1570
electrophoresis. London: National Heart and Lung Institute, 1991: 46-48. 10. Dunn MJ, Rose ML, Latif N, et al. Demonstration by Western blotting of antiheart antibodies before and after cardiac transplantation. Transplantation 1991; 51: 806-12. 11. Platt JL, Lindman BJ, Chen H, Spitalnik SL, Bach FH. Endothelial cell antigens recognised by xenoreactive human natural antibodies. Transplantation 1990; 50: 817-22. 12. Page C, Rose ML, Yacoub MH, Pigott R. Antigenic heterogeneity of vascular endothelium. Am J Pathol (in press). SHORT REPORTS
Prediction of normal-tissue tolerance to radiotherapy from invitro 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 normal-tissue variation among patients in radiosensitivity, we have tested the hypothesis that in-vitro cellular radiosensitivity is correlated with in-vivo 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 normaltissue reactions, especially acute effects. Assessment of radiosensitivity could lead to improved tumour cure rates by enabling radiation doses to be tailored to the individual.
is the most important non-surgical in cancer and success depends mainly on the total radiation dose. This dose is limited by the tolerance of normal tissues surrounding the tumour. There is wide variation among patients in normal-tissue tolerance and hence in reactions to the same curative dose.12 Severe damage to normal tissue can cause substantial morbidity and may even be life-threatening. Selection of appropriate radiation dose is based on a balance between keeping the rate of severe normal-tissue complications acceptably low and increasing the probability of local control. Much of the variation in normal-tissue reactions is due to differences in sensitivity, rather than other factors such as uncontrolled differences in delivered dose.2-4 Identification of individuals with the most sensitive normal tissues might allow the dose to be increased in the majority of patients, which in turn might increase local control and cure.5,6 Improved local control might also reduce the incidence of metastatic disease.7 We report the preliminary results of a study that aims to develop a predictive assay of normal-tissue response. We tested the hypothesis that in-vitro cellular radiosensitivity is correlated with in-vivo normal-tissue response .8 We studied fibroblasts, which have a wide range of in-vitro radiosensitivity,9 and are the only normal cell type amenable to mass culture.
Radiotherapy
treatment
13.
Springer TA. Adhesion receptors of the immune system. Nature 1990; 346: 425-34.
RN, Hughes CCW, Schoen FJ, et al. Human coronary transplantation associated arteriosclerosis: evidence for a chronic immune reaction to activated graft endothelial cells. Am J Pathol 1991;
14. Salomon
138: 791-98. 15. Heurkens AHM, Gorter A, de Vreede TM, et al. Methods for the detection of anti-endothelial antibodies by enzyme-linked immunosorbent assay. J Immunol Methods 1991; 141: 33-39.
The subjects were selected from those in the Gothenburg fractionation trials2-4 who were receiving radiotherapy after mastectomy for early breast cancer. All had parasternal nodal irradiation, with direct anterior fields; skin doses were measured and skin changes afterwards were observed. Reflectance spectrophotometry was used to measure peak acute skin reactions (erythema) at the time of radiotherapy, and late changes were quantified in terms of the degree of telangiectasia. We selected patients with a range of late normal-tissue reactions from no telangiectasia (grade 0) to confluent telangiectasia (grade 5; group CRE IVb2). The whole group received the same treatment regimen in 1978-79, so follow-up is longer than 10 years, which is necessary in view of the progression with time of late skin changes. 3,4 At first, ten fibroblast strains were established from six patients with the full range of skin changes; duplicates were produced for four. Clonogenic assays were used to assess intrinsic cellular radiosensitivity. Fibroblasts were grown to confluence in Dulbecco’s modified Eagle’s minimum essential medium plus 10% fetal calf serum, then plated out 4 h before irradiation with Cobalt-60 y-rays at low dose rate (LDR; 0.01 Gy/min) and high dose rate (HDR; 1-2 Gy/min). LDR has the practical advantage of increasing small differences between cell survival curves, and a theoretical advantage of more closely simulating clinical fractionated radiotherapy, at least for tumours.1o A range of doses was chosen to produce a maximum of 3 orders of magnitude of cell kill (0-8 Gy at HDR and 0-12 Gy at LDR). Survival curves were fitted by non-linear regression with the linear quadratic model. All experiments were carried out without knowledge of the clinical response or of which strains were duplicates.
