Chromosomal aberrations in peripheral blood lymphocytes exposed to a mixed beam of low energy neutrons and gamma radiation.

Cells exposed to thermal neutrons are simultaneously damaged by radiations with high and low linear energy transfer (LET). A question relevant for the assessment of risk of exposure to a mixed beam is whether the biological effect of both radiation types is additive or synergistic. The aim of the present investigation was to calculate whether the high and low LET components of a thermal neutron field interact when damaging cells. Human peripheral blood lymphocytes were exposed to neutrons from the HB11 beam at the Institute for Energy and Transport, Petten, Netherlands, in a 37 °C water phantom at varying depths, where the mix of high and low LET beam components differs. Chromosomal aberrations were analysed and the relative biological effectiveness (RBE) values as well as the expected contributions of protons and photons to the aberration yield were calculated based on a dose response of aberrations in lymphocytes exposed to (60)Co gamma radiation. The RBE for 10 dicentrics per 100 cells was 3 for mixed beam and 7.2 for protons. For 20 dicentrics per 100 cells the respective values were 2.4 and 5.8. Within the limitations of the experimental setup the results indicate that for this endpoint there is no synergism between the high and low LET radiations.

Recovery capacity of glial progenitors after in vivo fission-neutron or X irradiation: age dependence, fractionation and low-dose-rate irradiations.

Previous experiments on the radiosensitivity of O-2A glial progenitors determined for single-dose fission-neutron and X irradiation showed log-linear survival curves, suggesting a lack of accumulation of recovery of sublethal damage. In the present study, we addressed this question and further characterized the radiobiological properties of these glial stem cells by investigating the recovery capacity of glial stem cells using either fractionated or protracted whole-body irradiation. Irradiations were performed on newborn, 2-week-old or 12-week-old rats. Fractionated irradiations (four fractions) were performed with 24-h intervals, followed by cell isolations 16- 24 h after the last irradiation. Single-dose irradiations were followed by cell isolation 16-24 h after irradiation or delayed cell isolation (4 days after irradiation) of the O-2A progenitor cells from either spinal cord (newborns) or optic nerve (2- and 12-week-old rats). Results for neonatal progenitor cell survival show effect ratios for both fractionated fission-neutron and X irradiation of the order of 1.8 when compared with single-dose irradiation. A similar ratio was found after single-dose irradiation combined with delayed plating. Comparable results were observed for juvenile and adult optic nerve progenitors, with effect ratios of the order of 1.2. The present investigation clearly shows that fractionated irradiation regimens using X rays or fission neutrons and CNS tissue from rats of various ages results in an increase in O-2A progenitor cell survival while repair is virtually absent. This recovery of the progenitor pool after irradiation can be observed at all ages but is greatest in the neonatal spinal cord and can probably be attributed to repopulation.

Analysis of loss of heterozygosity in lymphoma and leukaemia arising in F1 hybrid mice locates a common region of chromosome 4 loss.

Previous studies have identified five lymphoma-related tumour suppressor gene regions on murine chromosome 4. Using detailed allelotype analysis on a range of lympho-haematopoietic tumour types arising in F1 hybrid mice, we now show a consistent pattern of loss of heterozygosity (LOH) which identifies a common region of loss delineated by microsatellites D4Mit21 and D4Mit53 on proximal chromosome 4. This critical segment corresponds to the thymic lymphoma tumour suppressor region 5 (TLSR5) identified in an earlier study. Tumours of this type have also been reported as showing allelic loss from the Trp53 and Ikaros regions on chromosome 11. In the present study, only a small fraction of tumours showed LOH in the Ikaros region, while a minority of lymphomas, but not acute myeloid leukaemias, showed allelic loss of the chromosome 11 segment encoding Trp53. These and other data indicate strongly that the genomic regions identified as showing recurrent LOH depend on the genetic background of the mice. Overall, the results indicate a key role for a tumour suppressor gene(s) encoded in an approximately 3 cM segment on proximal chromosome 4 and provide an experimental basis for the further investigation of the functional role of candidate genes which include Pax5 and Tgfbr1.

