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Editorial radiotherapy and oncology 2002: predictive assays for normal tissue damage
Radiotherapy and Oncology 64 (2002) 125–129 www.elsevier.com/locate/radonline
Editorial
Editorial radiotherapy and oncology 2002: predictive assays for normal tissue damage Nicola S. Russell, Adrian C. Begg 14 May 2002; 9 July 2002
In this issue of radiotherapy and oncology, two groups present their work on developing a predictive assay for the degree of radiation-induced normal tissue damage. Both groups have utilised in vitro tests using patients’ lymphocytes. The rational behind these studies is based on the fact that for curative radiotherapy, the tolerance of patients with the most severe normal tissue reactions determines the dose level for the patient group as a whole for a given tumour site and type. The ultimate aim is to develop a sufficiently robust predictive assay to enable individual dose adjustment, and thus improve the therapeutic ratio for the patient population as a whole [1,6,34–36,51]. Such studies can indeed provide information on the role of cell kill and/or DNA damage in determining morbidity, but are time consuming and unlikely to be used routinely in the clinic. The questions therefore arise as to the value of these studies for the future, and what the best way forward is.
1. History A number of laboratories demonstrated a significant variation in cellular radiosensitivity between donors using either lymphocytes/lymphoblasts or fibroblasts [13,17,21,33,57]. There is clear evidence that the variation in radiation sensitivity is, at least in part, determined by genetic factors. There are a number of genetic syndromes, of which ataxia telangiectasia is probably the best example, which are characterised by increased cellular radiosensitivity and an extreme response to therapeutic radiation [43,49]. However, studies on non-syndromic patients that compared the in vitro radiosensitivity of lymphocytes and skin fibroblasts from the same donor were unable to find a correlation between them [20,23,31]. This lack of correlation suggests that modifying factors play a role depending on the tissue and cell type. On the clinical side, for over half a century, radiotherapists have recognised that patients show an individual variation in their normal tissue reactions to radiotherapy [38]. Quantification of variation of normal tissue reactions has
mainly been limited to easily observable sites such as skin (acute and late reactions) and subcutaneous and breast fibrosis [8,15,22,52,53]. Inter-patient variation has also been demonstrated for lung damage following radiation [50]. The prediction of the extent of clinical normal tissue radiation reactions by determining an in vitro radiosensitivity parameter (or surrogate thereof) has been the subject of research by several groups over the last decade or so (e.g. refs. [12,30,39,45,59]). However, it is now becoming apparent that such tests will never actually be clinically implemented due to both methodological and biological reasons.
2. Biological and laboratory factors There are a number of factors that will influence the correlation between an in vitro test and the clinical endpoint. Firstly, there are several aspects of the laboratory protocol that could influence the result. This includes the cell type used, dose, dose rate, type of medium, consistent quality of the medium or serum, immediate or delayed plating, use of feeder cells, and so on. The type of assay is also important. Colony formation has been regarded as the gold standard for assessing cell kill, although due to its time consuming nature, surrogate assays have also been investigated. These include chromosome damage (assessed by cytogenetics, fluorescence in situ hybridisation, micronucleus formation and others) and DNA damage (initial DNA damage, residual DNA damage, using pulsed or constant field electrophoresis, alkaline elution, comet assay, halo assay, etc.). There are also factors other than cell kill that can effect response (see below) and which should be taken into account. Although there is often a demonstrable correlation between the various in vitro assays, for example for colony formation and chromosome damage [44], this is often thanks to the inclusion of cell types from patients with a highly radiosensitive syndrome such as Ataxia Telangiectasia. For cells from normal individuals, or the majority of cancer patients that do not demonstrate extreme radiosensi-
0167-8140/02/$ - see front matter q 2002 Published by Elsevier Science Ireland Ltd. PII: S 0167-814 0(02)00189-5