Features of the survival curves for the ten strains at LDR summarised in the table. The dose required to reduce the surviving fraction to 0.01 (DO.01) ranged from 6.43 to 8.12 Gy. All the strains showed almost linear survival curves, shown by the low values of 0. In the figure, the ranking of the strains’ sensitivity at LDR is related to rank of acute or late normal-tissue reactions. There was a correlation between in-vitro fibroblast cellular sensitivity and in-vivo tissue response, stronger for acute than for late reactions. Statistical analysis is difficult with so few subjects. However, there is a striking relation between in-vitro are
CELL SURVIVAL DATA FOR TEN FIBROBLAST STRAINS AT LDR I
I
-
I
I
I
*From semi-logarithmic survival curve, surviving fraction=exp (-CLD-&bgr;D2), where D= dose. SF,= surviving fraction after dose of 2 Gy tMean mactivation dose= area under survival curve on linear scale. tDose of radiation to reduce SF to 0 01.
1571
3. Turesson I. The progression rate of late radiation effects in normal tissue and its impact on dose-response relationships. Radiother Oncol 1989;
15: 217-26. 4. Turesson I. Individual variation and dose dependency in the progression rate of skin telangiectasia. Int J Radiat Oncol Biol Phys 1990; 19: 1569-74. 5. Ågren A, Brahme A, Turesson I. Optimisation of uncomplicated control for head and neck tumours. Int J Radiat Oncol Biol Phys 1990; 19: 1077-85. 6. West CML, Hendry JH, Scott D, Davidson SE, Hunter RD. 25th Paterson Symposium—is there a future for radiosensitivity testing? Br J Cancer 1991; 64: 197-99. 7. Fuks Z, Leibel SA, Wallner KE, et al. The effect of local control on metastatic dissemination in carcinoma of the prostate: long-term results in patients treated with 125I implantation. Int J Radiat Oncol Biol Phys
1991; 21: 537-47. J, Peckham MJ, Steel GG. The radioresponsiveness of human
8. Deacon
and the initial slope of the survival curve. Radiother Oncol 1984; 2: 317-23. 9. Fertil B, Deschavanne PJ, Debieu D, Malaise EP. Correlation between PLD repair capacity and the survival curve of human fibroblasts in exponential growth phase: analysis in terms of several parameters. Radiat Res 1988; 116: 74-88. 10. Steel GG. Cellular sensitivity to low dose-rate irradiation focuses the problem of tumour radioresistance. Radiother Oncol 1991; 20: 71-83. tumours
Rank of in-vitro fibroblast radiosensitivity rank of healthy-tissue reactions.
(Do 01)
at LDR and
Duplicate strains indicated by connecting lines. In-vitro sensitivity ranked from least (1 )to most (10) sensitive; tissue effects from least (1) to greatest (10) reaction. Erythema at time of radiotherapy= early skin response; telangiectasia score 10 yr after treatment= late response.
sensitivity and normal-tissue reactions, especially acute effects. Several conditions must be met if an association between in-vitro fibroblast sensitivity and in-vivp skin response to treatment given 10 years previously is to be detected: fibroblasts grown from different pieces of tissue must have similar radiation sensitivity; different cell types from the same patient must have the same relative sensitivity, so that fibroblasts can be used to determine the relative sensitivity of the critical target cells whose damage brings about tissue effects; cellular sensitivity must correlate with tissue response; and there must be no effect of time or ageing on cellular sensitivity. Also, the inevitable experimental variation must be small enough not to obscure these relations. Telangiectasia has been validated as a model of late normal-tissue damage,2 but the scale is limited in that it is not possible to score higher than grade 5 confluent telangiectasia, or lower than grade 0, and there are probably even greater degrees of sensitivity and resistance. The range of fibroblast sensitivity among our subjects is narrower than previously reported;9 it is possible, therefore, that there were no patients in the tested group with extremely sensitive or resistant tissues. The finding of a correlation with this narrow range of cellular sensitivities suggests that the method may be stronger still in detecting patients at extremes of the clinical spectrum. Our findings raise the possibility that cellular sensitivity can be used to predict normal-tissue response to curative radiotherapy. The clonogenic assay system we used is slow, requires a high level of expertise, and is labour intensive, so it could not be used as a predictive assay in clinical practice. However, the study provides a basis for evaluation of other assay systems; a DNA damage assay is being assessed as a possible alternative. A simple assay to measure tissue radiosensitivity could lead to improved tumour cure rates through individual prescription of radiation dose. This study was supported by the Cancer Research Campaign. R. W. is supported by by British Nuclear Fuels Ltd.