Microdosimetry model for boron neutron capture therapy: I. Determination of microscopic quantities of heavy particles on a cellular scale.

Due to the limitations of existing microdosimetry models, a new model called MICOR has been developed to analyze the spatial distribution of microscopic energy deposition for boron neutron capture therapy (BNCT). As in most existing models, the reactions independent of the incident neutron energy such as the boron and the nitrogen capture reactions can be considered. While other models do not include reactions that are dependent on the neutron energy such as the proton recoil reaction, the present model is designed so that the energy deposition resulting from these reactions is included. The model MICOR has been extended to enable the determination of the biological effects of BNCT, which cannot be done with the existing models. The present paper describes the determination of several microscopic quantities such as the number of hits, the energy deposition in the cell nucleus, and the distribution of lineal and specific energy deposition. The companion paper (Radiat. Res. 155, 000-000 2001) deals with the conversion of these microscopic quantities into biological effects. The model is used to analyze the results of a radiobiological experiment performed at the HB11 facility in the HFR in Petten. This analysis shows the value of the model in determining the dose depositions on a cellular scale and the importance of the extension to the energy deposition of the proton recoil.

Microdosimetry model for boron neutron capture therapy: II. Theoretical estimation of the effectiveness function and surviving fractions.

A model has been developed to obtain a better understanding of the effects of boron neutron capture therapy (BNCT) on a cellular scale. This model, the microdosimetry model MICOR, has been developed to include all reactions important for BNCT. To make the model more powerful in the translation from energy deposition to biological effect, it has been designed to be capable of calculating the effectiveness function. Based on this function, the model can calculate surviving fractions, RBE values and boron concentration distributions. MICOR has been used to analyze an extensive set of biological experiments performed at the HB11 beam in Petten. For V79 Chinese hamster cells, the effectiveness function is determined and used to generate surviving fractions. These fractions are compared with measured surviving fractions, which results in a good agreement between the measured and calculated surviving fractions (within the uncertainties of the measurements).

Age dependence of the radiosensitivity of glial progenitors for In vivo fission-neutron and X irradiation.

O-2A progenitor cells are the stem cells of the myelin-forming oligodendrocytes in the central nervous system. In the epithermal reactor beams used for boron neutron capture therapy (BNCT) for treatment of brain tumors, fission neutrons are a contaminating component. To estimate the radiosensitivity of the O-2A progenitors for fission neutrons, an in vivo-in vitro clonogenic assay was used. Radiosensitivity of progenitors obtained from the spinal cord of 1- or 5-day-old rats or the optic nerve of 2- or 12-week-old rats for 1 MeV fission neutrons was compared to that for 300 kVp X rays. Dose-survival curves were fitted according to the linear-quadratic model. The resulting beta component was very small to negligible. Progenitor cells obtained from rats of different ages show differences in radiosensitivity, characterized by different alpha values. RBE values for fission neutrons were 3.5 for 1-day-old spinal cord, 3.2 for 5-day-old spinal cord, 3.0 for 2-week-old optic nerve, and 4.3 for 12-week-old optic nerve. These high RBE values indicate the importance of minimizing the fast-neutron component in the epithermal neutron beams used for BNCT.

Postoperative treatment of glioblastoma with BNCT at the petten irradiation facility (EORTC protocol 11,961).

The boron neutron capture therapy is based on the reaction occurring between the isotope 10B and thermal neutrons. A low energy neutron is captured by the nucleus and it disintegrates into two densely ionising particles, Li nucleus and He nucleus (alpha particle), with high biological effectiveness. On the basis of comprehensive preclinical investigations in the frame of the European Collaboration with Na2B12H11SH (BSH), as boron delivery agent, the first European phase I, clinical trial was designed at the only available epithermal beam in Europe, at the High Flux Reactor, Petten, in the Netherlands. The goal of this study is to establish the safe BNCT dose for cranial tumors under defined conditions. BNCT is applied as postoperative radiotherapy in 4 fractions, after removal of the tumor for a group of patients suffering from glioblastoma, who would have no benefit from conventional treatment, but have sufficient life expectancy to detect late radiation morbidity due to BNCT. The starting dose is set at 80% of the dose where neurological effects occurred in preclinical large animal experiments following a single fraction. The radiation dose will be escalated, by constant boron concentration in blood, in 4 steps for cohorts of ten patients, after an observation period of at least 6 months after the end of BNCT of the last patient of a cohort. The adverse events on healthy tissues due to BSH and due to the radiotherapy will be analysed in order to establish the maximal tolerated dose and dose limiting toxicity. Besides of the primary aim of this study the survival will be recorded. The first patient was treated in October 1997, and further four patients have been irradiated to-date. The protocol design proved to be well applicable, establishing the basis for scientific evaluation, for performance of safe patient treatment in a very complex situation and for opening the possibility to perform further clinical research work on BNCT.