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tivity, there is usually considerable scatter in the correlation between two in vitro end points for a group of individuals [4,58]. This is probably because, of the individual parameters tested, none is the main determinant of clonogenic cell survival in all individuals. For example, the level of DNA damage can be modified by variations in cell cycle delay/checkpoint control, repair capacity and fidelity, differences in chromatin structure influencing the conversion of DNA damage into chromosome breaks, apototic tendency, etc. We can therefore conclude that strong correlations between assays would not necessarily be expected. Furthermore, finding a correlation with cell kill may not be important, since cell kill may not be the only important determinant of radiation-induced morbidity. There are a number of biological factors that influence treatment response, which are not directly related to intrinsic radiosensitivity, including the remodelling of tissues in vivo [47]. For several tissues (e.g. lung, skin and intestinal mucosa), models have been developed showing the involvement of cytokine-mediated multicellular interactions in the radiation response [27]. Currently the most interesting developments are related to cytokines such as interleukins 2 and 6 (IL-2, IL-6), interferon alpha and in particular transforming growth factor beta (TGF-b) and its role in generating and modulating tissue fibrosis in many tissues and organs (e.g. refs. [9,18]). In mouse mammary tissue, radiation has been shown to induce rapid increases in the level of TGF-b (particularly in adipose stroma), and radiation induced oxidative activation of latent TGF-b has also been demonstrated [5]. Another example of humoural effects is the effect of the renin-angiotensin system on the renal vasculature and its influence on the expression of radiation induced renal damage, probably also involving TGF-b [14]. Further, Travis and colleagues have shown that some mice strains have a greater genetic predisposition than others to develop radiation-induced lung and rectal fibrosis [26,48]. Interstrain variation in the development of radiation-induced lung fibrosis has also been shown in rats [55]. Identification of the determinant genes, their function and their human homologues will increase our understanding of the mechanism of radiation induced fibrosis. These and similar studies also give us insights into mechanisms of radiation response other than the conventional radiobiological paradigm of target cell death. Whichever processes are the most important in determining radiation morbidity, whether cell kill, fibrogenesis, vascular mediated injury or other processes, each is dependent on the interplay of multiple gene products. Genetic testing for only one or even a few mutant genes is therefore unlikely to be of sufficient predictive power. Some groups have been able to demonstrate a relationship between a genetic determinant of radiosensitivity (such as ATM heterozygosity or rare polymorphisms in the DNA repair genes XRCC1 and XRCC3) and clinical over-reactions to radiotherapy [24,41]. However, their findings could not always be confirmed by others [3]. Successful genetic test-
ing to predict normal tissue damage therefore most probably requires screening for expression or mutations in multiple genes related to radiosensitivity and development of tissue reactions.
3. Clinical factors Clinical factors that influence the clinical end point used in studies designed to develop a predictive assay can obscure the relationship between results of an in vitro assay and the degree of normal tissue damage observed. Firstly, radiotherapy related factors such as total dose and fractionation schedule can confuse the relationship between different normal tissue reactions in the same patient. For example two fractionation schedules can give the same level of acute skin reaction but very different incidence rates of various late complications [37]. We therefore cannot expect a correlation between acute and late reactions for both schedules, even if the individual variation between patients is determined by intrinsic radiosensitivity. The same is true to some extent for the correlation between different late normal tissue end points if the relevant tissues vary in fractionation sensitivity. This could explain why some authors have been able to demonstrate a correlation between different late normal tissue end-points, but others have not [7,19,32,54]. Secondly, the follow-up duration and number of observations per patient can influence the relative relationship between two end-points. Consideration has to be given to latency in the development of complications such as fibrosis and telangiectasia that can bias the estimate of incidence and severity [8]. Saturation of a clinical endpoint, e.g. maximum induration and fibrosis or confluent telangiectasia, may lead to underestimation of the degree of underlying tissue damage and reduce the discrimination between individual patients [45,46]. Thirdly, other identified factors that influence the development of a given normal tissue reaction have to be accounted for in any analysis of the relationship to an assay parameter. These include treatment factors such as volume irradiated, dose distribution, medication, surgery and also co-morbidity, age, anatomical site and ‘life-style’ factors such as smoking, etc. [10,15,16,45,54]. Ideally, investigations into the value of a biological assay as a predictive test should therefore preferably be conducted using a homogeneously treated group of patients with accurately known treatment parameters such as dose volume histograms for the normal tissue of interest. This is not a trivial matter. The clinical end point should be selected on the basis of clinical relevance, be amenable to objective scoring using a carefully selected scoring system [11,25] and should have a significant inter-patient variation. Factors other than the parameter being tested that are known to influence the outcome should be incorporated into a multivariate analysis. In the real world these criteria have yet to be met.