REFERENCES 1. Sutton ML. Some clinical aspects of radiobiology. In: Hope-Stone HF, ed. Radiotherapy in clinical practice. London: Butterworth, 1986: 1-18. 2. Turesson I, Thames H. Repair capacity and kinetics of human skin
during fractionated radiotherapy: erythema, desquamation and telangiectasia after 3 and 5 years’ follow-up. Radiother Oncol 1989; 15: 169-88.
ADDRESSES:
Radiotherapy Research Unit, Institute of Cancer Research, Sutton, Surrey, UK (N. G. Burnet, FRCR, R. Wurm, MD, J. R. Yarnold, FRCR, J. H. Peacock, CiBiol), and Department of Oncology, Sahlgren’s Hospital, Gothenburg, Sweden (J. Nyman, MD, I. Turesson, PhD). Correspondence to Dr N. G. Burnet, Radiotherapy Research Unit, Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey SM2 5NG, UK.
Intravascular ultrasound imaging combined with coronary
angioplasty
A novel catheter combining ultrasound imaging and coronary balloon angioplasty was used in the treatment of 69 coronary-artery lesions in 51 patients. The ultrasound transducer enables realtime cross-sectional imaging and qualitative and quantitative assessment of the vessel wall before and after angioplasty. The combination catheter successfully dilated 67 lesions. There was a characteristic three-layered appearance, and in 60 media, intima, adventitia, representing Intravascular information cases. imaging provided on the vessel wall unobtainable by standard contrast angiogaphy in 28 cases and influenced our management in 6 cases.
Technological advances have enabled miniaturisation of ultrasound transducers to such an extent that they can now be mounted in catheters and introduced into the body percutaneously.1 Intravascular ultrasonography allows direct visualisation, in vivo, of vessel walls and assessment of the results of interventional procedures. We report here our experience with a catheter that combines coronary ultrasound imaging and balloon angioplasty; the vessel wall can be viewed before and after angioplasty. 51 were
patients (69 lesions) of mean age 59-5 (range 41-81) years, who undergoing clinically indicated coronary angioplasty, gave
electrophoresis. London: National Heart and Lung Institute, 1991: 46-48. 10. Dunn MJ, Rose ML, Latif N, et al. Demonstration by Western blotting of antiheart antibodies before and after cardiac transplantation. Transplantation 1991; 51: 806-12. 11. Platt JL, Lindman BJ, Chen H, Spitalnik SL, Bach FH. Endothelial cell antigens recognised by xenoreactive human natural antibodies. Transplantation 1990; 50: 817-22. 12. Page C, Rose ML, Yacoub MH, Pigott R. Antigenic heterogeneity of vascular endothelium. Am J Pathol (in press). SHORT REPORTS
Prediction of normal-tissue tolerance to radiotherapy from invitro 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 normal-tissue variation among patients in radiosensitivity, we have tested the hypothesis that in-vitro cellular radiosensitivity is correlated with in-vivo 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 normaltissue reactions, especially acute effects. Assessment of radiosensitivity could lead to improved tumour cure rates by enabling radiation doses to be tailored to the individual.
is the most important non-surgical in cancer and success depends mainly on the total radiation dose. This dose is limited by the tolerance of normal tissues surrounding the tumour. There is wide variation among patients in normal-tissue tolerance and hence in reactions to the same curative dose.12 Severe damage to normal tissue can cause substantial morbidity and may even be life-threatening. Selection of appropriate radiation dose is based on a balance between keeping the rate of severe normal-tissue complications acceptably low and increasing the probability of local control. Much of the variation in normal-tissue reactions is due to differences in sensitivity, rather than other factors such as uncontrolled differences in delivered dose.2-4 Identification of individuals with the most sensitive normal tissues might allow the dose to be increased in the majority of patients, which in turn might increase local control and cure.5,6 Improved local control might also reduce the incidence of metastatic disease.7 We report the preliminary results of a study that aims to develop a predictive assay of normal-tissue response. We tested the hypothesis that in-vitro cellular radiosensitivity is correlated with in-vivo normal-tissue response .8 We studied fibroblasts, which have a wide range of in-vitro radiosensitivity,9 and are the only normal cell type amenable to mass culture.
Radiotherapy
treatment
13.
