Gerrit W. Barendsen

DOSE FRACTIONATION, DOSE RATE AND ISO-EFFECT RELATIONSHIPS FOR NORMAL TISSUE RESPONSES

An analysis is,,presented of responses of a variety of normal tissues in animals to fractionated irradiations. It is shown that the influence of fractionation can be described on the basis of a simple formula relating the effectiveness for induction of cellular effects to the dose per fraction: F(D) = 1D + a2D2. The ratio a1a2 is derived as an essential parameter for, the description of fractionation effects. It is concluded that the values of a1a2 for responses of various tissues range widely from 2 to 10 Gy. On the basis of the review of radiobiological data, a formalism is developed for the analysis and prediction of iso-effect relations for tissue tolerance, which can be used as an alternative to the nominal standard dose (NSD) formula of Ellis and its derived equations. An essential characteristic of the formalism is that three groups of tissue responses are distinguished which can be described with respect to fractionation effects by average values of ala2 = 10; 5 and 2.5 Gy, respectively. These groups comprise a l: a.o. skin and intestine; 2: connective tissue; 3:a.o. lung and vascular system. Dose rate effects can be described by a similar formalism. For the calculation of equivalent total doses to be applied in clinical treatments, a concept denoted Extrapolated Tolerance Dose (ETD) of Extrapolated Response Dose (ERD) is introduced. ETD is the tolerance dose for an infinite number of very small fractions. This concept is shown to be useful for the summation of different fractionated schedules and of low dose rate treatments. A number of examples is presented illustrating similarities and differences in comparison with calculations based on the NSD formula. An important feature of the described formalism is that it is directly based on radiobiological insights and it provides a more logical concept to account for the diversity of tissue responses than the assumption of different exponents of N and T in the NSD formula.

Comparison of RBE values of high-LET α-particles for the induction of DNA-DSBs, chromosome aberrations and cell reproductive death

BackgroundVarious types of radiation effects in mammalian cells have been studied with the aim to predict the radiosensitivity of tumours and normal tissues, e.g. DNA double strand breaks (DSB), chromosome aberrations and cell reproductive inactivation. However, variation in correlations with clinical results has reduced general application. An additional type of information is required for the increasing application of high-LET radiation in cancer therapy: the Relative Biological Effectiveness (RBE) for effects in tumours and normal tissues. Relevant information on RBE values might be derived from studies on cells in culture.MethodsTo evaluate relationships between DNA-DSB, chromosome aberrations and the clinically most relevant effect of cell reproductive death, for ionizing radiations of different LET, dose-effect relationships were determined for the induction of these effects in cultured SW-1573 cells irradiated with gamma-rays from a Cs-137 source or with α-particles from an Am-241 source. RBE values were derived for these effects. Ionizing radiation induced foci (IRIF) of DNA repair related proteins, indicative of DSB, were assessed by counting gamma-H2AX foci. Chromosome aberration frequencies were determined by scoring fragments and translocations using premature chromosome condensation. Cell survival was measured by colony formation assay. Analysis of dose-effect relations was based on the linear-quadratic model.ResultsOur results show that, although both investigated radiation types induce similar numbers of IRIF per absorbed dose, only a small fraction of the DSB induced by the low-LET gamma-rays result in chromosome rearrangements and cell reproductive death, while this fraction is considerably enhanced for the high-LET alpha-radiation. Calculated RBE values derived for the linear components of dose-effect relations for gamma-H2AX foci, cell reproductive death, chromosome fragments and colour junctions are 1.0 ± 0.3, 14.7 ± 5.1, 15.3 ± 5.9 and 13.3 ± 6.0 respectively.ConclusionsThese results indicate that RBE values for IRIF (DNA-DSB) induction provide little valid information on other biologically-relevant end points in cells exposed to high-LET radiations. Furthermore, the RBE values for the induction of the two types of chromosome aberrations are similar to those established for cell reproductive death. This suggests that assays of these aberrations might yield relevant information on the biological effectiveness in high-LET radiotherapy.

Parameters of linear-quadratic radiation dose-effect relationships: dependence on LET and mechanisms of reproductive cell death

An analysis of mammalian cell radiation-dose survival curves, based on the linear-quadratic formalism, is shown to yield insights in the various components of damage that contribute to cell reproductive death. RBE-LET relationships of single-track lethal damage, sublethal damage, potentially lethal damage and DNA double-strand breaks are compared. Single-track lethal damage is derived to be composed of two components: (1) damage that remains unrepaired in an interval between irradiation and assay for proliferative capacity, with a very strong dependence on LET, and (2) potentially lethal damage that is only weakly dependent on LET, similar to sublethal damage and DNA double-strand breaks. The results of this analysis lead to new interpretations of published experimental results and to suggestions for applications in radiotherapy.

Cell survival and radiosensitisation: modulation of the linear and quadratic parameters of the LQ model (Review).

