John H. Peacock

The dose-rate effect in human tumour cells

The radiation response of 12 cell lines derived from a variety of human tumours has been investigated over the dose-rate range from 150 to 1.6 cGy/min. As the dose rate was lowered, the amount of sparing varied widely; in 2 cell lines it was zero, in the other cell lines the dose required for 10(-2) survival ranged up to twice the value at high dose rate. Low dose-rate irradiation discriminates better than high dose rate between tumour cell lines of differing radiosensitivity. The data are equally well fitted by two mathematical models of the dose-rate effect: the LPL model of Curtis and the Incomplete Repair model of Thames. Analysis by the LPL model leads to the conclusion that the theoretical radiosensitivity in the total absence of repair was rather similar among the 7 cell lines on which this analysis was possible. What differs among these cell lines is the extent of repair and/or the probability of direct infliction of a non-repairable lesion. Recovery from radiation damage was also examined by split-dose experiments in a total of 17 human tumour cell lines. Half-time values ranged from 0.36 to 2.3 h and there was a systematic tendency for split-dose halving times to be longer than those derived from analysis of the dose-rate effect. This could imply that cellular recovery is a two-component process, low dose-rate sparing being dominated by the faster component. The extent of low dose-rate sparing shows some tendency to correlate with the magnitude of split-dose recovery; in our view the former is the more reliable measure of cellular recovery. The clinical implication of these studies is that some human tumour types may be well treated by hyperfractionation or low dose-rate irradiation, while for others these may be poor therapeutic strategies.

Prediction of normal-tissue tolerance to radiotherapy from in-vitro cellular radiation sensitivity.

The success of radiotherapy depends on the total radiation dose, which is limited by the tolerance of surrounding normal tissues. Since there is substantial variation among patients in normal-tissue radiosensitivity, we have tested the hypothesis that in-vitro cellular radiosensitivity is correlated with in-vitro normal-tissue responses. We exposed skin fibroblast cell lines from six radiation-treated patients to various doses of radiation and measured the proportions surviving. There was a strong relation between fibroblast sensitivity in vitro and normal-tissue reactions, especially acute effects. Assessment of radiosensitivity could lead to improved tumour cure rates by enabling radiation doses to be tailored to the individual.

Cellular radiosensitivity and complication risk after curative radiotherapy

PURPOSE To test for an association between in vitro fibroblast radiosensitivity and complication risk in a case-control study of breast cancer patients treated under standard conditions in a clinical trial of radiotherapy dose fractionation. PATIENTS AND METHODS A cohort of patients participating in a randomised clinical trial of radiotherapy dose fractionation was selected on the basis of treatment-induced changes in the breast several years later. Thirty-nine cases with marked normal tissue changes were matched on several variables with 65 controls with no changes attributable to radiotherapy. Dermal fibroblast strains were established from duplicate skin biopsies, and clonogenic cell survival assays performed in triplicate after both high ( approximately 1.6 Gy/min) and low ( approximately 1 cGy/min) dose-rate irradiation. Laboratory studies were blind to patient identity, treatment outcome and radiotherapy schedule. RESULTS Analysis of 1128 clonogenic survival curves confirmed significant inter-patient variation in fibroblast radiosensitivity as measured by clonogenic survival. However, no association between fibroblast radiosensitivity and the development of late radiotherapy normal tissue effects was detected. CONCLUSIONS Inter-individual variation in cellular radiosensitivity may not be the main determinant of complication risk in patients undergoing radiotherapy for breast cancer. Other biological and technical factors may be more important in explaining the marked inter-patient differences in normal tissue damage evident several years after curative radiotherapy.

The relationship between cellular radiation sensitivity and tissue response may provide the basis for individualising radiotherapy schedules

There is a wide variation in normal tissue reactions to radiotherapy and in many situations the severity of these reactions limits radiotherapy dose. Clinical fractionation studies carried out in Gothenburg have demonstrated that a large part of the spectrum of normal tissue reactions is due to differences in individual normal tissue sensitivity. If this variation in normal tissue reactions is due to differences in intrinsic cellular radiosensitivity, it should be possible to predict tissue response based on measurement of cellular sensitivity. Here we report the initial results of a study aimed at establishing whether a direct relationship exists between cellular radiosensitivity and tissue response. Ten fibroblasts strains, including four duplicates, were established from a group of patients in the Gothenburg fractionation trials who had received radiotherapy following mastectomy. Skin doses were measured and both acute and late skin changes were observed following radiotherapy. Right and left parasternal areas were treated with different dose fractionation schedules. Clonogenic assays were used to assess intrinsic cellular radiosensitivity, and all experiments were carried out without prior knowledge of the clinical response, or which strains were duplicates. Irradiation was carried out using 60Co gamma-rays at high dose-rate (HDR) of 1-2 Gy/min and low dose-rate (LDR) of 1 cGy/min. A spectrum of sensitivity was seen, with SF2 values of 0.17-0.28 at HDR and 0.25-0.34 at LDR, and values of D0.01 of 5.07-6.38 Gy at HDR and 6.43-8.12 Gy at LDR. Comparison of the in vitro results with the clinical normal tissue effects shows a correlation between cellular sensitivity and late tissue reactions, which is highly significant with p = 0.02. A correlation between cellular sensitivity and acute effects was noted in the left-sided parasternal fields, but not the right. This is thought to be coincidental, and without biological significance. Our results suggest that cellular sensitivity might form the basis for the development of an assay system capable of predicting late normal tissue effects to curative radiotherapy, which might allow dose escalation in some patients. Increased local control and cure, with unchanged or improved normal tissue complications, could result from such individualised radiotherapy prescriptions.

