René Huiskamp

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 ∼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 1 cM interval between markers D2Mit126 and D2Mit185 by microsatellite analysis of 21 primary radiation‐induced F1 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. Genes Chromosomes Cancer 24:95–104, 1999.

Clinical phase-I study of NA2B12H11SH (BSH) in patients with malignant glioma as precondition for boron neutron capture therapy (BNCT)

PURPOSE Within the European collaboration on boron neutron capture therapy (BNCT), a clinical Phase I study is being carried out to establish BNCT as an alternative treatment modality for malignant glioma (WHO III/IV). Data about the pharmacokinetics, biodistribution and toxicity of the boron compound Na2B12H11SH (BSH) are of great importance to avoid radiation damage of healthy tissue and to deliver a sufficient radiation dose. METHODS AND MATERIALS Twenty four patients suffering from a glioblastoma multiforme entered the study to date, infused with a maximum concentration of up to 50 mg BSH/kg. Boron concentration measurements in tissues, urine, and blood were carried out, using inductively coupled plasma-atomic spectroscopy (ICP-AES) and quantitative neutron capture radiography (QNCR). A cross-calibration of these boron determination techniques was carried out. RESULTS In tumor tissue, confirmed by histopathology of small biopsies, we found a consistently high but heterogeneous boron uptake. Necrotic parts contain much lower amounts of boron; normal brain tissue has shown no significant uptake. In skin, bone, muscle, and dura mater only small amounts of boron were found. In blood samples, we found biphasic kinetics, but with variations of the half-lives from patient to patient. The compound is mainly excreted through the urine, but an additional entero-hepatic pathway can be demonstrated. Systematic investigations revealed no toxic side effect of the intravenously administered BSH. Comparable data were obtained by using ICP-AES and QNCR for boron concentration measurements. CONCLUSION Taking into account the radiobiological considerations of the neutron beam source, we found promising facts that BNCT could be a useful irradiation method for highly malignant brain tumors. Favorable amounts of the boron compound BSH were found in tumor tissue, whereas healthy brain tissue has shown no significant uptake.

Pre-exposure to Low Doses: Modulation of X-Ray-Induced DNA Damage and Repair?

Abstract Cramers, P., Atanasova, P., Vrolijk, H., Darroudi, F., van Zeeland, A. A., Huiskamp, R., Mullenders, L. H. F. and Kleinjans, J. C. S. Pre-exposure to Low Doses: Modulation of X-Ray-Induced DNA Damage and Repair? Radiat. Res. 164, 383–390 (2005). The adaptive response to ionizing radiation may be mediated by the induction of antioxidant defense mechanisms, accelerated repair or altered cell cycle progression after the conditioning dose. To gain new insight into the mechanism of the adaptive response, nondividing lymphocytes and fibroblasts were used to eliminate possible contributions of cell cycle effects. The effect of conditioning doses of 0.05 or 0.1 Gy followed by challenging doses up to 8 Gy (with a 4-h interval between exposures) on induction and repair of DNA damage was determined by single-cell gel electrophoresis (comet assay), premature chromosome condensation, and immunofluorescence labeling for γ-H2AX. The conditioning dose reduced the induction of DNA strand breaks, but the kinetics of strand break rejoining was not influenced by the conditioning dose in nondividing cells of either cell type. We conclude that adaptation in nondividing cells is not mediated by enhanced strand break rejoining and that protection against the induction of DNA damage is rather small. Therefore, the adaptive response is most likely a reflection of perturbation of cell cycle progression.

Uptake of two 10B-compounds in liver metastases of colorectal adenocarcinoma for extracorporeal irradiation with boron neutron capture therapy (EORTC Trial 11001).

Disseminated metastases of colorectal cancer in liver are incurable. The trial EORTC 11001 investigates whether autotransplantation after extracorporeal irradiation of the liver by boron neutron capture therapy (BNCT) might become a curative treatment option because of selective uptake of the compounds sodium mercaptoundecahydro‐closo‐dodecaborate (BSH) or L‐para‐boronophenylalanine (BPA). BSH (50 mg/kg bw) or BPA (100 mg/kg bw) were infused into patients who subsequently underwent resection of hepatic metastases. Blood and tissue samples were analyzed forthe 10B‐concentration with prompt gamma ray spectroscopy (PGRS). Three patients received BSH and 3 received BPA. Adverse effects from the boron carriers did not occur. For BSH, the highest 10B‐concentration was observed in liver (31.5 ± 2.7 μg/g) followed by blood (24.8 ± 4.7 μg/g) and tumor (23.2 ± 2.1 μg/g) with a mean 10B‐concentration ratio metastasis/liver of 0.72 ± 0.07. For BPA, the highest 10B‐concentration was measured in metastases (12.1 ± 2.2 μg/g) followed by liver (8.5 ± 0.5 μg/g) and blood (5.8 ± 0.8 μg/g). As BPA is transported actively into cells, viable, metabolically active cells accumulate exclusively this compound. Consequently, a model is proposed to adjust the values measured by PGRS for the proportion of viable cells to express the relevant 10B‐concentration in the tumor cells, revealing a 10B‐concentration ratio metastasis/liver of 6.8 ± 1.7. In conclusion, BSH is not suitable as 10B‐carrier in liver metastases as the 10B‐concentration in liver was higher compared to metastasis. BPA accumulates in hepatic metastases to an extent that allows for extracorporeal irradiation of the liver with BNCT.