The linear-quadratic model (LQ model) provides a biologically plausible and experimentally established method to quantitatively describe the dose-response to irradiation in terms of clonogenic survival. In the basic LQ formula, the clonogenic surviving fraction Sd/S₀ following a radiation dose d (Gy) is described by an inverse exponential approximation: Sd/S₀ = e-(αd+βd²), wherein α and β are experimentally derived parameters for the linear and quadratic terms, respectively. Radiation is often combined with other agents to achieve radiosensitisation. In this study, we reviewed radiation enhancement ratios of hyperthermia (HT), halogenated pyrimidines (HPs), various cytostatic drugs and poly(ADP-ribose) polymerase‑1 (PARP1) inhibitors expressed in the parameters α and β derived from cell survival curves of various mammalian cell cultures. A significant change in the α/β ratio is of direct clinical interest for the selection of optimal fractionation schedules in radiation oncology, influencing the dose per fraction, dose fractionation and dose rate in combined treatments. The α/β ratio may increase by a mutually independent increase of α or decrease of β. The results demonstrated that the different agents increased the values of both α and β. However, depending on culture conditions, both parameters can also be separately influenced. Moreover, it appeared that radiosensitisation was more effective in radioresistant cell lines than in radiosensitive cell lines. Furthermore, radiosensitisation is also dependent on the cell cycle stage, such as the plateau or exponentially growing phase, as well as on post-treatment plating conditions. The LQ model provides a useful tool in the quantification of the effects of radiosensitising agents. These insights will help optimize fractionation schedules in multimodality treatments.

RBE-LET relationships for different types of lethal radiation damage in mammalian cells: comparison with DNA dsb and an interpretation of differences in radiosensitivity.

Relative biological effectiveness (RBE), as a function of linear energy transfer (LET), is evaluated for different types of damage contributing to mammalian cell reproductive death. Survival curves are analysed assuming a linear-quadratic dose dependence of lethal lesions. The linear term represents lethal damage due to single particle tracks, the quadratic term represents lethality due to interaction of lesions from independent tracks. RBE-LET relationships of single-track lethal damage, sublethal damage, potentially lethal damage and DNA double-strand breaks (dsb) are compared. Single-track lethal damage is shown to be composed of two components: damage that remains unrepaired in an interval between irradiation and assay, characterized by a very strong dependence on LET, with RBEs up to 20, and potentially lethal damage, which is weakly dependent on LET with RBEs < 3. Potentially lethal damage and sublethal damage depend similarly on LET as DNA dsb. The identification of these different components of damage leads to an interpretation of differences in radiosensitivity and in RBEs among various types of cells.

Induction of linear tracks of DNA double-strand breaks by alpha-particle irradiation of cells

Understanding how cells maintain genome integrity when challenged with DNA double-strand breaks (DSBs) is of major importance, particularly since the discovery of multiple links of DSBs with genome instability and cancer-predisposition disorders. Ionizing radiation is the agent of choice to produce DSBs in cells; however, targeting DSBs and monitoring changes in their position over time can be difficult. Here we describe a procedure for induction of easily recognizable linear arrays of DSBs in nuclei of adherent eukaryotic cells by exposing the cells to α particles from a small Americium source (Box 1). Each α particle traversing the cell nucleus induces a linear array of DSBs, typically 10–20 DSBs per 10 μm track length. Because α particles cannot penetrate cell-culture plastic or coverslips, it is necessary to irradiate cells through a Mylar membrane. We describe setup and irradiation procedures for two types of experiments: immunodetection of DSB response proteins in fixed cells grown in Mylar-bottom culture dishes (Option A) and detection of fluorescently labeled DSB-response proteins in living cells irradiated through a Mylar membrane placed on top of the cells (Option B). Using immunodetection, recruitment of repair proteins to individual DSB sites as early as 30 s after irradiation can be detected. Furthermore, combined with fluorescence live-cell microscopy of fluorescently tagged DSB-response proteins, this technique allows spatiotemporal analysis of the DSB repair response in living cells. Although the procedures might seem a bit intimidating, in our experience, once the source and the setup are ready, it is easy to obtain results. Because the live-cell procedure requires more hands-on experience, we recommend starting with the fixed-cell application.

Correlation between cell reproductive death and chromosome aberrations assessed by FISH for low and high doses of radiation and sensitization by iodo-deoxyuridine in human SW-1573 cells

PURPOSE To study the relationship between cell reproductive death and exchange frequency in SW-1573 human lung tumour cells with and without incorporated iodo-deoxyuridine (IdUrd) following irradiation of plateau-phase cultures with y-rays. METHOD Linear-quadratic (LQ) analysis was performed for the data on clonogenic survival and on the frequency of chromosomal exchanges studied with fluorescence in situ hybridization in chromosomes X and 2. RESULTS Differences in the LQ parameters alpha and beta of both non-sensitized and sensitized chromosomes were found. In both chromosomes an increase in the number of chromosomal exchanges in IdUrd-radiosensitized cells compared with non-sensitized cells was observed. The alpha-enhancement factors of 1.7 and 1.9 for the X-chromosome and for chromosome 2, respectively, are similar. For the X-chromosome, the beta coefficient increased by a factor of 3.9 and for chromosome 2 by a factor of 1.4. After correction to a full genome equivalence, no significant difference in alpha was found between chromosomes X and 2 for both control and sensitized cells. In contrast, an almost 2.8 times higher beta was found for the sensitized X-chromosome compared to this value for chromosome 2. CONCLUSIONS It can be concluded that the linear-quadratic analysis of dose-response relationships offers insights into the correlation between cell survival and induction of exchanges in non-sensitized and radiosensitized cells.

