RBE of the MIT epithermal neutron beam for crypt cell regeneration in mice.

The RBE of the new MIT fission converter epithermal neutron capture therapy (NCT) beam has been determined using intestinal crypt regeneration in mice as the reference biological system. Female BALB/c mice were positioned separately at depths of 2.5 and 9.7 cm in a Lucite phantom where the measured total absorbed dose rates were 0.45 and 0.17 Gy/ min, respectively, and irradiated to the whole body with no boron present. The gamma-ray (low-LET) contributions to the total absorbed dose (low- + high-LET dose components) were 77% (2.5 cm) and 90% (9.7 cm), respectively. Control irradiations were performed with the same batch of animals using 6 MV photons at a dose rate of 0.83 Gy/min as the reference radiation. The data were consistent with there being a single RBE for each NCT beam relative to the reference 6 MV photon beam. Fitting the data according to the LQ model, the RBEs of the NCT beams were estimated as 1.50 +/- 0.04 and 1.03 +/- 0.03 at depths of 2.5 and 9.7 cm, respectively. An alternative parameterization of the LQ model considering the proportion of the high- and low-LET dose components yielded RBE values at a survival level corresponding to 20 crypts (16.7%) of 5.2 +/- 0.6 and 4.0 +/- 0.7 for the high-LET component (neutrons) at 2.5 and 9.7 cm, respectively. The two estimates are significantly different (P = 0.016). There was also some evidence to suggest that the shapes of the curves do differ somewhat for the different radiation sources. These discrepancies could be ascribed to differences in the mechanism of action, to dose-rate effects, or, more likely, to differential sampling of a more complex dose-response relationship.

Fifteen symposia on microdosimetry: implications for modern particle-beam cancer radiotherapy.

The objective of microdosimetry was, and still is, to identify physical descriptions of the initial physical processes of ionising radiation interacting with biological matter which correlate with observed radiobiological effects with a view to improve the understanding of radiobiological mechanisms and effects. The introduction of therapy with particles starting with fast neutrons followed by negative pions, protons and light ions necessitated the application of biological weighting factors for absorbed dose in order to account for differences of the relative biological effectiveness (RBE). Dedicated radiobiological experiments in therapy beams with mammalian cells and with laboratory animals provided sets of RBE values which are used to evaluate empirical 'clinical RBE values'. The combination of such experiments with microdosimetric measurements in identical conditions offered the possibility to establish semi-empirical relationships between microdosimetric parameters and results of RBE studies.

Proton relative biological effectiveness (RBE) for survival in mice after thoracic irradiation with fractionated doses.

PURPOSE: This study aims at providing relative biological effectiveness (RBE) data under reference conditions accounting for the determination of the "clinical RBE" of protons. METHODS AND MATERIALS: RBE (ref. (60)Co gamma-rays) of the 200 MeV clinical proton beam produced at the National Accelerator Centre (South Africa) was determined for lung tolerance assessed by survival after selective irradiation of the thorax in mice. Irradiations were performed in 1, 3, or 10 fractions separated by 12 h. Proton irradiations were performed at the middle of a 7-cm spread out Bragg peak (SOBP). Control gamma irradiations were randomized with proton irradiations and performed simultaneously. A total of 1008 mice was used, of which 96 were assessed for histopathology. RESULTS: RBEs derived from LD50 ratios were found not to vary significantly with fractionation (corresponding dose range, approximately 2-20 Gy). They, however, tend to increase with time and reach (mean of the RBEs for 1, 3 and 10 fractions) 1.00, 1.08, 1.14, and 1.25 for LD50 at 180, 210, 240, and 270 days, respectively (confidence interval approximately 20%). alpha/beta ratios for protons and gamma are very similar and average 2.3 (0.6-4.8) for the different endpoints. Additional irradiations in 10 fractions at the end of the SOBP were found slightly more effective ( approximately 6%) than at the middle of the SOBP. A control experiment for intestinal crypt regeneration in mice was randomized with the lung experiment and yielded an RBE of 1.14 +/- 0.03, i.e., the same value as obtained previously, which vouches for the reliability of the experimental procedure. CONCLUSION: There is no need to raise the clinical RBE of protons in consideration of the late tolerance of healthy tissues in the extent that RBE for lung tolerance was found not to vary with fractionation nor to differ significantly from those of the majority of early- and late-responding tissues.