Mutations of the PU.1 Ets domain are specifically associated with murine radiation-induced, but not human therapy-related, acute myeloid leukaemia

Murine radiation-induced acute myeloid leukaemia (AML) is characterized by loss of one copy of chromosome 2. Previously, we positioned the critical haematopoietic-specific transcription factor PU.1 within a minimally deleted region. We now report a high frequency (>65%) of missense mutation at codon 235 in the DNA-binding Ets domain of PU.1 in murine AML. Earlier studies, outside the context of malignancy, determined that conversion of arginine 235 (R235) to any other amino-acid residue leads to ablation of DNA-binding function and loss of expression of downstream targets. We show that mutation of R235 does not lead to protein loss, and occurs specifically in those AMLs showing loss of one copy of PU.1 (P=0.001, Fishers exact test). PU.1 mutations were not found in the coding region, UTRs or promoter of human therapy-related AMLs. Potentially regulatory elements upstream of PU.1 were located but no mutations found. In conclusion, we have identified the cause of murine radiation-induced AML and have shown that loss of one copy of PU.1, as a consequence of flanking radiation-sensitive fragile domains on chromosome 2, and subsequent R235 conversion are highly specific to this mouse model. Such a mechanism does not operate, or is extremely rare, in human AML.

The sensitivity of quiescent and proliferating mouse spermatogonial stem cells to X irradiation

The radiosensitivity of spermatogonial stem cells to X rays was determined in the various stages of the cycle of the seminiferous epithelium of the CBA mouse. The numbers of undifferentiated spermatogonia present 10 days after graded doses of X rays (0.5-8.0 Gy) were taken as a measure of stem cell survival. Dose-response relationships were generated for each stage of the epithelial cycle by counting spermatogonial numbers and also by using the repopulation index method. Spermatogonial stem cells were found to be most sensitive to X rays during quiescence (stages IV-VII) and most resistant during active proliferation (stages IX-II). The D0 for X rays varied from 1.0 Gy for quiescent spermatogonial stem cells to 2.4 Gy for actively proliferating stem cells. In most epithelial stages the dose-response curves showed no shoulder in the low-dose region.

Tissue uptake of BSH in patients with glioblastoma in the EORTC 11961 phase I BNCT trial.

Purpose: The uptake of the boron compound Na2B12H10-SH (BSH) in tumor and normal tissues was investigated in the frame of the EORTC phase I trial ‘Postoperative treatment of glioblastoma with BNCT at the Petten Irradiation Facility’ (protocol 11961).Methods and Materials: The boron concentration in blood, tumor, normal brain, dura, muscle, skin and bone was detected using inductively coupled plasma-atomic emission spectroscopy in 13 evaluable patients. In a first group of 10 patients 100 mg BSH/kg bodyweight (BW) were administered; a second group of 3 patients received 22.9 mg BSH/kg BW. The toxicity due to BSH was evaluated.Results: The average boron concentration in the tumor was 19.9±9.1 ppm (1 standard deviation (SD)) in the high dose group and 9.8±3.3 ppm in the low dose group, the tumor/blood ratios were 0.6±0.2 and 0.9±0.2, respectively. The highest boron uptake has been detected in the dura, very low uptake was found in the bone, the cerebro-spinal fluid and especially in the brain (brain/blood ratio 0.2±0.02 and 0.4±0.2). No toxicity was detected except flush-like symptoms in 2 cases during a BSH infusion at a much higher speed than prescribed.Conclusion: BSH proved to be safe for clinical application at a dose of 100 mg BSH/kg infused and at a dose rate of 1 mg/kg/min. The study underlines the importance of a further investigation of BSH uptake in order to obtain enough data for significant statistical analysis. The boron concentration in blood seems to be a quite reliable parameter to predict the boron concentration in other tissues.

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

Large animal studies have been utilized to definetolerance of normal brain to irradiation and verifytreatment planning programs with two recently installed epithermalneutron beams.The normal brain tolerance studies utilized two biologicalendpoints, magnetic resonance visible damage only and neurologicsigns progressing to death. The studies focused ondefining the proton RBE for the contaminant fastneutrons, and from nitrogen capture of thermal neutronsand boron capture reaction biologic effect. The protonRBE was approximately 3.0 to 6.7, depending onwhether a dose reduction factor for the lowgamma dose rate was employed.The microscopic distribution of the boron compounds, coupledwith the extremely short length of the fissionfragments from thermal neutron capture by 10B yieldsan observed biologic effect much less than wouldbe expected from such high LET irradiation. Thisobserved biologic effect, which is a product ofthe microdistribution of the boron atom and therelative biologic effect of the fission fragments hasbeen termed compound factor. The compound factor wasbased on the calculated physical dose from thefission fragment in blood based on measured blood10B concentration. The approximate compound factor for BSHwas studied at the two institutions and itranged from 0.27 to 0.55, depending on thesite and the endpoint chosen. The mean compoundfactor for BPA was only studied at onesite and was found to be 1.1 forboth endpoints. The increase in the compound factorfor BPA is in keeping with previous calculationsbased on the differences in compound distribution. Resultsof these studies has helped the initiation ofphase I and phase II clinical trials atBrookhaven National Laboratory and the planned European clinicaltrials at Petten, The Netherlands.

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.

Postoperative treatment of glioblastoma with BNCT at the Petten Irradiation Facility (EORTC Protocol 11961)

SummaryThe boron neutron capture therapy is based on the reaction occuring between the isotope10B and thermal neutrons. A low energy neutron is captured by the nucleus and it disintegrates into two densly ionising particles, Li nucleus and He nucleus (α 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 Netherland. 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 occured 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 todate. 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.