Radiobiological intercomparisons of fast neutron beams used in therapy

A research program was performed at Louvain-la-Neuve to systematically determine the RBE of fast neutrons for the growth inhibition in Vicia faba bean roots and for the regeneration of the intestinal crypts in mice. The following neutron beams were compared p(75) + Be, p(65) + Be, p(45) + Be, p(34) + Be, d(20) + Be, and d(50) + Be. The RBE-variation as a function of neutron energy is larger for the Vicia faba system than for the regeneration of the intestinal crypt cells. This can be related to the inherent differency of the biological systems, but also to the different dose ranges involved (0.33 to 0.56 Gy and 7.66 to 8.56 Gy, respectively). In the high energy range explored, defined by the reactions p(75) + Be to p(34) + Be RBE varies only between 0.92 and 1.28 for Vicia faba and 0.96 and 1.12 for crypt cells normalized to the p(65) + Be beam. By contrast the RBE at lower energy beams (d(20) + Be and d(14.5) + Be) reaches values between 1.5 and 1.6 Finally fractionation has shown to be likely more important at the high energy beams.     

RBE and OER measurements on the p(66) + Be neutron beam at Faure, South Africa.

Results reported are for single dose exposures and refer to 60Co-gamma-irradiation. The RBE determined by V79 cell survival and based on the Do ratio was found to be 1.70 +/- 0.4 ranging from 1.5 to 1.8. In the case of the regeneration of mouse jejunal crypts the RBE was calculated at ten cell survival and was found to be 1.68. The maximum acute mouse skin reaction at a skin score of 2.0 was found to be 2.1 while the average skin reaction was 1.7. Growth retardation of Vicia faba bean roots measured at the level of 50% indicated an average RBE of 3.0 and a range of 2.7 to 3.7. The OER obtained for V79 cell survival was found to be 1.7 to 1.8. Comparison is made with the RBE and OER measurements for the neutron facilities at Clatterbridge, Fermilab and Louvain-la-Neuve which produce neutrons by the same nuclear reaction and whose physical specifications closely resemble those of the Faure neutrons. This comparison indicates that the Faure beam shows no unusual biological features and that its biological effectiveness is in line with that expected from its physical characteristics.     

Radiobiological studies with therapeutic neutron beams generated by p+ leads to Be or d+ leads to Be

Mammalian cells cultured in vitro were used to study the radiobiological characteristics of neutron beams generated by 43 MeV protons on beryllium or 25 MeV deutrons on beryllium. For an unfiltered beam of neutrons generated by 43 MeV p+ leads to Be the relative biological effectiveness was found to be 8-12% higher at a depth of 2 cm than at a depth of 12 cm due to the presence of a large component of low-energy neutrons. The addition of a hydrogenous filter 4 cm thick preferentially removed the low-energy neutrons from the beam and, as a result, the neutron RBE was independent of depth. There was no significant difference in the oxygen enhancement ratio between the filtered neutrons produced by 43 Mev p+ leads to Be and neutrons produced by 25 MeV d+ leads to Be; for both beams the OER value was about 1.6.     

Effects of boron neutron capture irradiation on the normal lung of rats.

The whole lung of rats was irradiated with X-rays, thermal neutrons, or thermal neutrons in the presence of p-boronophenylalanine (BPA). A >/= 20% increase in breathing rate, in the period 40-80 days after irradiation, was indicative of radiation-induced pneumonitis. The ED(50) (+/-SE) for a >/= 20% increase in breathing rate, relative to age-matched controls, was 11.6 +/- 0.13 Gy for X-rays and 9.6 +/- 0.08 Gy for neutrons only. This indicated a thermal neutron beam RBE of 1.2 and an RBE of 2.2 for the high-LET components of the dose, assuming a dose reduction factor of 1.0 for gamma rays. Preliminary data indicate the compound biological effectiveness factor for BPA in the lung is approximately 1.5.     