N.S. Russell, A.C. Begg / Radiotherapy and Oncology 64 (2002) 125–129
Finally, one should consider the work of Jung and colleagues [29] who studied the kinetics of the development of late complications after radiotherapy. The incidence of late effects in several organs appears to occur with exponential kinetics, implying a random process in the timing of the occurrence of late radiation sequelae. Thus patients who develop a late effect early on may not necessarily be more sensitive than an individual who develops the same complication at a longer follow-up. This effect could interfere with the correct ranking of patients when correlating with an in vitro parameter. It would therefore be advisable to test the chosen purported low and high risk groups for their rates of development of reactions according to the log-linear plots advocated by Jung et al, to ensure that these clinical groups are indeed different and suitable for comparisons with cellular or genetic predictors.
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primarily on cytokine responses suggests that the historical emphasis on cell kill and/or DNA damage may be inappropriate and could be misleading for normal tissues. What is clear is that cell kill/DNA damage of target or surrogate cells, while showing mildly encouraging results in some studies, will not provide a clinically useable or robust predictor. Furthermore, these techniques do not indicate future directions leading to greater understanding of the relevant biology and pathology, whereas expression profiling does offer this possibility by directing attention to specific genes and pathways. Further biological and radiobiological research on normal tissue reactions to radiation must lead to a better understanding of their pathogenesis and enable the development of strategies directed at intervention and modulation of the normal tissue response. We believe this approach will help improve the therapeutic ratio for curative radiotherapy schedules.
4. Future On the basis of the arguments discussed above, it is becoming apparent that finding a good predictor for normal tissue damage is an extremely difficult task. Even though intrinsic radiosensitivity (determined in vitro) may influence the extent of the normal tissue radiation response, it is clear it is not the only determinant. The same argument can also be applied to other factors that modulate the tissue response. For example if a gene contributing to radiation fibrosis is found for humans, a test for gene expression or a certain mutation or polymorphism is unlikely to give sufficient information to enable dose adjustment. With enough knowledge of the relevant genes and pathways, looking at expression or polymporphisms in a far wider range of genes than is now being done may provide a viable approach. Microarray technology allows simultaneous measurement of the expression of many thousands of genes, and thus overcomes the problem of restricting investigations to one or a few genes only. Several studies on have shown the predictive power of pretreatment expression profiling for human tumours [2,40,56]. Similar large studies on normal tissues to predict treatment-induced morbidity have not yet been reported. However, ‘proof-of-principle’ has recently been shown by the studies of Quarmby et al. [42] using a cytokine gene array on fibroblasts derived from patients with minimal and severe radiation reactions, and from Johnston et al. [28] who showed expression differences in fibrosis-resistant and sensitive mouse strains. Other promising methodologies rapidly being developed for high throughput analysis are protein expression profiling (proteomics) and single nucleotide polymorphisms, the latter having the advantage that these DNA alterations will occur in all tissues, even readily accessible lymphocytes. Whether these methodologies will provide better predictive power than messenger RNA expression profiles remains to be seen. The fact that both the Quarmby and Johnston studies showed significant correlations with morbidity by focusing
References [1] Agren A, Brahme A, Turesson I. Optimisation of uncomplicated control for head and neck tumours. Int J Radiat Oncol Biol Phys 1990;19:1077–1085. [2] Alizadeh AA, Eisen MB, Davis RE, et al. Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature 2000;403:503–511. [3] Appleby JM, Barber JB, Levine E, et al. Absence of mutations in the ATM gene in breast cancer patients with severe responses to radiotherapy. Br J Cancer 1997(76):1546–1549. [4] Barber JB, Burrill W, Spreadborough AR, et al. Relationship between in vitro chromosomal radiosensitivity of peripheral blood lymphocytes and the expression of normal tissue damage following radiotherapy for breast cancer. Radiother Oncol 2000;55:179–186. [5] Barcellos-Hoff MH, Derynck R, Tsang ML, Weatherbee JA. Transforming growth factor-beta activation in irradiated murine mammary gland. J Clin Invest 1994;93:892–899. [6] Bentzen SM. Potential clinical impact of normal-tissue intrinsic radiosensitivity testing. Radiother Oncol 1997(43):121–131. [7] Bentzen SM, Overgaard M, Overgaard J. Clinical correlations between late normal tissue endpoints after radiotherapy: implications for predictive assays of radiosensitivity. Eur J Cancer 1993;29A:1373–1376. [8] Bentzen SM, Overgaard M, Thames HD, Christensen JJ, Overgaard J. Early and late normal-tissue injury after postmastectomy radiotherapy alone or combined with chemotherapy. Int J Radiat Biol 1989;56:711–715. [9] Border WA, Noble NA. Transforming growth factor beta in tissue fibrosis. N Engl J Med 1994;331:1286–1292. [10] Borger JH, Kemperman H, Smitt HS, et al. Dose and volume effects on fibrosis after breast conservation therapy. Int J Radiat Oncol Biol Phys 1994;30:1073–1081. [11] Burnet NG, Johansen J, Turesson I, Nyman J, Peacock JH. Describing patients’ normal tissue reactions: concerning the possibility of individualising radiotherapy dose prescriptions based on potential predictive assays of normal tissue radiosensitivity. Steering committee of the BioMed2 European Union concerted Action Programme on the Development of Predictive tests of normal tissue response to radiation therapy. Int J Cancer 1998;79:606–613. [12] Burnet NG, Nyman J, Turesson I, et al. Prediction of normal-tissue tolerance to radiotherapy from in-vitro cellular radiation sensitivity. Lancet 1992(339):1570–1571.