Dose-rate effects and the repair of radiation damage

The extent of dose-sparing that occurs in a variety of cell lines and in vivo cell systems as a result of a reduction in dose-rate is reviewed. The emphasis is on the range from around 200 cGy/min down to 5 cGy/min, in which the predominant reason for dose-sparing is the repair of radiation damage. Dose-rate dependence is considered in relation to the Lethal-Potentially Lethal model of cell inactivation, which satisfactorily fits 4 sets of data that we have tested; estimates of half-time for repair varied from 0.07 to 1.4 h. The model shows that in spite of these short half-times, repair will often continue to influence response down to dose-rates below 5 cGy/min. The steepness of the dose-rate dependence varies widely among in vitro cell lines and among mouse normal tissues, indeed the ranges in vitro and in vivo are similar. Haemopoietic tissues are much less spared by a lowering of dose-rate than are other normal tissues. Uncertainties about the rate of reoxygenation preclude similar considerations in experimental tumours in vivo. There is a need for detailed studies of dose-rate dependence in human tumour cell lines, and the present review outlines the basis (including the optimum dose-rate range) for such studies.

DESCRIBING PATIENTS' NORMAL TISSUE REACTIONS: CONCERNING THE POSSIBILITY OF INDIVIDUALISING RADIOTHERAPY DOSE PRESCRIPTIONS BASED ON POTENTIAL PREDICTIVE ASSAYS OF NORMAL TISSUE RADIOSENSITIVITY

Clinical radiotherapeutic doses are limited by the tolerance of normal tissues. Patients given a standard treatment exhibit a range of normal tissue reactions, and a better understanding of this individual variation might allow for individualisation of radiotherapeutic prescriptions, with consequent improvement in the therapeutic ratio. At present, there is no simple way to describe normal tissue reactions, which hampers communication between clinic and laboratory and between groups from different centres. There is also no method for comparing the severity of reactions in different normal tissues. This arises largely because there is no definition of a “normal” reaction, an “extreme” reaction or the particular term “over‐reactor” (OR). This report proposes definitions for these terms, as well as a simple terminology for describing normal tissue reactions in patients having radiotherapy. The “normal” range represents the individual variation in normal tissue reactions amongst large numbers of patients treated in the same way which is within clinically acceptable limits. The term “OR” is applied to an individual whose reaction is more severe than the normal range but also implies that this forced a major change in the radiotherapeutic prescription or that the reactions were very severe or fatal. A “severe OR” would develop serious problems with a typical radical dose, while an “extreme OR” would have such difficulties at a much lower dose. To describe the normal range, a numerical scale is suggested, from 1 to 5, resistant to sensitive. The term “highly radiosensitive” (HR) is suggested for category 5. An “informal” relative scale, as suggested here, is quick and simple. It should allow comparison between different hospitals, compensate for differences in radiotherapeutic dose and technique and allow comparison of reactions between different anatomical sites. It should be adequate for discriminating patients at the extremes of the normal range from those at the centre. It is hoped that the definitions and terminology proposed here will aid communication in the field of predictive testing of normal tissue radiosensitivity. Int. J. Cancer (Pred. Oncol.) 79:606–613, 1998.

Cellular radiosensitivity and dna damage in primary human fibroblasts

PURPOSE To evaluate the relationship between radiation-induced cell survival and DNA damage in primary human fibroblasts to decide whether the initial or residual DNA damage levels are the more predictive of normal tissue cellular radiosensitivity. METHODS AND MATERIALS Five primary human nonsyndromic and two primary ataxia telangiectasia fibroblast strains grown in monolayer were studied. Cell survival was assessed by clonogenic assay. Irradiation was given at high dose rate (HDR) 1-2 Gy/min. DNA damage was measured in stationary phase cells and expressed as fraction released from the well by pulsed-field gel electrophoresis (PFGE). For initial damage, cells were embedded in agarose and irradiated at HDR on ice. Residual DNA damage was measured in monolayer by allowing a 4-h repair period after HDR irradiation. RESULTS Following HDR irradiation, cell survival varied between SF2 0.025 to 0.23. Measurement of initial DNA damage demonstrated linear induction up to 30 Gy, with small differences in the slope of the dose-response curve between strains. No correlation between cell survival and initial damage was found. Residual damage increased linearly up to 80 Gy with a variation in slope by a factor of 3.2. Cell survival correlated with the slope of the dose-response curves for residual damage of the different strains (p = 0.003). CONCLUSION The relationship between radiation-induced cell survival and DNA damage in primary human fibroblasts of differing radiosensitivity is closest with the amount of DNA damage remaining after repair. If assays of DNA damage are to be used as predictors of normal tissue response to radiation, residual DNA damage provides the most likely correlation with cell survival.