Comparison of the effects of neutron and/or photon irradiation on spontaneous squamous-cell carcinoma in mice

The effect of 30MeV D+ leads to Be NIRS cyclotron neutrons on a spontaneous NR-S1 squamous-cell carcinoma was studied. The TD50 (number of tumor cells required for transplantation in half the sites) and tumor growth delay time (TGD) were determined. The RBE of 200-kVp x rays at the 10% survival level was 1.9 for aerobic cells and 2.0 for hypoxic cells; the OER at the same level was congruent to 1.7. Tumor cells irradiated with neutrons were unable to repair potentially lethal damage, in contrast to x-irradiated tumor cells. The RBE of 50-MeV D+ leads to Be neutrons was inversely related to deuteron energy for hypoxic cells. The relationship between TGD and peak skin indicated no benefit from mixed beams or 5 neutron doses.     

Kinetics of sublethal damage recovery in mouse lip mucosa comparing low and high-LET radiation

The effects of d(50) + Be neutrons on the lip mucosa in mice were investigated as a model of early effects. The biological endpoint eas the incidence of desquamation in the lower lip after selective irradiation of the snout of the animals. ED 50 (dose leading to desquamation in 50% of the animals) were calculated by probit analysis. Fractionated (two, four and ten fractions) and protracted (43.5, 11.5 and 0.88 Gy.h-1) irradiations have been carried out. Results were analysed using the mathematical method of Dale. An alpha/beta of 39.6 Gy and a t 1/2 of recovery of sublethal damage of 47 min have been derived. These results have been compared to data previously obtained with cobalt-60 gamma rays. Using the same mathematical approach, and comparing similar fractionated and protracted experiments, an alpha/beta of 7.4 Gy and a t 1/2 of recovery of 47 min have been calculated. There was no significant difference in the repair kinetics after irradiations with gamma rays or d(50) + Be neutrons.     

Comparison of repair of DNA double-strand breaks caused by neutron or gamma radiation in cultured human cells.

The dose-response for the induction of initial double-strand breaks (dsb) in DNA of human epithelioid cells by JANUS 0.85 MeV fission-spectrum neutrons was parabolic as assayed by a calibrated neutral filter elution technique. The relative biological effectiveness (RBE) of these neutrons relative to 60Co gamma-rays was unity. The kinetics of repair after a 60 Gy gamma-ray exposure were biphasic. About 65% of these dsb were rapidly repaired (T 1/2 of approximately 2 min), and the remainder were almost completely removed after 150 min at a slower rate (T 1/2 = 30 min). After the same dose of JANUS neutrons, the rapid repair component was markedly reduced (possibly not a significant repair component), and the bulk of the dsb were sealed more slowly (T 1/2 = 90 min). After 150 min, 25% remained unsealed. Even after a lower neutron dose (20 Gy), a proportion of the dsb were refractory to repair. Thus, unrepaired (or irreparable) dsb induced by high energy neutrons might explain the high RBE of neutrons for cell killing.     

Characterization of ultra high energy neutron beam generated by 500 MeV proton beam

An ultra high energy neutron facility was constructed at PARMS, University of Tsukuba, to produce a neutron beam superior to an X-ray beam generated by a modern linac in terms of dose distribution. This has been achieved using the reaction on a thick uranium target struck by 500 MeV proton beam from the booster-synchrotron of High Energy Physics Laboratory. The percentage depth dose of this neutron beam is nearly equivalent to that of X-rays at around 20 MV and the dose rate of 15 cGy per minute. Relative biological effectiveness of this neutron beam has been estimated on the cell killing effect by the use of HMV-I cell line. Resultant survival curve of cells after the neutron irradiation shows the shoulder with n and Dq of 8 and 2.3 Gy, respectively. RBE value at 10(-2) survival level for the present neutron, compared with 137Cs gamma-rays is 1.24. The result suggests that the biological effects of high energy neutrons are not practically large enough whenever the depth dose distribution of neutrons becomes superior to high energy linac X-rays.     

The relative biological effectiveness of fractionated doses of fast neutrons (42 MeVd----Be) for normal tissues in the pig. IV. Effects on renal function