Influence of cisplatinum on intestinal tolerance to photon and neutron irradiation in mice.

The hypothesis that cisplatinum (c-DDP) interacts with radiation by inhibiting the cellular repair capacities, was tested by comparing the interaction of c-DDP with low-LET (60Co gamma-rays) and high-LET radiation (d(50) + Be neutrons) in mice. The biological endpoint was lethality, 6 days after total body irradiation (early intestinal tolerance). The dose modifying factor was 1.80 +/- 0.25 for c-DDP plus 60Co gamma-rays, and 1.97 +/- 0.3 for c-DDP plus neutrons. As less repairable damage is induced by fast neutrons than by photons, this suggests that, in this system, the interaction between radiation and c-DDP is not explained by repair inhibition but is purely additive.

Proton RBE for early intestinal tolerance in mice after fractionated irradiation.

BACKGROUND AND PURPOSE: To determine the influence of the number of fractions (or the dose per fraction) on the proton relative biological effectiveness (RBE). MATERIALS AND METHODS: Intestinal crypt regeneration in mice was used as the biological endpoint. RBE was determined relative to cobalt-60 gamma rays for irradiations in one, three and ten fractions separated by a time interval of 3.5h. Proton irradiations were performed at the middle of a 7-cm Spread Out Bragg Peak (SOBP). RESULTS: Proton RBEs (and corresponding gamma dose per fraction) at the level of 20 regenerated crypts per circumference were found equal to 1.15+/-0.04 (10.0 Gy), 1.15+/-0.05 (4.8 Gy) and 1.14+/-0.07 (1.7 Gy) for irradiations in one, three and ten fractions, respectively. Alpha/beta ratios as derived from direct analysis of the 'quantal radiation response data' were found to be 7.6 Gy for gamma rays and 8.2 Gy for protons. Additional proton irradiations in ten fractions at the end of the SOBP were found to be more effective than at the middle of the SOBP by a factor of 1.14 (1.05-1.23). CONCLUSION: Proton RBE for crypt regeneration was found to be independent of fractionation up to ten fractions. One can expect that it remains unchanged for higher number of fractions as the lethalities for doses smaller than 3 Gy are exclusively due to direct lethal events. As a tendency for increased effectiveness at the end of the SOBP is reported in the majority of the studies, for clinical applications it would be advisable to allow for by arranging a sloping depth dose curve in the deeper part of the target volume. Finally, it must be noticed that most of in vitro and in vivo RBE values for protons are larger than the current clinical RBE (RBE=1.10).

RBE variation as a function of depth in the 200-MeV proton beam produced at the National Accelerator Centre in Faure (South Africa).

BACKGROUND AND PURPOSE: Thorough knowledge of the RBE of clinical proton beams is indispensable for exploiting their full ballistic advantage. Therefore, the RBE of the 200-MeV clinical proton beam produced at the National Accelerator Centre of Faure (South Africa) was measured at different critical points of the depth-dose distribution. MATERIAL AND METHODS: RBEs were determined at the initial plateau of the unmodulated and modulated beam (depth in Perspex = 43.5 mm), and at the beginning, middle and end of a 7-cm spread-out Bragg peak (SOBP) (depths in Perspex = 144.5, 165.5 and 191.5 mm, respectively). The biological system was the regeneration of intestinal crypts in mice after irradiation with a single fraction. RESULTS: Using 60Co gamma-rays as the reference, the RBE values (for a gamma-dose of 14.38 Gy corresponding to 10 regenerated crypts) were found equal to 1.16 +/- 0.04, 1.10 +/- 0.03, 1.18 +/- 0.04, 1.12 +/- 0.03 and 1.23 +/- 0.03, respectively. At all depths, RBEs were found to increase slightly (about 4%) with decreasing dose, in the investigated dose range (12-17 Gy). No significant RBE variation with depth was observed, although RBEs in the SOBP were found to average a higher value (1.18 +/- 0.06) than in the entrance plateau (1.13 +/- 0.04). CONCLUSION: An RBE value slightly larger than the current value of 1.10 should be adopted for clinical application with a 200-MeV proton beam.