Molecular mapping of chromosome 2 deletions in murine radiation-induced AML localizes a putative tumor suppressor gene to a 1.0 cM region homologous to human chromosome segment 11p11-12.

Radiation-induced acute myeloid leukemias (AMLs) in the mouse are characterized by chromosome 2 deletions. Previous studies showed that a minimal deleted region (mdr) of approximately 6.5 cM is lost from one homologue in chromosome 2-deleted AMLs. An AML tumor suppressor gene is proposed to map within this mdr. In this study, we refine the mdr to a I cM interval between markers D2Mit126 and D2Mit185 by microsatellite analysis of 21 primary radiation-induced F I AMLs. The construction of a partial yeast artificial chromosome (YAC) contig spanning the mdr and the location of six known genes indicated that the 1 cM mdr is homologous to human 11p11-12, a region implicated in some human AMLs. Screening of five cell lines derived from primary radiation-induced AMLs for homozygous loss of microsatellites and genes mapping within the mdr revealed loss of both copies of the hemopoietic tissue-specific transcription factor Sfpi1(PU.1/Spi1) in one cell line. Studies of primary and F1 AMLs failed to implicate Sfpi1 as the AML tumor suppressor gene. YAC contig construction, together with data suggesting that the critical gene flanks Sfpi1, represents significant progress toward identifying an AML tumor suppressor gene.

Interaction of mercaptoundecahydrododecaborate (BSH) with phosphatidylcholine: relevance to boron neutron capture therapy.

The interaction of mercaptoundecahydrododecaborate (B12H11SH2-, BSH) with phosphatidylcholine was investigated in this study in order to illuminate possible uptake mechanisms of BSH in tumor cells. BSH has been used clinically in Japan as a boron containing agent in patients with malignant brain tumors for boron neutron capture therapy (BNCT). After infusion, BSH accumulates selectively in tumor tissue. Little is known for the mechanism of boron uptake to tumor cells. Fourier transform infrared (FTIR) spectrometry was used to quantify BSH (at wavenumber 2490 cm-1) and phosphatidylcholine (at wavenumber 2850-2970 cm-1). After extraction into carbon tetrachloride (CCl4), we could find an absorbance maximum at 2490 cm-1 as a B-H band in the mixture of BSH with phosphatidylcholine, which is attributed to a BSH-phosphatidylcholine complex, which could dissolve well in CCl4. The molar ratio of BSH to phosphatidylcholine in the CCl4 solution was at most one mole of BSH to two moles of phosphatidylcholine independent of the excess BSH. The doubly negatively charged BSH can interact with two phosphatidylcholine molecules through their singly positively charged choline residues. These ion pairs could be responsible for membrane binding and penetration, and for cell internalization.

The compound factor of the 10B(n,alpha)7Li reaction from borocaptate sodium and the relative biological effectiveness of recoil protons for induction of brain damage in boron neutron capture therapy.