The effects of fractionated doses of fast neutrons (42 MeVd----Be) on the radiation response of the pig kidney have been assessed and compared with those observed after X irradiation. Following X irradiation there was a marked increase in the total dose at which renal function was preserved with decreasing fraction size. The rate of this increase was dependent on the overall treatment time; for fractionated irradiation given over 18 or 39 days the exponents related to fraction number, N, were 0.36 +/- 0.03 and 0.48 +/- 0.003, respectively. In contrast, there was no significant change in the iso-effect dose for renal injury following fractionated irradiation with fast neutrons where there was also little effect of varying the overall treatment time. Analysing these data by means of the linear-quadratic (LQ) model, using both an Fe-plot and the Tucker test, gave alpha/beta ratios of 2.42 +/- 0.06 Gy and 2.99 +/- 0.16 Gy, respectively, for X-ray doses given in 18 days. For fractionated doses of X rays given in 39 days the alpha/beta ratios were 0.40 +/- 0.01 Gy and 0.47 +/- 0.02 Gy, respectively. The alpha/beta ratios for renal tissue following fast neutron irradiation obtained by the two methods were also similar, i.e. 15.00 +/- 0.60 Gy and 15.72 +/- 3.76 Gy, respectively. The pronounced fractionation effect seen with X irradiation, particularly for doses administered over 39 days as opposed to 18 days, coupled with the absence of any such effect with fast neutrons, resulted in a marked increase in relative biological effectiveness (RBE) with decreasing X-ray dose/fraction. The slopes of the resulting regression lines were -0.73 +/- 0.05 and -0.33 +/- 0.02, respectively. The lack of dose sparing associated with fractionation, or variation of the overall treatment time for fast neutron irradiation, suggests that doses administered to tumours adjacent to the kidney can be given as a few relatively large dose/fractions in a short overall treatment time without an increased risk of complications related to renal tissue. This may be of therapeutic advantage in the treatment of rapidly proliferating tumours where dose may be wasted using more conventional protracted fractionated irradiation schedules.     

Effects of negative pions and neutrons on the growth of Vicia faba bean roots

Vicia faba bean roots have been irratiated with neutrons of various energies and with negative pi-mesons, and the effect on the ten-day growth of the roots has been determened. The neutron irratiations were made in beams of 400 and 600 MeV maximum energy, as well as with neutrons from a plutonium-beryllium source (mean energy 4.4 MeV) and from a 14 MeV neutron generator. The bean roots have also been irradiated at various points along the depth-dose curve of negative pi-mesons, including the gegion where the pions annihilate on coming to rest. The results show a maximum relative biological effectiveness (RBE) of 3.7 for 50% reduction in ten days growth for stopped negative pions and values up to 3.3 for high-energy neutrons, compared to 5.5 for 14 MeV neutrons. The biological effectiveness of high-energy neutrons and stopped pions shows a more pronounced dependence on dose than does the effect with lower-energy neutrons.     

Relationship between oocyte apoptosis and ovarian tumours induced by high and low LET radiations in mice

PURPOSE: To investigate the biological effectiveness of neutrons at the energy below 1 MeV on apoptosis and carcinogenesis in the mouse ovary. MATERIALS AND METHODS: Female mice were exposed to 1.0 Gy monoenergetic neutrons (0.317, 0.525 and 1.026 MeV), (252)Cf fission neutron (2.13 MeV) or (137)Cs gamma-rays at 7 days of age. Apoptosis of the oocyte and pregranulosa cells, and ovarian carcinogenesis were compared between the radiations. The efficiency of gamma-rays for granulosa cell tumorigenesis was tested by transplantation of the irradiated ovaries into non-irradiated mice. RESULTS: The cumulative apoptotic index of oocytes was 77.9%, 65.6% and 41.6% for the 0.525 MeV neutron, 2.13 MeV neutron and gamma-rays, respectively. Follicles with apoptotic pregranulosa cells were 53.0%, 18.3% and 22.8% of cumulative index for the three groups. Tubular adenomas developed in the groups of monoenergetic neutrons (26.1%) and gamma-ray (35.5%), whereas granulosa cell tumours developed only in the gamma-ray groups (3.2% for 1.0 Gy and 15.6% for 3.0 Gy). Partial-body irradiation with 3 Gy gamma-rays to the ovaries induced granulosa cell tumours with an incidence of 27.3%. CONCLUSION: Effectiveness of neutrons to cause apoptosis was higher for 0.525 MeV than for 2.13 MeV. The pregranulosa cell apoptosis occurred in an oocyte-prone manner. The higher effectiveness of neutrons than gamma-rays to induce oocyte and pregranulosa cell apoptosis correlates with the inhibition of granulosa cell tumour development.     