Radiosensitive Human Tumour Cell Lines May Not Be Recovery Deficient

Split-dose studies have been performed on four human tumour cell lines of widely differing radiosensitivity in order to characterize the relationship between cellular recovery and radiation dose. Previous studies using the split-dose experiment have usually measured recovery at a single dose level and assumed an underlying multi-target model of radiation effect. This predicts that the recovery ratio should reach a plateau when the dose used per fraction is beyond the shoulder of the acute survival curve. In contrast, the linear-quadratic model predicts that the recovery ratio will increase steeply as a function of dose and will never reach a plateau. Our results show that recovery increases with increasing dose and therefore no single value of the recovery ratio can be used for comparative purposes. Using these data, we have derived a value for the beta-component of the linear-quadratic model that is independent of alpha. In addition we propose that the beta-parameter derived in this way provides the most satisfactory basis for intercomparison of cellular recovery between cell lines of differing radiosensitivity. Cellular recovery at any given dose was greatest in the most radiosensitive cell line, suggesting that increased radiosensitivity does not result from decreased recovery capacity. The results suggest that cells with steep acute radiation survival curves and which show little split-dose recovery may not be recovery deficient. Consequently, using such cells in attempts to correlate recovery with the underlying molecular processes of radiation damage repair could lead to misleading results.

The Picture Has Changed in the 1980s

SummarySubstantial developments have been made during the 1980s in the radiobiology of human tumours, in particular in studies of the radiosensitivity of human tumour cells. It is now clear that tumour cells differ considerably in radiosensitivity, to an extent that by itself is capable of explaining the clinical response of tumours to radiotherapy. There also is evidence that the radiosensitivity of human tumour cell lines to low radiation doses correlates with clinical experience. Irradiation at low dose rate amplifies the differences between cell lines. In conjunction with mathematical modelling, a study of the dose-rate effect also allows a distinction to be drawn between repairable and non-repairable damage. The differences seen between cell lines at low acute doses or low dose rates are associated with the non-repairable component. The most radiosensitive cell lines have a steep component of non-repairable damage and they give the impression of being recovery-deficient; this may, however, be incorre...

Why recent studies relating normal tissue response to individual radiosensitivity might have failed and how new studies should be performed.

PURPOSE New insights into the kinetics of late complications occurring after radiation therapy indicated that all patients have a constant risk of developing late tissue complications. These observations might have a great impact on studies relating normal tissue complications to individual radiosensitivity. METHODS AND MATERIALS Data previously published by Peacock et al. were used for analysis. In this study, 39 breast cancer patients with severe reactions (responders) were compared with 65 matched patients showing no reactions (nonresponders). Cellular radiosensitivity as measured in vitro in terms of D(0.01) did not show significant differences between the two groups, both for high-dose-rate (5.84 +/- 0.06 vs. 5.85 +/- 0.07 Gy) and low-dose-rate (7.44 +/- 0.10 vs. 7.56 +/- 0.09 Gy) irradiation. RESULTS A theoretical distribution was calculated for the individual radiosensitivity of patients with Grade <or= 1, Grade 2, or Grade 3 reactions under the following assumptions: (1). The variation of the individual radiosensitivity is described by a normal distribution. (2). All patients and not only a subgroup have a risk of developing late complications. Based on the normal distribution of low-dose-rate data (mean value [MV] = 7.56 Gy, standard deviation [SD] = 0.5 Gy), a total of 200 hypothetical patients were divided into three groups: a resistant group with a sensitivity >or=(MV + SD), a normal group with a sensitivity between MV - SD and MV + SD, and a sensitive group <or=(MV - SD), the relative fractions being 16%, 68%, and 16%, respectively. It was assumed that these groups differed in the risk of developing late complication; for Grade 3 the annual incidence rate was set at 1%, 2%, and 4% and for Grade 2 at 5%, 10%, and 20% per year, respectively. It was shown that the mean cellular sensitivity calculated for Grade 3 (7.39 +/- 0.10 Gy) or Grade 2 patients (7.46 +/- 0.06 Gy) was slightly but not significantly lower than that of Grade <or= 1 patients (7.65 +/- 0.04 Gy). This result demonstrated that even if the risk was assumed to depend clearly on the individual radiosensitivity, significant differences in the mean cellular sensitivity between responders and nonresponders were not expected, just as found by Peacock et al. It was shown that a significant correlation between these two parameters could be detected only when the risk was analyzed separately for each group of patients. CONCLUSION Most data published so far aiming at establishing a relationship between cellular radiosensitivity and the risk of late complications might need to be reevaluated, because the questions asked up to now were inadequate to arrive at a meaningful answer.