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[13] Cox R, Masson WK. Radiosensitivity in cultured human fibroblasts. Int J Radiat Biol Relat Stud Phys Chem Med 1980;38:575–576. [14] Datta PK, Moulder JE, Fish BL, Cohen EP, Lianos EA. TGF-beta 1 production in radiation nephropathy: role of angiotensin II. Int J Radiat Biol 1999;75:473–479. [15] Denham JW, Hamilton CS, Simpson SA, et al. Acute reaction parameters for human oropharyngeal mucosa. Radiother Oncol 1995(35):129–137. [16] Denham JW, Hamilton CS, Simpson SA, et al. Factors influencing the degree of erythematous skin reactions in humans. Radiother Oncol 1995;36:107–120. [17] Deschavanne PJ, Debieu D, Fertil B, Malaise EP. Re-evaluation of in vitro radiosensitivity of human fibroblasts of different genetic origins. Int J Radiat Biol Relat Stud Phys Chem Med 1986(50):279–293. [18] Franko AJ, Sharplin J, Ghahary A, Barcellos-Hoff MH. lmmunohistochemical localisation of transforming growth factor beta and tumour necrosis factor alpha in the lungs of fibrosis-prone and ‘non-fibrosing’ mice during the latent period and early phase after irradiation. Radiat Res 1997;147:245–256. [19] Geara FB, Peters U, Ang KK, Wike JL, Brock WA. Prospective comparison of in vitro normal cell radiosensitivity and normal tissue reactions in radiotherapy patients. Int J Radiat Oncol Biol Phys 1993;27:1173–1179. [20] Geara FB, Peters U, Ang KK, et al. Intrinsic radiosensitivity of normal human fibroblasts and lymphocytes after high- and low-dose-rate irradiation. Cancer Res 1992;52:6348–6352. [21] Gentner NE, Morrison DP, Myers DK. Impact on radiogenic cancer risk of persons exhibiting abnormal sensitivity to ionising radiation. Health Phys 1988;55:415–425. [22] Glicksman AS, Chu FCH, Bane HN, Nickson JJ. Quantitative and qualitative evaluation of skin erythema. Clinical study in patients on a standardised irradiation schedule. Radiology 1960;75:411–414. [23] Green MH, Arlett CF, Cole J, et al. Comparative human cellular radiosensitivity: III. Gamma-radiation survival of cultured skin fibroblasts and resting T-lymphocytes from the peripheral blood of the same individual. Int J Radiat Biol 1991;59:749–765. [24] Hall EJ, Schiff PB, Hanks GE, et al. A preliminary report: frequency of A-T heterozygotes among prostate cancer patients with severe late responses to radiation therapy. Cancer J Sci Am 1998;4:385– 389. [25] Hanlon AL, Schultheiss TE, Hunt MA, et al. Chronic rectal bleeding after high-dose conformal treatment of prostate cancer warrants modification of existing morbidity scales. Int J Radiat Oncol Biol Phys 1997;38:59–63. [26] Haston CK, Travis EL. Murine susceptibility to radiation-induced pulmonary fibrosis is influenced by a genetic factor implicated in susceptibility to bleomycin-induced pulmonary fibrosis. Cancer Res 1997(57):5286–5291. [27] Herskind C, Bamberg M, Rodemann HP. The role of cytokines in the development of normal-tissue reactions after radiotherapy. Strahlenther Onkol 1998;174(Suppl. 3):12–15. [28] Johnston CJ, Williams JP, Okunieff P, Finkelstein JN. Radiationinduced pulmonary fibrosis: examination of chemokine and chemokine receptor families. Radiat Res 2002;157:256–265. [29] Jung H, Beck-Bornholdt HP, Svoboda V, Alberti W, Herrmann T. Quantification of late complications after radiation therapy. Radiother Oncol 2001(61):233–246. [30] Kiltie AE, Ryan AJ, Swindell R, et al. A correlation between residual radiation-induced DNA double-strand breaks in cultured fibroblasts and late radiotherapy reactions in breast cancer patients. Radiother Oncol 1999;51:55–65. [31] Kushiro J, Nakamura N, Kyoizumi S, et al. Absence of correlations between radiosensitivities of human T-lymphocytes in G0 and skin fibroblasts in log phase. Radiat Res 1990;122:326–332. [32] Lartigau E, Saunders MI, Dische S, Warburton MF, Des RC. A comparison of the late radiation changes after three schedules of radiotherapy. Radiother Oncol 1991;20:139–148.