Lack of inverse dose-rate effect on fission neutron induced transformation of C3H/10T1/2 cells

Exponential and density-inhibited cultures of C3H/10T1/2 cells were exposed to a single dose of 0.3 Gy of fission neutrons delivered at rates ranging from 0.005 to 0.1 Gy/min. No discernible effect upon cell survival or transformation was observed by a lowering of the fission neutron dose rate in either exponential or plateau cultures. At the level of 2.3 x 10(-4) transformants per surviving cell, the RBE for neoplastic transformation was three at acute dose rates and ten at the lowest dose rate studied (0.005 Gy/min for neutrons and 0.01 Gy/min for X-rays).     

Repair in mouse lung of multifraction X rays and neutrons: extension to 40 fractions

We have extended our previous multiple irradiations of mouse lung from 20 to 40 fractions of both X-ray and neutron radiation in order to test whether the repair parameters previously derived will hold for lower doses per fraction, down to 1.1 Gy of X rays and 0.18 Gy of 3 MeV neutrons per fraction. Repair parameters were calculated from measurements of breathing rate and lethality at monthly intervals up to 17 months after irradiation with 1, 10, 20 or 40 equal fractions. Sparing of neutron damage was negligible when the neutron dose was divided into multiple fractions, but progressively greater repair of lung damage was seen after increasing numbers of X-ray fractions. A significant increase in the iso-effect dose for 40 fractions of X rays was found compared with 20 fractions, even when two fractions per day were given at intervals of about 6 hours, as was the case in the 40 fraction experiment. The data were well fitted by the linear quadratic formula for response vs. dose per fraction and the ratio alpha/beta yielded values of approximately 3 Gy after X rays and 30 to 40 Gy after neutron irradiation; these values are not different from alpha/beta ratios found for up to 20 fractions. The single dose RBE was less than 2, increasing to about 6 at the lowest dose per fraction measured, in agreement with previous results. The ratio of the alpha component for neutrons to that for X rays was about 8, which is therefore the limiting RBE predicted for infinitely small doses per fraction.     

Lung Tumour Induction in Mice after X-rays and Neutrons

Summary

Dose–response curves were determined for pulmonary adenomas and adenocarcinomas in mice after single acute doses of 200 kVp X-rays and cyclotron neutrons (E¯ = 7·5 MeV). A serial-killing experiment established that the radiation induction of chromosome aberrations. The validity of the concept of PLD neous cancers. The dose versus incidence (I) of tumours in male and female mice for X-ray doses between 0·25 and 7·5 Gy is ‘bell-shaped’ and best fitted with a purely quadratic induction and exponential inactivation terms, i.e. I = A + BD²e^?αD. In contrast, the tumour dose–response after 0·1–4·0 Gy of neutrons is best fitted by I = A + BD e^?αD and is steeply linear ≤ 1 Gy, peaks between 1 and 3 Gy and sharply declines at 4·0 Gy. The data for the female mice ≤ 1 Gy neutrons are best fitted to the square root of the dose.

A major objective of the experiments was to derive neutron RBE values. Because of the differences between the X-ray (quadratic) and neutron (linear) curves, the RBE_n will vary inversely with decreasing X-ray dose. The RBE values at 1 Gy of X-rays derived from the B coefficients in the above equations are 7·4 ± 3·2 (male and female); 8·6 ± 3·6 (female) and 4·7 ± 1·8 (male). These are high values and imply even higher values at the doses of interest to radiation protection. If, however, one restricts the analysis to the initial, induction side of the response (≤ 1 Gy neutrons, ≤ 3 Gy X-rays) then good linear fits are obtainable for both radiations and indicate neutron RBE values of 7·4 ± 2·3 for female mice and 4·5 ± 1·8 for males, and these are independent of dose level.     