To make clinical trials of boron neutron capture therapy safe for patients, it is necessary to know the relative biological effectiveness (RBE) of the radiation components and the compound factor of the boron carrier to be used. Here a method is derived to determine the RBE of recoil protons and the compound factor of compounds from in vivo experiments with different concentrations of boron. The method uses a simultaneous fit of both these parameters to all experimental data. This method is applied to the studies of tolerance of healthy tissue in dogs at the High Flux Reactor in Petten, The Netherlands. The RBE for the recoil protons generated by the neutrons present in the epithermal neutron beam [together with the RBE of the protons from the 14N(n,p)14C reaction] for induction of severe neurological symptoms was found to be 3.93+/-0.43 (95% confidence limits 3.06-4.79), and 2.33+/-0.14 (2.04-2.61) for induction of changes detectable by magnetic resonance imaging. The compound factor for Na2B12H11SH in brain tissue, using severe neurological symptoms as end point, was determined to be 0.37+/-0.06 (95% confidence limits 0.24-0.50). For changes detectable by magnetic resonance imaging, the value was found to be 0.65+/-0.04 (0.58-0.73).

A review: CNS effects and normal tissue tolerance in dogs.

Large animal studies have been utilized to define tolerance of normal brain to irradiation and verify treatment planning programs with two recently installed epithermal neutron beams. The normal brain tolerance studies utilized two biological endpoints, magnetic resonance visible damage only and neurologic signs progressing to death. The studies focused on defining the proton RBE for the contaminant fast neutrons, and from nitrogen capture of thermal neutrons and boron capture reaction biologic effect. The proton RBE was approximately 3.0 to 6.7, depending on whether a dose reduction factor for the low gamma dose rate was employed. The microscopic distribution of the boron compounds, coupled with the extremely short length of the fission fragments from thermal neutron capture by 10B yields an observed biologic effect much less than would be expected from such high LET irradiation. This observed biologic effect, which is a product of the microdistribution of the boron atom and the relative biologic effect of the fission fragments has been termed compound factor. The compound factor was based on the calculated physical dose from the fission fragment in blood based on measured blood 10B concentration. The approximate compound factor for BSH was studied at the two institutions and it ranged from 0.27 to 0.55, depending on the site and the endpoint chosen. The mean compound factor for BPA was only studied at one site and was found to be 1.1 for both endpoints. The increase in the compound factor for BPA is in keeping with previous calculations based on the differences in compound distribution. Results of these studies has helped the initiation of phase I and phase II clinical trials at Brook haven National Laboratory and the planned European clinical trials at Petten, The Netherlands.

Sensitivity of murine haemopoietic stem cell populations to X-rays and 1 MeV fission neutrons in vitro and in vivo under hypoxic conditions.

The radiosensitivity of primitive haemopoietic stem cells that repopulate the bone marrow with precursors of granulocytes and macrophages (MRA[CFU-C]), mature stem cells capable of forming spleen colonies in lethally irradiated recipients (CFU-S-7) and colony-forming units in culture (CFU-C) were determined in vitro and under hypoxic conditions in vivo for 1 MeV fission neutrons and 300 kV X-rays. The obtained D0's were compared with previously observed D0's after irradiation in vivo under normal oxic conditions. With 1 MeV fission neutron irradiation no significant difference in radiosensitivity of the cell populations was observed between normal in vivo irradiation and in vitro irradiation. With 300 kV X-rays a lower radiosensitivity for all three cell populations was observed after in vitro compared to in vivo irradiation. In vivo irradiation with fission neutrons under hypoxic conditions led to a small decrease in radiosensitivity. The obtained oxygen enhancement ratio (OER) for fission neutrons varied from 1.2 for MRA[CFU-C] to 1.5 for CFU-C. After in vivo irradiation with 300 kV X-rays under hypoxic conditions much higher OERs were observed. An OER = 1.8 was obtained for CFU-S and for MRA[CFU-C] and for CFU-C OER 3.0 and 2.9 were observed. These results indicate that the radioresistance of primitive haemopietic stem cells (MRA[CFU-C]) compared to mature stem cells (CFU-S-7) is mainly due to intrinsic factors and not to differences in localization or oxygenation between primitive and mature stem cells.

Chromosomal abnormalities in neutron-induced acute myeloid leukemias in CBA/H mice.

