided a basic understanding of the processes producing hypoxia in tumors and of the characteristics of hypoxic cells, and have established the importance of hypoxia in producing tumor radioresistance. Studies of tumor hypoxia now in progress should be of great value in examining approaches to improving radiotherapy that are based on either exploiting or eliminating hypoxia. The use of animal tumor systems should extend past the screening of new agents. Animal tumors can be used in biological, physiological, and pharmacologic studies to elucidate the biological factors influencing the efficacy of the agents. T u m o r studies can be combined with studies of normal tissues to predict toxicities to be anticipated in clinical trials and to assess the potential for therapeutic gain. Animal studies can also provide data which are useful in designing optimal clinical trials of new agents and maximizing the potential for successful clinical application of new approaches. The successful application of animal tumor models in this process requires a relatively sophisticated approach to experimental radiotherapy, and an understanding and integration of data obtained in vitro, in animals, and in the clinic. Transplantable tumors of rodents provide valuable models for analyzing the mechanisms underlying the response of tumors to fractionated irradiation. However, the quantitative effect of fractionation is different for the various types of tumors. Most experimental tumors studied to date respond to fractionation in a fashion similar to acutely reacting normal tissues; however, some animal and human tumors will probably be found to react like late reacting tissues. Thus, no general statement can be made concerning the therapeutic benefit of using small fractions. Optimization of fractionation in radiotherapy must be considered individually for each tumor type; animal data can provide useful concepts, but can not compensate for the present shortage of human data. Animal model studies can provide significant information concerning combined modality treatments, particularly when assessing normal tissue toxicities or the role of tumor cell heterogeneity in the overall response of tumors to therapies. However, it is clearly difficult to translate results obtained in model studies directly to man. Part of the difficulty arises because combined modality

924

I.J. Radiation Oncology • Biology • Physics

investigations are difficult to m o d e l in the laboratory. Exp e r i m e n t a l investigations have often focused o n o p t i m i z ing p r o t o c o l s (timing, sequencing) to m a x i m i z e t u m o r responses in a p a r t i c u l a r m o d e l system. In general, it is n o t possible to a p p l y such specific l a b o r a t o r y d a t a directly to m a n . T o translate, rather t h a n transpose, i n f o r m a t i o n

May 1988, Volume 14, Number 5 f r o m the l a b o r a t o r y to the clinic, the m o d e l studies m u s t be directed at evaluating principles rather t h a n o b t a i n i n g detailed d a t a on one system. O n l y t h r o u g h studies o f m e c h a n i s m s , b y designing e x p e r i m e n t s to test or refute a hypothesis, will it be possible to a p p l y m o d e l studies to m a n .

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1. Adams, G.E., Flockhart, I.R., Smithen, C.E., Stratford, I.J., Wardman, P., Watts, M.E.: Electron-affinic sensitization: VII. A correlation between structures, one-electron reduction potentials, and efficiencies of nitroimidazoles as hypoxic cell radiosensitizers. Radiat. Res. 67: 9-20, 1976. 2. AUalunis-Turner, M.J., Siemann, D.W.: Potentially lethal damage (PLD) repair in human epidermoid tumor cells grown in vivo and in vitro (Abstr.). In Proceeding of the 8th International Congress of Radiation Research, Vol. 1, Fielden, E.M., Fowler, J.F., Hendry, J.H., Scott, D. (Eds.). London, Taylor and Francis. 1987, p. 157. 3. Arndt-Jovan, D.J., Jovan, T.M.: Analysis and sorting of living cells according to deoxyribonucleic acid content. J. Histochem. Cytochem. 25: 585-589, 1977. 4. Barendsen, G.W.: Variations in radiation response among experimental tumors. In Radiation Biology in Cancer Research, Meyn, R.E., Withers, H.R. (Eds.). New York, Raven Press. 1980, pp. 333-343. 5. Barendsen, G.W.: Dose fractionation, dose rate and isoeffect relationships for normal tissue response. Int. J. Radiat. Oncol. Biol. Phys. 8: 1981-1997, 1982. 6. Barendsen, G.W., Broerse, J.J.: Experimental radiotherapy of a rat rhabdomyosarcoma with 15 MeV neutrons and 300 kV X-rays. II. Effects of fractionated treatments applied five times a week for several weeks. Eur. J. Cancer 6: 89109, 1970. 7. Bates, T.D., Peters, L.J.: Dangers of the clinical use of the NSD formula for small fraction numbers. Brit. J. Radiol. 48: 773, 1975. 8. Bauer, K.D., Keng, P.C., Sutherland, R.M.: Isolation of quiescent cells from multicellular tumor spheroids using centrifugal elutriation. Cancer Res. 42: 72-78, 1982. 9. Bedford, J.S., Mitchell, J.B.: The effect of hypoxia on the growth and radiation response of mammalian cells in culture. Brit. J. Radiol. 47: 687-696, 1974. 10. Berry, R.J., Wiernik, G., Patterson, R.J.S., Hopewell, J.W.: Excess late subcutaneous fibrosis after irradiation of pig skin consequent upon the application of the NSD formula. Brit. J. Radiol. 47: 277-281, 1974. 11. Born, R., Hug, O., Trott, K.R.: The effect of prolonged hypoxia on growth and viability of Chinese hamster cells. Int. J. Radiat. Oncol. Biol. Phys. 1: 687-697, 1976. 12. Brown, J.M.: Drug or radiation changes to the host which could affect the outcome of combined modality therapy. Int. J. Radiat. Oncol. Biol. Phys. 5:1151-1163, 1979. 13. Brown, J.M.: Evidence for acutely hypoxic cells in mouse tumors, and a possible mechanism of reoxygenation. Brit. J. Radiol. 52: 650-656, 1979. 14. Brown, J.M.: Clinical trials ofradiosensitizers: What should we expect? Int. J. Radiat. Oncol. Biol. Phys. 10: 425-429, 1984. 15. Bush, R.S., Hill, R.P.: Biological discussions augmenting radiation effects and model systems. Laryngoscope 85: 1119-1133, 1975. 16. Bush, R.S., Jenkin, R.D.T., Allt, W.E.C., Bean, F.A., Behne, H., Dembo, A.J., Pringle, J.F.: Definitive evidence

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