Radiobiological intercomparison of clinical neutron beams for growth inhibition in Vicia faba bean roots.

Relative biological effectiveness (RBE) and oxygen enhancement ratio (OER) values of different neutron beams produced at the variable energy cyclotron "Cyclone" of Louvain-la-Neuve (Belgium) were determined. The neutrons were obtained by bombarding a beryllium target with 34-, 45-, 65-, or 75-MeV protons or with 50-MeV deuterons. The biological system was growth inhibition in Vicia faba bean roots. Taking the p(65) + Be neutron beam as a reference, RBE values were found equal to 1.36 +/- 0.2, 1.20 +/- 0.1, 1.00 (ref), 0.98 +/- 0.1, and 1.18 +/- 0.1, respectively; the doses corresponding to 50% growth inhibition were 0.39, 0.44, 0.53, 0.54, and 0.45 Gy. For the same beams, OER values were found equal to 1.55 +/- 0.1, 1.38 +/- 0.1, 1.29 +/- 0.1, 1.41 +/- 0.1, and 1.60 +/- 0.2, respectively.

Variation of RBE between p(75) + Be and d(50) + Be neutrons determined for chromosome aberrations in Allium cepa.

The RBE of p(75) + Be neutrons relative to d(50) + Be neutrons has been determined for chromosome aberrations induced in Allium cepa (onion) roots. Two biological criteria were selected: the average number of aberrations (mainly fragments) per cell in anaphase and telophase, and the percentage of aberration-free cells. The influence of sampling time (3 to 7 h incubation) between irradiation and fixation was investigated systematically. This factor did not significantly influence the results. The RBE values of p(75) + Be neutrons compared to those of d(50) + Be neutrons were 0.85 (0.79-0.91) and 0.87 (0.80-0.95) for the first and the second criteria, respectively. In previous experiments for the same beams, we found an RBE of 0.90 (0.86-0.94) for survival of V79 cells (D0 ratio), 0.96 (0.93-0.99) for the intestinal crypt cell system, and 0.83 (0.70-0.96) for Vicia faba growth delay.

Measurements of radiobiological effectiveness in the 85 MeV proton beam produced at the cyclotron CYCLONE of Louvain-la-Neuve, Belgium.

The RBE of the 85 MeV proton beam produced at the cyclotron of Louvain-la-Neuve using 60Co gamma rays as the reference radiation was determined for survival of Chinese hamster ovary cells in vitro and for intestinal crypt regeneration in mice in vivo. Cell survival curves determined at different depths yielded, for a surviving fraction (SF) of 0.01, RBE values of 1.11 +/- 0.05 at the initial plateau of the unmodulated beam, 1.10 +/- 0.03 at the middle of a 0.5-cm spread-out Bragg peak (SOBP), 1.03 +/- 0.03 at the beginning of a 3-cm SOBP and 1.07 +/- 0.03 at the end of a 3-cm SOBP. The highest RBE values were obtained at the middle of the 0.5-cm SOBP and at the end of the 3-cm SOBP (RBE = 1.22 and 1.16, respectively, at SF = 0.5), although the variations are not statistically significant. Irradiations with 3-Gy fractions separated by an interval of 3.5 h yielded RBEs of 1.11 +/- 0.30 and 0.90 +/- 0.32 at the initial plateau and at the middle of the 0.5-cm SOBP, respectively. Irradiations of mice at the middle of the 3-cm SOBP yielded an RBE of 1.08 +/- 0.03 for 20 regenerated crypts at a proton dose of 12.3 Gy.

Ex vivo determination of the effect of whole-body exposure to fast neutrons on murine spleen cell viability and apoptosis.