Acute myeloid leukemias (AMLs) induced in CBA/H mice by 1 MeV fission neutrons have been examined for chromosomal abnormalities by G-band analysis. In common with X-ray- and alpha-particle-induced AMLs in CBA/H mice, more than 90% (16/17) of the myeloid leukemias had chromosome 2 abnormalities, in this case, all interstitial deletions. Chromosome 2 breakpoints were not wholly consistent, but clustering in three specific G-band regions was observed. Very distal (H-region) breakpoints were more common in the neutron AMLs than in X-ray- or alpha-particle-induced leukemias. These data indicate that neutron-induced AMLs in CBA/H mice are not characterized by a specific chromosome deletion but that a variety of chromosome 2 deletion types are associated with the disease. Trisomy of chromosome 1(12.5% AMLs) and aneusomy of chromosomes 6 (31% AMLs) and Y (37.5% AMLs) were noted. While chromatid breakage was observed occasionally in neutron-induced AML, no clear indications of persistent chromosomal instability or high levels of stable chromosomal change were apparent.

Role of telomeric sequences in murine radiation-induced myeloid leukaemia.

A previous study indicated that a highly inbred CBA/H mouse colony contained four genotypic variants for telomere-like repeat (TLR) sequence arrays and that one variant subpopulation that constituted 20% of the colony contributed the vast majority (> 90%) of radiation-induced acute myeloid leukaemias (AMLs). Through screening of a satellite CBA/H colony and rescreening of the original colony, we show that, whereas germline telomere sequence polymorphism is frequent in CBA/H mice, there is no genetic link between a specific TLR locus variant and susceptibility to AML. Studies on telomere-hybridising fragments between 200 bp and 150 kb revealed that the germline telomere mutation frequency was highest for restriction fragments > 50 kb. The hypervariability of these high-molecular-weight fragments resulted in each CBA/H mouse from the highly inbred colony having a different genotype. Although it was not possible to ascribe a specific somatic telomere mutation to AML development, telomere rearrangements were common in induced AMLs. Some terminal telomere-hybridising restriction fragments were shortened in AML samples in comparison with normal tissue, but, insofar as the reduction in size was relatively small, it seems unlikely that telomere erosion is a major contributor to the molecular pathology of murine radiation-induced AML.

Microsatellite analysis of recurrent chromosome 2 deletions in acute myeloid leukaemia induced by radiation in F1 hybrid mice.

Deletions and/or rearrangements involving one copy of chromosome 2 are consistent and early events in the development of murine acute myeloid leukaemia (AML) by radiation. More than 90% of AMLs induced in the CBA strain of mice express such cytogenetic alterations, with chromosome 2 breakpoints clustering in the C and F regions of the chromosome. In inbred mouse strains, the molecular resolution of these breakpoints is problematic. However, by using x-ray-induced AMLs in FI progeny of genetically divergent CBA/H x C57BI, it has been possible to show region-specific loss of heterozygosity (LOH) in genetically linked sets of chromosome 2 microsatellite alleles from one of the two parental chromosomes. In the majority of cases, an acceptable concordance was shown for AML chromosome 2 deletion, as defined by microsatellites and as revealed by G-band cytogenetics. A degree of breakpoint clustering was found, but the identification of a number of deletion types, based on the position of proximal and distal breakpoints as defined by microsatellite analysis, strongly supports a leukaemogenic mechanism involving gene deletion. No bias towards loss of CBA or C57BI alleles was observed, and the gender of AML-presenting animals did not appear to influence the parental origin of the deletions. A molecular map of chromosome 2 breakpoints has now been established in FI AMLs as a first step towards the molecular cloning of breakpoint sequences.

A stochastic model for subcellular dosimetry in boron neutron capture therapy.

The therapeutic effectiveness of boron neutron capture therapy is highly dependent on the microscopic distribution of the administered boron compound. Two boron compounds with different uptake mechanisms in the tumour cells may thus cause effects of different degrees even if the macroscopic boron concentrations in the tumour tissue are the same. This difference is normally expressed quantitatively by the so-called relative local efficiency (RLE). In this work, a stochastic model for the subcellular dosimetry has been developed. This model can be used to calculate the probability for an energy deposition above a certain threshold level in the cell nucleus due to a single neutron capture reaction. If a threshold cell-kill function is assumed, and if the dose is low enough that multiple energy depositions are rare, the model can also be applied to calculations of the survival probability for a cell population. Subcellular boron distributions in rats carrying RG 2 rat gliomas were measured by subcellular fractionation after administration of two different boron compounds: a sulphydryl boron hydride (BSH) and a boronated porphyrin (BOPP). Based on these data, the RLE factors were then calculated for these compounds using the stochastic model.