[33] Little JB, Nove J, Strong LC, Nichols WW. Survival of human diploid skin fibroblasts from normal individuals after X-irradiation. Int J Radiat Biol 1988;54:899–910. [34] MacKay RI, Hendry JH. The modelled benefits of individualizing radiotherapy patients’ dose using cellular radiosensitivity assays with inherent variability. Radiother Oncol 1999;50:67–75. [35] MacKay RI, Niemierko A, Goitein M, Hendry JH. Potential clinical impact of normal-tissue intrinsic radiosensitivity testing. Radiother Oncol 1998;46:215–216. [36] Norman A, Kagan AR, Chan SL. The importance of genetics for the optimisation of radiation therapy. A hypothesis. Am J Clin Oncol 1988;11:84–88. [37] Overgaard M, Bentzen SM, Christensen JJ, Madsen EH. The value of the NSD formula in equation of acute and late radiation complications in normal tissue following 2 and 5 fractions per week in breast cancer patients treated with postmastectomy irradiation. Radiother Oncol 1987;9:1–11. [38] Paterson R. The treatment of malignant disease by radium and X-rays. Edward Arnold & Co., London, 1949. [39] Peacock J, Ashton A, Bliss J, et al. Cellular radiosensitivity and complication risk after curative radiotherapy. Radiother Oncol 2000;55:173–178. [40] Pomeroy SL, Tamayo P, Gaasenbeek M, et al. Prediction of central nervous system embryonal tumour outcome based on gene expression. Nature 2002;415:436–442. [41] Price EA, Bourne SU, Radbourne R, et al. Rare microsatellite polymorphisms in the DNA repair genes XRCC1, XRCC3 and XRCC5 associated with cancer in patients of varying radiosensitivity. Somat Cell Mol Genet 1997;23:237–247. [42] Quarmby S, West C, Magee B, et al. Differential expression of cytokine genes in fibroblasts derived from skin biopsies of patients who developed minimal or severe normal tissue damage after radiotherapy. Radiat Res 2002;157:243–248. [43] Rogers PB, Plowman PN, Harris SJ, Arlett CF. Four radiation hypersensitivity cases and their implications for clinical radiotherapy. Radiother Oncol 2000;57:143–154. [44] Russell NS, Arlett CF, Bartelink H, Begg AC. Use of fluorescence in situ hybridisation to determine the relationship between chromosome aberrations and cell survival in eight human fibroblast strains. Int J Radiat Biol 1995;68:185–196. [45] Russell NS, Grummels A, Hart AA, et al. Low predictive value of intrinsic fibroblast radiosensitivity for fibrosis development following radiotherapy for breast cancer. Int J Radiat Biol 1998;73:661– 670. [46] Russell NS, Uara PC, Grummels A, et al. In vitro differentiation characteristics of human skin fibroblasts: correlations with radiotherapy-induced breast fibrosis in patients. Int J Radiat Biol 2000;76:231– 240. [47] Sassi M, Jukkola A, Riekki R, et al. Type I collagen turnover and cross-linking are increased in irradiated skin of breast cancer patients. Radiother Oncol 2001;58:317–323. [48] Skwarchuk MW, Travis EU. Changes in histology and fibrogenic cytokines in irradiated colorectum of two murine strains. Int J Radiat Oncol Biol Phys 1998;42:169–178. [49] Taylor AM, Harnden DG, Arlett CF, et al. Ataxia telangiectasia: a human mutation with abnormal radiation sensitivity. Nature 1975;258:427–429. [50] Theuws JC, Kwa SL, Wagenaar AC, et al. Dose-effect relations for early local pulmonary injury after irradiation for malignant lymphoma and breast cancer. Radiother Oncol 1998;48:33–43. [51] Tucker SL, Geara FB, Peters U, Brock WA. How much could the radiotherapy dose be altered for individual patients based on a predictive assay of normal-tissue radiosensitivity? Radiother Oncol 1996;38:103–113. [52] Tucker SU, Turesson I, Thames HD. Evidence for individual differences in the radiosensitivity of human skin. Eur J Cancer 1992;28A:1783–1791.