The effects of high-linear energy transfer (LET) radiations on lymphoid tissues and lymphocytes are not well understood. As a first approach to delineate these effects, the present work was conducted to assess the effects of high-LET radiations on murine spleen cells ex vivo and in vitro. BALB/c mice were irradiated whole-body with 65 MeV neutrons or 15 MV X rays at doses ranging from 0.2 to 3 Gy. Spleens were removed 1 day postirradiation and weighed, and single cell suspensions were prepared and cultured for several days. Apoptosis occurring in vitro was determined at different times by flow cytometry analysis of cells labeled with propidium iodide. It was found that irradiation with fast neutrons reduced spleen weight and cellularity to a greater extent than photons. Considering the spleen cellularity as end point, the relative biological effectiveness (RBE) of fast neutrons was 2. However, for both modes of irradiation, apoptosis of recovered spleen cells in vitro increased as a function of dose and the duration of culture. The level of apoptosis occurring at various times postirradiation was found to be identical for high- and low-LET radiations. Taken together, these results suggest that external as well as cellular factors might differentially modulate the sensitivity of lymphocytes to fast neutrons and photons.

The effects of a fractionated gamma irradiation on life shortening and disease incidence in BALB/c mice.

BALB/c male mice (12 weeks old) were exposed to a single or fractionated exposure of 137Cs gamma rays. The fractionated dose was split into 10 equal doses delivered at an interval of 1 day. The causes and possible causes of spontaneous death were ascertained by autopsy and histological examination, and the data were treated by competing risk analysis. Life shortening followed a linear dose dependency and was about the same for fractionated (38.1 +/- 3.1 days/Gy) as for single (46.2 +/- 4.3 days/Gy) exposure. Death from tumor disease was enhanced and that from nonstochastic lung and kidney diseases was reduced after fractionated compared to single exposure.

Life shortening and disease incidence in BALB/c mice following a single d(50)-Be neutron or gamma exposure.

Male BALB/c mice, 12 weeks old, were given a single exposure of either 137Cs gamma rays or d(50)-Be neutrons at a dose rate of 3 Gy/min. The animals were kept until death, and causes of death or possible causes of death were ascertained by autopsy and histology. The data were evaluated by competing risk methods. The survival time dose-effect curve for both types of exposure was linear and did not differ significantly (slopes: 55.8 +/- 4.0 days/Gy for neutrons and 46.2 +/- 4.3 days/Gy for gamma rays). The incidence of different diseases also was similar for both groups except that more carcinomas, sarcomas, and myeloid leukemias seemed to occur after neutron exposure and that nonstochastic lung and kidney diseases seemed to arise at lower doses.

Effect of gemcitabine on the tolerance of the lung to single-dose irradiation in C3H mice.

In an early phase II trial combining gemcitabine (dFdC) and radiotherapy for lung carcinomas, severe pulmonary toxicity was observed. In this framework, the objective of this study was to investigate the effect of dFdC on the tolerance of the lungs of C3H mice to single-dose irradiation. The thoraxes of C3H mice were irradiated with a graded single dose of 8 MV photons; dFdC (150 mg/kg) or saline (control animals) was administered i.p. 3 or 48 h prior to irradiation. Lung tolerance was assessed by the LD50 at 7-180 days after irradiation. For irradiation alone, the LD50 reached 14.45 Gy (95% CI 13.33-15.66 Gy). With a 3-h interval between administration of dFdC and irradiation, the LD50 reached 13.29 (95% CI 12.26-14.44 Gy); the corresponding value with a 48-h interval reached 13.01 Gy (95% CI 11.92-14.20 Gy). Our data also suggested a possible effect of dFdC on radiation-induced esophageal toxicity. dFdC has a minimal effect on lung tolerance after single-dose irradiation. However, a proper phase I-II trial should be designed before any routine use of combined dFdC and radiotherapy in the thoracic region.

Life-shortening and disease incidence in C57Bl mice after single and fractionated gamma and high-energy neutron exposure.