Performance of sulfhydryl boron hydride in patients with grade III and IV astrocytoma: a basis for boron neutron capture therapy.

This study investigated the rationale of boron neutron capture therapy (BNCT) for the treatment of Grade III and IV astrocytoma. The European Community joint research program on BNCT plans to use sulfhydryl boron hydride (BSH) in clinical trials. The work presented here, examines the performance of BSH in eight patients with Grade III and IV astrocytoma using a measurement technique which precisely correlates the boron uptake with the histology of the tumor and the peritumoral brain. Astrocytomas are exceptionally heterogeneous and spread migrating tumor cells into the surrounding brain. The patients were infused with 50 mg BSH per kilogram of body weight at 12, 18, 24 or 48 hours before surgery. At the time of operation, specimens were obtained of the tumor, skin, muscle, dura, blood, urine, and, when surgically possible, the brain adjacent to tumor. In three patients the intracellular boron distribution was investigated by subcellular fractionation. The blood clearance was biphasic with half-lives of 0.6 and 8.2 hours. After 3 days, approximately 70% of the dose injected was excreted in the urine. The maximum boron concentration in the tumor was 20 ppm, 12 hours after the infusion. The tumor-to-blood ratios ranged between 0.2 and 1.4, with the highest values after 18 to 24 hours. In the brain specimens the boron concentration never exceeded 1 ppm. This work confirms a selective uptake of boron in the tumor compared to the surrounding brain and that boron, to some extent, is incorporated in the tumor cells.

Monitoring of blood-10B concentration for boron neutron capture therapy using prompt gamma-ray analysis.

The aim of the present study was to monitor the blood-10B concentration of laboratory dogs receiving boron neutron capture therapy, in order to obtain optimal agreement between prescribed and actual dose. A prompt gamma-ray analysis system was developed for this purpose at the High Flux Reactor in Petten. The technique was compared with inductively coupled plasma-atomic emission spectrometry and showed good agreement. A substantial variation in 10B clearance pattern after administration of borocaptate sodium was found between the different dogs. Consequently, the irradiation commencement was adjusted to the individually determined boron elimination curve. Mean blood-10B concentrations during irradiation of 25.8 +/- 2.2 micrograms/g (1 SD, n = 18) and 49.3 +/- 5.3 micrograms/g (1 SD, n = 17) were obtained for intended concentrations of 25 micrograms/g and 50 micrograms/g, respectively. These variations are a factor of two smaller than irradiations performed at a uniform post-infusion irradiation starting time. Such a careful blood-10B monitoring procedure is a prerequisite for accurately obtaining such steep dose-response curves as observed during the dog study.

Pharmacokinetics in melanoma-bearing mice of 5-dihydroxyboryl-6-propyl-2-thiouracil (BPTU), a candidate compound for boron neutron capture therapy.

Blood pharmacokinetics and tissue distribution of 5-dihydroxyboryl-6-propyl-2-thiouracil (BPTU), a boron carrier with postulated melanin-seeking properties for boron neutron capture therapy, were determined in C57/BL mice with subcutaneous pigmented or non-pigmented B16 melanomas. Borocaptate sodium (BSH) was used as a boron compound without melanin-seeking properties in a comparative biodistribution study in the same animal tumour models. Administration of single doses showed that BPTU was retained better in the pigmented B16 tumour than in the non-pigmented variant. BPTU was found in large concentrations in kidney and liver. Brain boron was approximately 10-fold lower than tumour boron. On a molar basis, BPTU demonstrated higher affinity for B16 tumours than BSH. Owing to solubility limits, tumour boron concentrations in this mouse study were too low for effective application of BNCT. However, the high tumour-to-blood and tumour-to-normal tissues ratios indicate that, with appropriate formulation, BPTU could be a promising candidate for clinical BNCT.