The radiobiology of 24 keV neutrons. Measurement of the relative biological effect free-in-air, survival and cytogenetic analysis of the biological effect at various depths in a polyethylene phantom and modification of the depth-dose profile by boron 10 for V79 Chinese hamster and HeLa cells

HeLa human carcinoma cells and V79 Chinese hamster fibroblasts have been irradiated in vitro with a beam of neutrons with a nearly pure 24 keV spectrum. The relative biological effectiveness (RBE) of the filtered neutron beam relative to 60Co gamma radiation was determined by irradiation of cell cultures "free-in-air". The values obtained for the RBE at 37% survival were 5.8 +/- 0.8, at a dose of 0.69 +/- 0.06 Gy for the HeLa cells and 3.14 +/- 1.1 at a dose of 1.09 +/- 0.091 Gy for the V79 cells. Cytogenetic analysis of the damage in irradiated V79 cells gave an RBE of 6.7 +/- 1.4. Irradiation in a polyethylene phantom markedly attenuated the beam's biological effect. For both cell lines 2 cm of polyethylene virtually eliminated cell killing. Addition of boron 10 to the medium led to increased cell killing and a value of 4 was obtained for the RBE of the 10B(n, alpha)7Li reaction in HeLa cells.     

The relative biological effectiveness of fractionated doses of fast neutrons (42 MeVd----Be) for normal tissues in the pig. I. Effects on the epidermis and dermal vascular/connective tissues

The effects of fractionated doses of fast neutrons (42 MeVd----Be) on the early epithelial and later dermal response of pig skin have been assessed and compared with those after X irradiation. For the early epithelial reaction, i.e. moist desquamation, the relative biological effectiveness (RBE) of the neutron beam increased with the decreasing size of the X-ray dose/fraction. There was an experimentally observed upper RBE value of approximately 2.75 for X-ray doses/fraction of between 2 and 5 Gy. For the late reaction of ischaemic dermal necrosis the RBE was greater than 3.0 for X-ray doses/fraction of less than 3 Gy and, based on the assumptions made in the linearquadratic model of cell survival, an upper limiting RBE of 4.32 +/- 0.39 was calculated for infinitely small doses/fraction. These findings were compared with other radiobiological data and the conclusions drawn from the results of clinical trials. It was concluded that for the sparing of late effects in skin and subcutaneous tissues, relative to acute reactions, a relatively small number of fractions in a short overall treatment time may be optimal for fast neutron therapy.     

Induction and disappearance of G2 chromatid breaks in lymphocytes after low doses of low-LET gamma-rays and high-LET fast neutrons

PURPOSE: To determine by means of the G2 assay the number of chromatid breaks induced by low-LET gamma-rays and high-LET neutrons, and to compare the kinetics of chromatid break rejoining for radiations of different quality. MATERIALS AND METHODS: The G2 assay was performed on blood samples of four healthy donors who were irradiated with low-LET gamma-rays and high-LET neutrons. In a first set of experiments a dose-response curve for the formation of chromatid breaks was carried out for gamma-rays and neutrons with doses ranging between 0.1 and 0.5 Gy. In a second set of experiments, the kinetics of chromatid break formation and disappearance were investigated after a dose of 0.5 Gy using post-irradiation times ranging between 0.5 and 3.5 h. For the highest dose of 0.5 Gy, the number of isochromatid breaks was also scored. RESULTS: No significant differences in the number of chromatid breaks were observed between low-LET gamma-rays and high-LET neutrons for the four donors at any of the doses given. The dose-response curves for the formation of chromatid breaks are linear for both radiation qualities and RBEs = 1 were obtained. Scoring of isochromatid breaks at the highest dose of 0.5 Gy revealed that high-LET neutrons were, however, more effective at inducing isochromatid breaks (RBE = 6.2). The rejoining experiments further showed that the kinetics of disappearance of chromatid breaks following irradiation with low-LET gamma-rays or high-LET neutrons were not significantly different. Half-times of 0.92 h for gamma-rays and 0.84 h for neutrons were obtained. CONCLUSIONS: Applying the G2 assay, the results demonstrate that at low doses of irradiation, the induction as well as the disappearance of chromatid breaks is independent of the LET of the radiation qualities used (0.24 keV x microm(-1) 60Co gamma-rays and 20 keV x microm(-1) fast neutrons). As these radiation qualities produce the same initial number of double-strand breaks, the results support the signal model that proposes that chromatid breaks are the result of an exchange process which is triggered by a single double-strand break.     

Changes in RBE of 14-MeV (d+T) neutrons for V79 cells irradiated in air and in a phantom: Is RBE enhanced near the surface?