N.S. Russell, A.C. Begg / Radiotherapy and Oncology 64 (2002) 125–129 [53] Turesson I. Individual variation and dose dependency in the progression rate of skin telangiectasia. Int J Radiat Oncol Biol Phys 1990;19:1569–1574. [54] Turesson I, Nyman J, Holmberg E, Oden A. Prognostic factors for acute and late skin reactions in radiotherapy patients. Int J Radiat Oncol Biol Phys 1996;36:1065–1075. [55] van Eerde MR, Kampinga HH, Szabo BG, Vujaskovic Z. Comparison of three rat strains for development of radiation-induced lung injury after hemithoracic irradiation. Radiother Oncol 2001;58:313–316. [56] van’t Veer U, Dai H, van de Vijver MJ, et al. Gene expression profil-
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ing predicts clinical outcome of breast cancer. Nature 2002;415:530– 536. [57] Weeks DE, Paterson MC, Lange K, et al. Assessment of chronic gamma radiosensitivity as an in vitro assay for heterozygote identification of ataxia-telangiectasia. Radiat Res 1991;128:90–99. [58] West CM. Invited review: intrinsic radiosensitivity as a predictor of patient response to radiotherapy. Br J Radiol 1995;68:827–837. [59] West CM, Davidson SE, Elyan SA, et al. Uymphocyte radiosensitivity is a significant prognostic factor for morbidity in carcinoma of the cervix. Int J Radiat Oncol Biol Phys 2001;51:10–15.
Editorial
Editorial radiotherapy and oncology 2002: predictive assays for normal tissue damage Nicola S. Russell, Adrian C. Begg 14 May 2002; 9 July 2002
In this issue of radiotherapy and oncology, two groups present their work on developing a predictive assay for the degree of radiation-induced normal tissue damage. Both groups have utilised in vitro tests using patients’ lymphocytes. The rational behind these studies is based on the fact that for curative radiotherapy, the tolerance of patients with the most severe normal tissue reactions determines the dose level for the patient group as a whole for a given tumour site and type. The ultimate aim is to develop a sufficiently robust predictive assay to enable individual dose adjustment, and thus improve the therapeutic ratio for the patient population as a whole [1,6,34–36,51]. Such studies can indeed provide information on the role of cell kill and/or DNA damage in determining morbidity, but are time consuming and unlikely to be used routinely in the clinic. The questions therefore arise as to the value of these studies for the future, and what the best way forward is.
1. History A number of laboratories demonstrated a significant variation in cellular radiosensitivity between donors using either lymphocytes/lymphoblasts or fibroblasts [13,17,21,33,57]. There is clear evidence that the variation in radiation sensitivity is, at least in part, determined by genetic factors. There are a number of genetic syndromes, of which ataxia telangiectasia is probably the best example, which are characterised by increased cellular radiosensitivity and an extreme response to therapeutic radiation [43,49]. However, studies on non-syndromic patients that compared the in vitro radiosensitivity of lymphocytes and skin fibroblasts from the same donor were unable to find a correlation between them [20,23,31]. This lack of correlation suggests that modifying factors play a role depending on the tissue and cell type. On the clinical side, for over half a century, radiotherapists have recognised that patients show an individual variation in their normal tissue reactions to radiotherapy [38]. Quantification of variation of normal tissue reactions has
mainly been limited to easily observable sites such as skin (acute and late reactions) and subcutaneous and breast fibrosis [8,15,22,52,53]. Inter-patient variation has also been demonstrated for lung damage following radiation [50]. The prediction of the extent of clinical normal tissue radiation reactions by determining an in vitro radiosensitivity parameter (or surrogate thereof) has been the subject of research by several groups over the last decade or so (e.g. refs. [12,30,39,45,59]). However, it is now becoming apparent that such tests will never actually be clinically implemented due to both methodological and biological reasons.