C57Bl Cnb mice were exposed to single or fractionated d(50)+Be neutrons or 137Cs gamma-ray exposure at 12 weeks of age and were followed for life-shortening and disease incidence. The data were analyzed by the Kaplan-Meier procedure using as criteria cause of death and possible cause of death. Individual groups were compared by a modified Wilcoxon test according to Hoel and Walburg, and entire sets of different doses from one radiation schedule were evaluated by the procedure of Peto and by the Cox proportional hazard model. No significant difference was found in life-shortening of C57Bl mice between a single gamma and neutron exposure. Gamma fractionation was clearly less effective in reducing survival time than a single exposure. On the contrary, fractionation of neutrons was slightly although not significantly more effective in reducing life span than a single exposure. Life-shortening appeared to be a linear function of dose in all groups studied. The data on causes of death show that malignant tumors, particularly leukemias including thymic lymphoma, and noncancerous late degenerative changes in lung were the principal cause of life-shortening after a high single gamma exposure. Exposure delivered in 8 fractions 3 h apart was more effective in causing leukemias and all carcinomas and sarcomas than one delivered in 10 fractions 24 h apart or in a single session. Following a single neutron exposure, leukemias and all carcinomas and sarcomas appeared to increase somewhat more rapidly with dose than after gamma irradiation. No significant difference in the incidence of leukemias and all carcinomas and sarcomas was noted between a single and a fractionated neutron exposure.

Radiobiological intercomparison of p(45)+Be and p(65)+Be neutron beams for lung tolerance in mice after single and fractionated irradiation.

The lung tolerance in mice after single and fractionated irradiations with p(45)+Be and p(65)+Be neutrons produced at the isochronous cyclotron "CYCLONE" of Louvain-la-Neuve (Belgium) was studied. Cobalt-60 gamma rays were used for control irradiations. The end point was the dose which was lethal to 50% of the mice by 180 days (LD50/180). On a log-log plot, the slope (+/- SE) of the relationship between total isoeffect dose and fraction number decreases from 0.34 +/- 0.01 for gamma rays to 0.19 +/- 0.01 for p(65)+Be and 0.12 +/- 0.01 for p(45)+Be neutrons. The data have been analyzed using the linear-quadratic (LQ) model. The alpha/beta ratio (+95% confidence interval) increases from 5.3 (4.3-6.4) for gamma rays to 20.7 (16.7-24.9) for p(65)+Be and 37.9 (25.8-65.8) for p(45)+Be. The RBEs of neutrons relative to gamma rays were estimated from the LQ parameters, to 1.15 and 1.19 for a dose of 14 Gy gamma rays and 2.02 and 2.47 for a dose of 2 Gy gamma rays for p(65)+Be and p(45)+Be neutrons, respectively. The neutron RBE of the p(45)+Be relative to the p(65)+Be calculated from the ratio of their respective RBEs relative to gamma rays reaches 1.03 and 1.23 for doses of 14 and 2 Gy gamma-ray equivalent, respectively. These data are compared with other published data on lung tolerance after irradiation with lower-energy neutrons and with data obtained previously in our laboratory on mouse jejunum and Vicia faba.

Life-shortening and disease incidence in mice after exposure to gamma rays or high-energy neutrons.

Male C57Bl/Cnb and BALB/c mice were exposed to single and fractionated d(50) + Be neutrons or 137Cs gamma rays at 12 weeks of age and were followed for life-shortening and disease incidence as ascertained by autopsy and histological examinations at the time of spontaneous death. Fractionation schedules used were 10 exposures at 24-h intervals and 8 exposures at 3-h intervals for gamma rays, and 8 exposures at 3-h intervals for neutrons. The data were analyzed by the Kaplan-Meier procedure using as criteria causes of death and possible causes of death. Individual groups were compared by a modified Wilcoxon test according to Hoel and Walburg (J. Natl. Cancer Inst. 49, 361-372 (1972)). No significant difference was found in C57Bl/Cnb and BALB/c male mice between a single gamma-ray exposure and a single neutron exposure. Gamma-ray fractionation was clearly less effective in reducing survival time than a single exposure. In contrast, fractionation of neutrons was slightly, although not significantly, more effective in reducing survival time than a single exposure. The relative biological effectiveness (RBE) for life-shortening for d(50)-Be neutrons compared to gamma rays is of the order of 1 to 2 for a single exposure to neutrons and between 2 and 3 for fractionated neutrons compared to a single exposure to gamma rays. Neutron irradiation caused somewhat more cancer than gamma irradiation, and the RBE for cancer induction may be higher, probably between 2 and 3 in the range of 1 to 3 Gy, although the present data do not allow a more precise assessment.

Involvement of TP53 in apoptosis induced in human lymphoblastoid cells by fast neutrons.