Purpose

The relative biological effectiveness (RBE) for inacivation of V79 cells was determined as function of dose at the Heidelberg 14-MeV (d+T) neutron therapy facility after irradiation with single doses in air and at different depths in a therapy phantom. Furthermore, to assess the reproducibility of RBE determinations in different experiments we examined the relationship between the interexperimental variation in radiosensitivity towards neutrons with that towards low LET⁶⁰Co photons.

Methods

Clonogenic survival of V79 cells was determined using the colony formation assay. The cells were irradiated in suspension in small volumes (1.2 ml) free in air or at defined positions in the perspex phantom. Neutron doses were in the range, D₁=0.5–4 Gy.⁶⁰Co photons were used as reference radiation.

Results

The radiosensitivity towards neutrons varied considerably less between individual experiments than that towards photons and also less than RBE. However, the mean sensitivity of different series was relatively constant. RBE increased with decreasing dose per fraction from RBE=2.3 at 4 Gy to RBE=3.1 at 0.5 Gy. No significant difference in RBE could be detected between irradiation at 1.6 cm and 9.4 cm depth in the phantom. However, an approximately 20% higher RBE was found for irradiation free in air compared with inside the phantom. Combining the two effects, irradiation with 0.5 Gy free in air yielded an approximately 40% higher RBE than a dose of 2 Gy inside the phantom

Conclusion

The measured values of RBE as function of dose per fraction within the phantom is consistent with the energy of the neutron beam. The increased RBE free in air, however, is greater than expected from microdosimetric parameters of the beam and may be due to slow recoil protons produced by interaction of multiply scattered neutrons or to an increased contribution of α particles from C(n,α) reactions near the surface. An enhanced RBE in subcutaneous layers of skin combined with an increase in RBE at low doses per fraction outside the target volume could potentially have significant consequences for normal tissue reactions in radiotherapy patients treated with fast neutrons.     

Radiobiological intercomparison of two clinical neutron beams using the regeneration of mouse intestinal crypts

Determination of dose modification factor greatly facilitates the introduction of clinically proven neutron therapy schedules at new installations. We have compared the biological performance of the p(66)+Be neutron facility at Faure, South Africa, with the established p(65)+Be installation at Louvain-la-Neuve, Belgium. Filtration, D gamma/DT, dose rate and HVT 5/15 for the Louvain and Faure beam are: 2 cm, 2.5 cm polyethylene; 3%, 5%; 0.2 Gy/min, 0.4 Gy/min; and 20 cm and 19 cm respectively. Dosimetry was done in A-150 plastic. Irradiation of BALB/C mice was carried on according to the dose accumulation method in a perspex phantom at 5 cm depth and at an SSD of 150 cm at a field size of 28 X 28 cm2. Sections of the jejunum were prepared at each centre and analyzed by both. The RBE of the Faure beam determined at a survival level of 50 crypts ranged from 1.64 to 1.69. The dose modification factor RBE of the Louvain beam given by Beauduin et al. was 1.61 +/- 0.14. The dose modification factor of the Faure beam relative to the Louvain beam is thus 1.03 +/- 0.13 which could be expected from the similarity of the physical characteristics. Independent RBE measurements in a variety of systems also suggest similar biological properties. The depth variation of the RBE was found to be 4% (mouse gut) using 3 cm polyethylene filter over the depth range of 2.5 to 13.5 cm. This is in agreement with microdosimetry measurements using polyethylene filters of various thicknesses and with V79 measurements reported by Slabbert et al.     

Clinical study of relative biological effectiveness for cervical carcinoma treated by 252Cf neutrons and assessed by histological tumour eradication

Clinical data of the University of Kentucky trial using californium (252Cf), or caesium (137Cs), are reviewed for dose-response based on the endpoint of tumour eradication estimated from hysterectomy specimens obtained 4-6 weeks after preoperative irradiation. These data are used to assess the relative biological effectiveness (RBE) for 252Cf neutrons compared with 137Cs gamma radiation. Tumours treated were of common stage but were of bulky or barrel shape suitable for "radiosurgical" therapy. Dose-response curves were constructed, and additional data from the literature used to analyse the curve shape. The photon dose-response curve is complex on a logarithmic plot, whereas the 252Cf neutron curve is exponential. This indicates that the RBE can be different depending on the number of implants, schedule and size of dose delivered per session. The RBE values were approximately 8.0 at low doses or for multiple implants but they may rise to approximately 16 at larger doses or for single 252Cf implants.