Springer TA. Adhesion receptors of the immune system. Nature 1990; 346: 425-34.
RN, Hughes CCW, Schoen FJ, et al. Human coronary transplantation associated arteriosclerosis: evidence for a chronic immune reaction to activated graft endothelial cells. Am J Pathol 1991;
14. Salomon
138: 791-98. 15. Heurkens AHM, Gorter A, de Vreede TM, et al. Methods for the detection of anti-endothelial antibodies by enzyme-linked immunosorbent assay. J Immunol Methods 1991; 141: 33-39.
The subjects were selected from those in the Gothenburg fractionation trials2-4 who were receiving radiotherapy after mastectomy for early breast cancer. All had parasternal nodal irradiation, with direct anterior fields; skin doses were measured and skin changes afterwards were observed. Reflectance spectrophotometry was used to measure peak acute skin reactions (erythema) at the time of radiotherapy, and late changes were quantified in terms of the degree of telangiectasia. We selected patients with a range of late normal-tissue reactions from no telangiectasia (grade 0) to confluent telangiectasia (grade 5; group CRE IVb2). The whole group received the same treatment regimen in 1978-79, so follow-up is longer than 10 years, which is necessary in view of the progression with time of late skin changes. 3,4 At first, ten fibroblast strains were established from six patients with the full range of skin changes; duplicates were produced for four. Clonogenic assays were used to assess intrinsic cellular radiosensitivity. Fibroblasts were grown to confluence in Dulbecco’s modified Eagle’s minimum essential medium plus 10% fetal calf serum, then plated out 4 h before irradiation with Cobalt-60 y-rays at low dose rate (LDR; 0.01 Gy/min) and high dose rate (HDR; 1-2 Gy/min). LDR has the practical advantage of increasing small differences between cell survival curves, and a theoretical advantage of more closely simulating clinical fractionated radiotherapy, at least for tumours.1o A range of doses was chosen to produce a maximum of 3 orders of magnitude of cell kill (0-8 Gy at HDR and 0-12 Gy at LDR). Survival curves were fitted by non-linear regression with the linear quadratic model. All experiments were carried out without knowledge of the clinical response or of which strains were duplicates.
Features of the survival curves for the ten strains at LDR summarised in the table. The dose required to reduce the surviving fraction to 0.01 (DO.01) ranged from 6.43 to 8.12 Gy. All the strains showed almost linear survival curves, shown by the low values of 0. In the figure, the ranking of the strains’ sensitivity at LDR is related to rank of acute or late normal-tissue reactions. There was a correlation between in-vitro fibroblast cellular sensitivity and in-vivo tissue response, stronger for acute than for late reactions. Statistical analysis is difficult with so few subjects. However, there is a striking relation between in-vitro are
CELL SURVIVAL DATA FOR TEN FIBROBLAST STRAINS AT LDR I
I
-
I
I
I
*From semi-logarithmic survival curve, surviving fraction=exp (-CLD-&bgr;D2), where D= dose. SF,= surviving fraction after dose of 2 Gy tMean mactivation dose= area under survival curve on linear scale. tDose of radiation to reduce SF to 0 01.
1571
3. Turesson I. The progression rate of late radiation effects in normal tissue and its impact on dose-response relationships. Radiother Oncol 1989;
15: 217-26. 4. Turesson I. Individual variation and dose dependency in the progression rate of skin telangiectasia. Int J Radiat Oncol Biol Phys 1990; 19: 1569-74. 5. Ågren A, Brahme A, Turesson I. Optimisation of uncomplicated control for head and neck tumours. Int J Radiat Oncol Biol Phys 1990; 19: 1077-85. 6. West CML, Hendry JH, Scott D, Davidson SE, Hunter RD. 25th Paterson Symposium—is there a future for radiosensitivity testing? Br J Cancer 1991; 64: 197-99. 7. Fuks Z, Leibel SA, Wallner KE, et al. The effect of local control on metastatic dissemination in carcinoma of the prostate: long-term results in patients treated with 125I implantation. Int J Radiat Oncol Biol Phys
1991; 21: 537-47. J, Peckham MJ, Steel GG. The radioresponsiveness of human
8. Deacon
and the initial slope of the survival curve. Radiother Oncol 1984; 2: 317-23. 9. Fertil B, Deschavanne PJ, Debieu D, Malaise EP. Correlation between PLD repair capacity and the survival curve of human fibroblasts in exponential growth phase: analysis in terms of several parameters. Radiat Res 1988; 116: 74-88. 10. Steel GG. Cellular sensitivity to low dose-rate irradiation focuses the problem of tumour radioresistance. Radiother Oncol 1991; 20: 71-83. tumours
Rank of in-vitro fibroblast radiosensitivity rank of healthy-tissue reactions.