We investigated the involvement of TP53 in apoptosis induced by fast neutrons in cells of three human B-lymphoblast cell lines derived from the same donor and differing in TP53 status: TK6 (wild-type TP53), WTK1 (mutant TP53) and NH32 (knockout TP53). Cells were exposed to X rays or to fast neutrons at doses ranging from 0.5 to 8 Gy. Apoptosis was determined by measurements of the sub-G0 /G1-phase DNA content and by the externalization of phosphatidylserine. Fast neutrons induced extensive apoptosis in TK6 cells, as shown by the formation of hypodiploid particles, the externalization of phosphatidylserine, and the activation of caspases. In contrast, cell death was triggered at a significantly lower rate in cells lacking functional TP53. However, TP53-independent cell death also expressed the morphological and biochemical hallmarks of apoptosis. Proliferation tests and clonogenic assays showed that fast neutrons can nevertheless kill WTK1 and NH32 cells efficiently. The absence of functional TP53 only delays radiation-induced cell death, which is also mediated by caspases. These results indicate that fast-neutron irradiation activates two pathways to apoptosis and that the greater relative biological effectiveness of fast neutrons reflects mainly an increase in clonogenic cell death.

Specification of radiation quality in fast neutron therapy: microdosimetric and radiobiological approach.

Specification of radiation quality is an important issue in fast neutron therapy since the biological effectiveness of the beams varies to a large extent with neutron energy. It must meet specific criteria, mainly derived from the accuracy requirement for absorbed dose delivery. A first approach to this problem consists in identifying physical parameters that can be related to Relative Biological Effectiveness (RBE) and which describe the beam production technique (e.g. neutron-producing reaction, p + Be or d + Be, energy of the incident particle). A second is based on microdosimetry, which provides a description of the secondary radiation components to which the biological consequences of irradiations are more directly correlated. A third approach consists in experimental RBE determinations in reference conditions: intestinal crypt regeneration in mice after irradiation to the whole body with single doses is proposed as a standard biological system for radiobiological calibrations of clinical fast neutron beams. Dosimetric, microdosimetric and radiobiological intercomparisons are encouraged since they provide a homogeneous set of data which facilitate the exchange of clinical information. They also constitute a basis for the clinical RBE approach and an overall check of the irradiation procedure. Therefore they should be recommended in every non-conventional radiation therapy facility.

No evidence for radiation-induced clastogenic factors after in vitro or in vivo exposure of human blood.

Experiments were performed with human plasma irradiated in vitro or in vivo in order to evaluate the extent to which clastogenic factors might disturb the adaptive response to DNA-damaging factors currently studied in our laboratory. The studies were carried out with plasma isolated from whole blood given 4 Gy of X-rays in vitro and with plasma from people receiving local radiotherapy at a total dose of about 60 Gy gamma rays. Addition of irradiated plasma to culture medium did not result in a statistically significant increase in structural aberrations in chromosomes of non-irradiated normal blood.

[Response of digestive tract mucosa to single and fractionated irradiation. Practical implications for radiotherapy]. / Réponse de la muqueuse digestive à une irradiation unique et fractionnée. Conséquences pratiques pour la radiothérapie

After a short description of the gastro-intestinal syndrome (for a single "acute" irradiation) and a discussion of the importance of fractionation in radiotherapy, the authors report their experimental results on intestinal tolerance in mice after fractionated irradiation. Intestinal tolerance was assessed from LD50 after abdomen irradiation. Variation of LD50 was first studied as a function of the fraction number N (keeping constant the overall time T). From these data iso-effect curves were calculated which can be used for clinical applications for correcting the total dose when the fraction number has to be modified (for T constant). In a second series of experiments, variation of LD50 was studied as a function of overall time T (keeping constant the fraction number N), this study confirms the large recovery capability of the intestinal mucosa, which is due to the combination of 2 mechanisms: shortening of the mitotic cycle of the surviving stem cells and increase of the size of the stem cell compartment. These 2 mechanisms were evaluated quantitatively for a fractionated irradiation. The data obtained for intestine are compared to similar data obtained for other tissues (skin, lung). The authors emphasize the risk of applying to late effects the conclusions obtained for early effets.