2. Biological and laboratory factors There are a number of factors that will influence the correlation between an in vitro test and the clinical endpoint. Firstly, there are several aspects of the laboratory protocol that could influence the result. This includes the cell type used, dose, dose rate, type of medium, consistent quality of the medium or serum, immediate or delayed plating, use of feeder cells, and so on. The type of assay is also important. Colony formation has been regarded as the gold standard for assessing cell kill, although due to its time consuming nature, surrogate assays have also been investigated. These include chromosome damage (assessed by cytogenetics, fluorescence in situ hybridisation, micronucleus formation and others) and DNA damage (initial DNA damage, residual DNA damage, using pulsed or constant field electrophoresis, alkaline elution, comet assay, halo assay, etc.). There are also factors other than cell kill that can effect response (see below) and which should be taken into account. Although there is often a demonstrable correlation between the various in vitro assays, for example for colony formation and chromosome damage [44], this is often thanks to the inclusion of cell types from patients with a highly radiosensitive syndrome such as Ataxia Telangiectasia. For cells from normal individuals, or the majority of cancer patients that do not demonstrate extreme radiosensi-
0167-8140/02/$ - see front matter q 2002 Published by Elsevier Science Ireland Ltd. PII: S 0167-814 0(02)00189-5
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tivity, there is usually considerable scatter in the correlation between two in vitro end points for a group of individuals [4,58]. This is probably because, of the individual parameters tested, none is the main determinant of clonogenic cell survival in all individuals. For example, the level of DNA damage can be modified by variations in cell cycle delay/checkpoint control, repair capacity and fidelity, differences in chromatin structure influencing the conversion of DNA damage into chromosome breaks, apototic tendency, etc. We can therefore conclude that strong correlations between assays would not necessarily be expected. Furthermore, finding a correlation with cell kill may not be important, since cell kill may not be the only important determinant of radiation-induced morbidity. There are a number of biological factors that influence treatment response, which are not directly related to intrinsic radiosensitivity, including the remodelling of tissues in vivo [47]. For several tissues (e.g. lung, skin and intestinal mucosa), models have been developed showing the involvement of cytokine-mediated multicellular interactions in the radiation response [27]. Currently the most interesting developments are related to cytokines such as interleukins 2 and 6 (IL-2, IL-6), interferon alpha and in particular transforming growth factor beta (TGF-b) and its role in generating and modulating tissue fibrosis in many tissues and organs (e.g. refs. [9,18]). In mouse mammary tissue, radiation has been shown to induce rapid increases in the level of TGF-b (particularly in adipose stroma), and radiation induced oxidative activation of latent TGF-b has also been demonstrated [5]. Another example of humoural effects is the effect of the renin-angiotensin system on the renal vasculature and its influence on the expression of radiation induced renal damage, probably also involving TGF-b [14]. Further, Travis and colleagues have shown that some mice strains have a greater genetic predisposition than others to develop radiation-induced lung and rectal fibrosis [26,48]. Interstrain variation in the development of radiation-induced lung fibrosis has also been shown in rats [55]. Identification of the determinant genes, their function and their human homologues will increase our understanding of the mechanism of radiation induced fibrosis. These and similar studies also give us insights into mechanisms of radiation response other than the conventional radiobiological paradigm of target cell death. Whichever processes are the most important in determining radiation morbidity, whether cell kill, fibrogenesis, vascular mediated injury or other processes, each is dependent on the interplay of multiple gene products. Genetic testing for only one or even a few mutant genes is therefore unlikely to be of sufficient predictive power. Some groups have been able to demonstrate a relationship between a genetic determinant of radiosensitivity (such as ATM heterozygosity or rare polymorphisms in the DNA repair genes XRCC1 and XRCC3) and clinical over-reactions to radiotherapy [24,41]. However, their findings could not always be confirmed by others [3]. Successful genetic test-
ing to predict normal tissue damage therefore most probably requires screening for expression or mutations in multiple genes related to radiosensitivity and development of tissue reactions.