Quantifying the Combined Effect of Radiation Therapy and Hyperthermia in Terms of Equivalent Dose Distributions

PURPOSE To develop a method to quantify the therapeutic effect of radiosensitization by hyperthermia; to this end, a numerical method was proposed to convert radiation therapy dose distributions with hyperthermia to equivalent dose distributions without hyperthermia. METHODS AND MATERIALS Clinical intensity modulated radiation therapy plans were created for 15 prostate cancer cases. To simulate a clinically relevant heterogeneous temperature distribution, hyperthermia treatment planning was performed for heating with the AMC-8 system. The temperature-dependent parameters α (Gy(-1)) and β (Gy(-2)) of the linear-quadratic model for prostate cancer were estimated from the literature. No thermal enhancement was assumed for normal tissue. The intensity modulated radiation therapy plans and temperature distributions were exported to our in-house-developed radiation therapy treatment planning system, APlan, and equivalent dose distributions without hyperthermia were calculated voxel by voxel using the linear-quadratic model. RESULTS The planned average tumor temperatures T90, T50, and T10 in the planning target volume were 40.5°C, 41.6°C, and 42.4°C, respectively. The planned minimum, mean, and maximum radiation therapy doses were 62.9 Gy, 76.0 Gy, and 81.0 Gy, respectively. Adding hyperthermia yielded an equivalent dose distribution with an extended 95% isodose level. The equivalent minimum, mean, and maximum doses reflecting the radiosensitization by hyperthermia were 70.3 Gy, 86.3 Gy, and 93.6 Gy, respectively, for a linear increase of α with temperature. This can be considered similar to a dose escalation with a substantial increase in tumor control probability for high-risk prostate carcinoma. CONCLUSION A model to quantify the effect of combined radiation therapy and hyperthermia in terms of equivalent dose distributions was presented. This model is particularly instructive to estimate the potential effects of interaction from different treatment modalities.

Thermal enhancement of the effectiveness of gamma radiation for induction of reproductive death in cultured mammalian cells.

The induction by gamma radiation of reproductive death in cultured cells derived from a rat ureter carcinoma (RUC-2) and from Chinese-hamster lung tissue (CH-V79) was shown to be enhanced by hyperthermic treatments at 41, 43 and 45 degrees C. The degree of enhancement was found to depend on the line of cells studied, the temperature employed and the level of damage considered. The influence of accumulating sublethal damage was decreased by hyperthermia, and the final slope of the radiation survival curve was increased. The degree of enhancement of lethal damage was found to depend on the time interval between the heat treatment and irradiation, especially at 41 degrees C.

Hyperthermia and incorporation of halogenated pyrimidines: radiosensitization in cultured rodent and human tumor cells.

PURPOSE To investigate the possible benefit of hyperthermia (HT) in combination with radiosensitization by halogenated pyrimidines (HPs) in rodent as well as in human tumor cells. METHODS AND MATERIALS Exponentially growing rodent cells, radiosensitive R-1 and MOS cells and radioresistant RUC-II and V79 cells, and human SW1573 cells, were exposed to 0, 1, 2, and 4 microM of chloro- (CldUrd), bromo- (BrdUrd), or iodo-deoxyuridine (IdUrd) in the culture medium. Survival after irradiation with gamma-rays from a 137Cs source and/or hyperthermic treatment (HT, 60 min at 42 degrees C) was determined by clonogenic assay. Linear-quadratic analyses of the radiation survival curves were performed to assess sensitization in the dose range 1 to 3 Gy relevant to radiotherapy. RESULTS The incorporation of HPs sensitized all cell lines to HT and resulted in radiosensitization dependent on the percentage of thymidine replacement. At equal levels of thymidine replacement, IdUrd was the most potent radiosensitizer. HT further increased radiation-induced lethality of cells that had incorporated HPs. Linear-quadratic analyses showed that HT further increased the linear parameter of the LQ formula while the quadratic parameter was not significantly changed. CONCLUSION The combination of HT and HPs act additively in increasing the radiosensitivity of rodent tumor cell lines with varying radiosensitivities as well as of a human tumor cell line. In particular, the ratio of the linear parameter to the quadratic parameter, relevant for fractionation effects in radiotherapy, was increased.