(Do 01)
at LDR and
Duplicate strains indicated by connecting lines. In-vitro sensitivity ranked from least (1 )to most (10) sensitive; tissue effects from least (1) to greatest (10) reaction. Erythema at time of radiotherapy= early skin response; telangiectasia score 10 yr after treatment= late response.
sensitivity and normal-tissue reactions, especially acute effects. Several conditions must be met if an association between in-vitro fibroblast sensitivity and in-vivp skin response to treatment given 10 years previously is to be detected: fibroblasts grown from different pieces of tissue must have similar radiation sensitivity; different cell types from the same patient must have the same relative sensitivity, so that fibroblasts can be used to determine the relative sensitivity of the critical target cells whose damage brings about tissue effects; cellular sensitivity must correlate with tissue response; and there must be no effect of time or ageing on cellular sensitivity. Also, the inevitable experimental variation must be small enough not to obscure these relations. Telangiectasia has been validated as a model of late normal-tissue damage,2 but the scale is limited in that it is not possible to score higher than grade 5 confluent telangiectasia, or lower than grade 0, and there are probably even greater degrees of sensitivity and resistance. The range of fibroblast sensitivity among our subjects is narrower than previously reported;9 it is possible, therefore, that there were no patients in the tested group with extremely sensitive or resistant tissues. The finding of a correlation with this narrow range of cellular sensitivities suggests that the method may be stronger still in detecting patients at extremes of the clinical spectrum. Our findings raise the possibility that cellular sensitivity can be used to predict normal-tissue response to curative radiotherapy. The clonogenic assay system we used is slow, requires a high level of expertise, and is labour intensive, so it could not be used as a predictive assay in clinical practice. However, the study provides a basis for evaluation of other assay systems; a DNA damage assay is being assessed as a possible alternative. A simple assay to measure tissue radiosensitivity could lead to improved tumour cure rates through individual prescription of radiation dose. This study was supported by the Cancer Research Campaign. R. W. is supported by by British Nuclear Fuels Ltd.
REFERENCES 1. Sutton ML. Some clinical aspects of radiobiology. In: Hope-Stone HF, ed. Radiotherapy in clinical practice. London: Butterworth, 1986: 1-18. 2. Turesson I, Thames H. Repair capacity and kinetics of human skin
during fractionated radiotherapy: erythema, desquamation and telangiectasia after 3 and 5 years’ follow-up. Radiother Oncol 1989; 15: 169-88.
ADDRESSES:
Radiotherapy Research Unit, Institute of Cancer Research, Sutton, Surrey, UK (N. G. Burnet, FRCR, R. Wurm, MD, J. R. Yarnold, FRCR, J. H. Peacock, CiBiol), and Department of Oncology, Sahlgren’s Hospital, Gothenburg, Sweden (J. Nyman, MD, I. Turesson, PhD). Correspondence to Dr N. G. Burnet, Radiotherapy Research Unit, Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey SM2 5NG, UK.
Intravascular ultrasound imaging combined with coronary
angioplasty
A novel catheter combining ultrasound imaging and coronary balloon angioplasty was used in the treatment of 69 coronary-artery lesions in 51 patients. The ultrasound transducer enables realtime cross-sectional imaging and qualitative and quantitative assessment of the vessel wall before and after angioplasty. The combination catheter successfully dilated 67 lesions. There was a characteristic three-layered appearance, and in 60 media, intima, adventitia, representing Intravascular information cases. imaging provided on the vessel wall unobtainable by standard contrast angiogaphy in 28 cases and influenced our management in 6 cases.
Technological advances have enabled miniaturisation of ultrasound transducers to such an extent that they can now be mounted in catheters and introduced into the body percutaneously.1 Intravascular ultrasonography allows direct visualisation, in vivo, of vessel walls and assessment of the results of interventional procedures. We report here our experience with a catheter that combines coronary ultrasound imaging and balloon angioplasty; the vessel wall can be viewed before and after angioplasty. 51 were
patients (69 lesions) of mean age 59-5 (range 41-81) years, who undergoing clinically indicated coronary angioplasty, gave