3. Clinical factors Clinical factors that influence the clinical end point used in studies designed to develop a predictive assay can obscure the relationship between results of an in vitro assay and the degree of normal tissue damage observed. Firstly, radiotherapy related factors such as total dose and fractionation schedule can confuse the relationship between different normal tissue reactions in the same patient. For example two fractionation schedules can give the same level of acute skin reaction but very different incidence rates of various late complications [37]. We therefore cannot expect a correlation between acute and late reactions for both schedules, even if the individual variation between patients is determined by intrinsic radiosensitivity. The same is true to some extent for the correlation between different late normal tissue end points if the relevant tissues vary in fractionation sensitivity. This could explain why some authors have been able to demonstrate a correlation between different late normal tissue end-points, but others have not [7,19,32,54]. Secondly, the follow-up duration and number of observations per patient can influence the relative relationship between two end-points. Consideration has to be given to latency in the development of complications such as fibrosis and telangiectasia that can bias the estimate of incidence and severity [8]. Saturation of a clinical endpoint, e.g. maximum induration and fibrosis or confluent telangiectasia, may lead to underestimation of the degree of underlying tissue damage and reduce the discrimination between individual patients [45,46]. Thirdly, other identified factors that influence the development of a given normal tissue reaction have to be accounted for in any analysis of the relationship to an assay parameter. These include treatment factors such as volume irradiated, dose distribution, medication, surgery and also co-morbidity, age, anatomical site and ‘life-style’ factors such as smoking, etc. [10,15,16,45,54]. Ideally, investigations into the value of a biological assay as a predictive test should therefore preferably be conducted using a homogeneously treated group of patients with accurately known treatment parameters such as dose volume histograms for the normal tissue of interest. This is not a trivial matter. The clinical end point should be selected on the basis of clinical relevance, be amenable to objective scoring using a carefully selected scoring system [11,25] and should have a significant inter-patient variation. Factors other than the parameter being tested that are known to influence the outcome should be incorporated into a multivariate analysis. In the real world these criteria have yet to be met.
N.S. Russell, A.C. Begg / Radiotherapy and Oncology 64 (2002) 125–129
Finally, one should consider the work of Jung and colleagues [29] who studied the kinetics of the development of late complications after radiotherapy. The incidence of late effects in several organs appears to occur with exponential kinetics, implying a random process in the timing of the occurrence of late radiation sequelae. Thus patients who develop a late effect early on may not necessarily be more sensitive than an individual who develops the same complication at a longer follow-up. This effect could interfere with the correct ranking of patients when correlating with an in vitro parameter. It would therefore be advisable to test the chosen purported low and high risk groups for their rates of development of reactions according to the log-linear plots advocated by Jung et al, to ensure that these clinical groups are indeed different and suitable for comparisons with cellular or genetic predictors.
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primarily on cytokine responses suggests that the historical emphasis on cell kill and/or DNA damage may be inappropriate and could be misleading for normal tissues. What is clear is that cell kill/DNA damage of target or surrogate cells, while showing mildly encouraging results in some studies, will not provide a clinically useable or robust predictor. Furthermore, these techniques do not indicate future directions leading to greater understanding of the relevant biology and pathology, whereas expression profiling does offer this possibility by directing attention to specific genes and pathways. Further biological and radiobiological research on normal tissue reactions to radiation must lead to a better understanding of their pathogenesis and enable the development of strategies directed at intervention and modulation of the normal tissue response. We believe this approach will help improve the therapeutic ratio for curative radiotherapy schedules.
4. Future On the basis of the arguments discussed above, it is becoming apparent that finding a good predictor for normal tissue damage is an extremely difficult task. Even though intrinsic radiosensitivity (determined in vitro) may influence the extent of the normal tissue radiation response, it is clear it is not the only determinant. The same argument can also be applied to other factors that modulate the tissue response. For example if a gene contributing to radiation fibrosis is found for humans, a test for gene expression or a certain mutation or polymorphism is unlikely to give sufficient information to enable dose adjustment. With enough knowledge of the relevant genes and pathways, looking at expression or polymporphisms in a far wider range of genes than is now being done may provide a viable approach. Microarray technology allows simultaneous measurement of the expression of many thousands of genes, and thus overcomes the problem of restricting investigations to one or a few genes only. Several studies on have shown the predictive power of pretreatment expression profiling for human tumours [2,40,56]. Similar large studies on normal tissues to predict treatment-induced morbidity have not yet been reported. However, ‘proof-of-principle’ has recently been shown by the studies of Quarmby et al. [42] using a cytokine gene array on fibroblasts derived from patients with minimal and severe radiation reactions, and from Johnston et al. [28] who showed expression differences in fibrosis-resistant and sensitive mouse strains. Other promising methodologies rapidly being developed for high throughput analysis are protein expression profiling (proteomics) and single nucleotide polymorphisms, the latter having the advantage that these DNA alterations will occur in all tissues, even readily accessible lymphocytes. Whether these methodologies will provide better predictive power than messenger RNA expression profiles remains to be seen. The fact that both the Quarmby and Johnston studies showed significant correlations with morbidity by focusing
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